WO2016051166A1

WO2016051166A1 - Devices and methods for selection and culture of microorganisms

Info

Publication number: WO2016051166A1
Application number: PCT/GB2015/052845
Authority: WO
Inventors: Brian Selby Hartley
Original assignee: Brian Selby Hartley
Priority date: 2014-10-01
Filing date: 2015-09-30
Publication date: 2016-04-07
Also published as: GB201417362D0

Abstract

Devices for selection of microbial strains, comprising a plurality of fermenter compartments for the culture of microbial cells, wherein the fermenter compartments are linked to provide a series of fermenter compartments in fluid connection, such that microbial cells are successively transferable through each fermenter compartment, in the series. Methods of selecting microbial strains using a device of the invention.

Description

DEVICES AND METHODS FOR SELECTION AND CULTURE OF MICROORGANISMS

Field of the invention

The invention relates to devices for selection of microbial strains, and in particular to methods and uses of the devices of the invention for selection of improved strains of Geobacillus for bioethanol production, and for isolation of microbial strains for antibiotic production.

Background to the invention

Strain improvement bv culture selection

Microbial cells can be grown in aqueous culture medium using batch, fed-batch, or continuous culture. In batch culture (e.g. shake flask culture), microbial cells are inoculated into a fixed volume of culture medium. In fed-batch culture, the medium is added gradually, In both batch culture and fed-batch culture systems, the medium composition changes continuously throughout growth.

By contrast, continuous culture is an open system in which the cells are grown in a flow system of constant volume, to which culture medium is continually added and from which the broth is continuously removed by an overflow. Eventually a steady state is reached in which the cell growth rate equals the cell overflow rate and the broth composition is constant.

Since its invention (Monod, 1949; Herbert et al. 1956) continuous culture has therefore been a useful tool for studying microbial physiology. Moreover, growth of microbial cells in continuous culture can rapidly select for the microbial strain that grows fastest on a particular feedstock under chosen culture conditions (Hartley et al., 1972). However, continuous culture has only occasionally been used for selection of improved microbial strains; for example, for selection of increased ethanol tolerance in strains of yeast (Brown & Oliver, 1982). This may be because standard laboratory continuous culture fermenters generally require large volumes of sterilised medium, and higher levels of technical supervision than batch cultures such as shake flask cultures. Furthermore, many sequential rounds of random mutagenesis and continuous culture selections with increasingly stringent selective pressure may be needed to achieve the desired result.

Consequently, microbial strain improvement has predominantly been by random

mutagenesis followed by batch culture and a selection step comprising plate selection and colony screening. This can be time-consuming and laborious, and a variety of mutants are simultaneously selected in the selection step, which must then be individually compared. Improved strains for bioethanol production

Bioethanol is a renewable additive or substitute for gasoline that can be produced from agricultural feedstocks. Current ethanol fermentations use yeasts or Zymomonas strains to convert certain sugars to ethanol and carbon dioxide. In 'First Generation' bioethanol production processes, the sugars are derived from cereal starches, sugar cane, sugar beet or sweet sorghum, all of which have high commercial value as human and/or animal foods and are therefore relatively expensive. Therefore there have been major efforts to develop 'Second Generation' processes that utilise instead sugars derived from waste biomass, most of which yeasts cannot use. Such waste biomass is composed mainly of lignocellulose, which includes cellulosic fibres (around 45% of the dry weight of the biomass) coated with a waterproof coat of lignin (around 10%), embedded in a loose pith of hemicellulose fibres (around 35%).

Most such research efforts into 'Second Generation' processes have focussed on using cellulose as the sugar source, since it can be hydrolysed to glucose which yeasts can ferment. However lignocellulose hydrolysis processes are energy consuming and expensive, since the fibres are waterproofed and rigidly cross-linked by hydrogen bonds. Mechanical degradation and/or strong alkali treatment are required to remove the lignin from the cellulose and separate the cellulose fibres (e.g. the Kraft alkali process, used in paper manufacture). Even then, the rigid cellulose fibres are only slowly hydrolysed by enzymes to glucose. The whole process is slow, energy intensive and highly polluting, so cellulosic ethanol is not even competitive with 'First Generation' bioethanol. Combustion for heat and/or electricity production remains the most commercially viable prospect for renewable energy from cellulose and lignin (Aristou er a/, 2012).

By contrast, hemicelluloses, which are almost as abundant as cellulose in waste biomass, are of little commercial value and are readily hydrolysed by enzymes. Hydrolysis of hemicelluloses yields a mixture of 'hemicellulosic sugars' comprising mainly C5 sugars such as xylose and arabinose, which yeasts and Zymomonas cannot ferment. Despite considerable efforts to discover or engineer yeast or Zymomonas strains that can ferment C5 sugars, none have so far been commercially successful.

An alternative strategy for producing ethanol from hemicelluloses is to use mutant strains of Geobacilli; a class of thermophilic bacteria that are abundant in compost heaps. Geobacilli are almost unique in that their sugar uptake is unregulated and they consume hemicellulosic sugars very rapidly in both aerobic and anaerobic conditions. They produce mainly carbon dioxide and lactic acid, plus energy which is dissipated as heat, by futile cycling of ATP synthesis via glycolysis and subsequent hydrolysis to ADP (Aristou, 2012).

In anaerobic conditions mutant Geobacilli that lack lactate dehydrogenase (LDH) activity (LDH^~ Geobacilli) can consume hemicellulosic sugars using the pyruvate-formate lyase (PFL) pathway which results in excretion of formate and acetate, and thereby produce high yields of ethanol from biomass by a pyruvate dehydrogenase (PDH) overflow pathway (Hartley, 1988) (Figure 1 ).

However a barrier to commercial exploitation of LDH- Geobacil \r\ bloethanol production is that they die rapidly at the high concentrations of sugar required for commercial production of bioethanol. This is because unregulated sugar uptake exceeds the maximum possible flux through the pyruvate dehydrogenase (PDH) pathway. In this situation, pyruvate accumulates and is excreted as illustrated in Figure 1. This causes metabolic imbalance which at a critical point results in a catastrophic drop in the NAD/NADH causing instant 'redox death' (Aristou et al. (2012).

Isolation of microbial strains for antibiotic production

Environmental microorganisms (e.g. those in soils) contain many as yet unknown microbial species that will not grow in standard media on Petri dishes (so-called "uncultivatable" microorganisms). Rondon et al., (2000) pioneered a novel approach for studying these by constructing a meta-genomic library of mixed soil DNAs in E.coii. From this mixture, Gillespie et al., (2002) isolated a pigmented colony and showed that purified pigments, which they named turbomycin A and B, had antibiotic activity against both gram-negative and gram-positive organism.

This discovery stimulated a search for other antibiotic-producing microorganisms in soils. A significant breakthrough to cultivation of these has been made by the iChip invention of Nichols ef al., (2010). The iChip is a stack of plastic plates containing hundreds of small holes which can be inoculated with soil cells simply by dipping them into a dilute suspension of soil microorganisms. The dilution is such that, on average, less than one cell is drawn into each hole, which is then sealed with liquid agar. Then the stack is tightly sandwiched between two dialysis membranes and suspended in wet soil, which will supply all necessary growth factors. These will diffuse into the holes so as to provide a set of micro fed-batch cultures. After suitable incubation, individual clones can be withdrawn and grown up in wet soil for further study. Use of this device has already yielded a promising new antibiotic, teixobactin (Ling ef al, 20 5).

Summary of the invention

The present invention provides devices and methods for the selection and culture of microorganisms. In particular, the invention provides devices and methods for the selection of microbial strains having desired characteristics.

The present invention allows continuous culture to be used efficiently for the selection of microbial strains; in particular it allows efficient selection of Geobacillus strains with characteristics useful for bioethanol production, such as resistance to "redox death". The invention also enables the culture of microorganisms which are difficult to grow on standard laboratory media, thereby facilitating the identification and isolation of strains capable of producing antibiotics as explained in more detail below.

Accordingly, the present invention provides a device for selection of microbial strains, comprising a plurality of fermenter compartments for the culture of microbial cells, wherein the fermenter compartments are linked to provide a series of fermenter compartments in fluid connection, such that microbial cells are successively transferable through each fermenter compartment in the series. A fermenter compartment may have a volume of about 5 μΙ to 200 ml.

In a development, the present invention provides a device for selection of microbial strains, which device may be referred to herein as an "autoselector" or "autoselector device". The device comprises a plurality of fermenter compartments for continuous culture of microbial cells. The fermenter compartments are linked to form a series of fermenter compartments in fluid connection, such that microbial cells are successively transferable through each fermenter compartment in the series. A fermenter compartment may have a volume of about 10 to about 200 ml, or about 40 to 200 ml.

In another development, the present invention provides a device for selection of microbial strains wherein the fermenter compartments comprise a coil suitable for segmented flow. These devices may be referred to herein generally as "segmented flow devices", or as a "miniselector" or "miniselector device". The device comprises a plurality of fermenter compartments each comprising a coil suitable for segmented flow. Cells are carried in segments of broth which flow through the coil. The fermenter compartments are linked to form a series of fermenter compartments in fluid connection, such that microbial cells are successively transferable through each fermenter compartment in the series. A segmented flow device compartment may have a volume of about 5μΙ - 50ml. A miniselector fermenter compartment may have a volume of about 5 -50ml, and may be suitable for holding segments having a volume of about 100 - 1000 μΙ. A microselector fermenter compartment may have a volume of about 5-1000 μΙ, and may be suitable for holding segments having a volume of about 1-10 μΙ. The present invention also provides a method of selecting a microbial strain by providing a device of the invention, adding microbial cells to a fermenter compartment, and applying a selection gradient across the series of fermenter compartments. The method may also comprise a step of initial picking (i.e. choosing, selecting) of an inoculum of microbial cells for addition to a fermenter compartment. The present invention also provides a use of a device according to the invention, for the selection of microbial strains.

Accordingly, the invention provides a method of selecting a microbial strain, the method comprising providing a device of the invention, picking an inoculum of microbial cells, and adding the inoculum of microbial cells to a first fermenter compartment of the series of fermenter compartments. The method may further comprise applying a selection gradient across the series of fermenter compartments.

In use, a device of the invention comprises microbial cells. In use, the culture conditions in each fermenter compartment are chosen such that a selection gradient is applied across the series of fermenter compartments. In a selection gradient a culture condition varies progressively between each fermenter in the series. A selection gradient may be an increasing or decreasing gradient of temperature, pH, aeration, dilution rate, feed rate or concentration of a feedstock, or concentration of a toxin or growth inhibitor.

In this way, the device enables the application of a selection gradient (a gradient of a condition which applies an evolutionary selection pressure) to a culture of microbial cells. In this way, the device enables the application of a selection gradient (a gradient of a condition which applies an evolutionary selection pressure) to a continuous culture of microbial cells. This allows for selection of microbial strains having characteristics that enable them to survive in particular culture conditions. In each fermenter compartment the culture conditions are substantially constant, but across the series of fermenter compartments the culture conditions vary in a progressive way. Preferably in each fermenter compartment in the series the culture conditions are substantially identical except for a single culture condition which varies in a progressive way across the series of fermenter compartments. This enables the selection of microbial strains having improved growth in particular culture conditions, for example high or low temperature, high or low pH, low aeration, high feedstock concentration, high dilution rate, or high toxin concentration.

A device of the invention automatically selects for microbial strains having a desired characteristic. Unlike batch culture, continuous culture automatically selects the single fastest growing mutant strain once a steady state is established. In use of a device of the invention, an inoculum of microorganisms are added to the first fermenter compartment in the series, a selection gradient is applied across the series, and culture broth is transferred from the first fermenter compartment through each fermenter compartment in the series. Preferably portions of culture broth are transferred from the first fermenter compartment through each fermenter compartment in the series. Preferably the selection gradient is applied such that the cells of the inoculum are capable of growing in the first fermenter compartment in the series, but are not capable of growing in the final fermenter

compartment in the series. During operation of the device, cells from the first fermenter compartment are continuously transferred through subsequent fermenter compartments until eventually a mutant cell arises that is capable of growing in the final fermenter compartment in the series. This mutant cell will form the basis of a mutant strain population that forms an essentially homogenous population in the final fermenter compartment in the series.

Preferably the selection gradient is applied such that the cells of the inoculum are capable of growing in the first fermenter compartments in the series until they reach a compartment in which the dilution rate exceeds the maximum growth rate under the increasingly stringent selection conditions. Thereafter the cell density will decline in successive compartments and residual feedstock concentration will rise. However any faster-growing mutant cell will then displace the displace the original inoculum cells until they take over the whole compartment, as demonstrated by Hartley et al. (1972). In this way, microbial strains capable of growing under particular conditions are selected with minimal human intervention or supervision.

A device of the present invention is "bench top" device, meaning that the overall size of the device is relatively small such that it can be placed on a standard laboratory bench during use.

An autoselector device of the present invention is a "bench top" device, meaning that the overall size of the device is relatively small such that it can be placed on a standard laboratory bench during use. The number of fermenters and the size and volume of each will govern its dimensions, but in some embodiments a device comprising three to seven fermenters each of working volume around 10 to 200 ml will be convenient, and a working volume of 40 to 100 ml may be particularly preferred. However for some purposes, such as Pilot Plant fermentations, larger commercial fermenters having volumes of around 1 to 20 litres may be used.

In a development of the invention a fermenter compartment comprises a coil of tubing, for segmented flow, through which flow segments of culture broth separated by bubbles of gas. Each segment acts as a tiny batch fermenter, so the residence time of each segment within the coil is inversely equivalent to the dilution rate in a conventional continuous culture. The volume of each segment may be about 250 μΙ (or about 100 μΙ to about 1000 μΙ), and the total working volume of the fermenter compartment may be about 5ml (or about 5 ml to about 50 ml), such a fermenter may be referred to as a miniselector. The volume of each segment may be about 2 μΙ (or about 1 μΙ to about 10 μΙ), and the total working volume of the fermenter compartment may be about 200 pi (or about 5 μΙ to about 1000 μΙ), such a fermenter may be referred to as a microselector. In embodiments of this development, the working volume of a fermenter compartment is preferably from about 5 μΙ to about 50 ml. The relatively small size of each device means that it can be used in a wide variety of research environments, because it occupies a relatively small space and requires less supervision than typical laboratory continuous culture fermenters, which have volumes of at least 1-10 litres. Moreover it can be operated using a relatively low volume of culture medium, which reduces the operating costs and supervision required.

Preferably the device is a modular assembly, wherein each module comprises a fermenter compartment. Such a modular assembly allows for ease of change of configuration of the device because if facilitates changing of the links between fermenter compartments. Such a modular assembly also allows for ease of disassembly of the device for sterilisation. A device which is a modular assembly is easily scalable, and the number of fermenter compartments can be varied according to the desired configuration.

The present invention provides a method of selecting a microbial strain. The method comprises providing a device of the invention and adding one or more microbial cells to a first fermenter compartment in the series of fermenter compartments. The cells added to the first fermenter compartment may be referred to as an inoculum. The method further comprises applying a selection gradient across the series of fermenter compartments. A selection gradient, or culture condition gradient, may be an increasing or decreasing gradient of temperature, pH, aeration, concentration of a feedstock, dilution rate, concentration of an environmental water, or concentration of a toxin. A toxin in this context may be an antibiotic, thus the present invention may be used for screening antibiotics against a pathogen. The present invention may be used for screening antibiotics against a pathogen from a patient's infection, thereby identifying a suitable antibiotic for treating the patient. The present invention also provides a use of a device of the invention for selecting a microbial strain, the use comprising the method steps disclosed herein.

Selection of microbial strains in the context of the present invention refers to the culture of microbial cells grown in particular culture conditions that permit only cells with certain characteristics to survive, thereby enabling their isolation (separation from cells that do not have those characteristics). In the context of the present invention the selection of microbial strains refers to the selection of improved microbial strains, that is, microbial strains having a desired characteristic. Preferably that characteristic improves the usefulness of the microbial strain in the bioethanol production. Alternatively, that characteristic may be the production of an antibiotic. A microbial strain is a group of microbial cells sharing a certain characteristic, which characteristic distinguishes the cells from other groups of microbial cells belonging to the same species or variety. In the present context, selection of microbial strains may also be referred to as selection of mutant strains, selection of mutant cells, or selection of mutants.

Selection of microbial strains in the context of the present invention may also refer to the culture of microbial cells from a population in particular culture conditions that enable their isolation and / or the identification of growth factors required for their culture. This is particularly useful in the culture of microorganisms that are difficult to grow on standard laboratory media, many environmental microorganisms fall into this category. It is a known phenomenon that only a small proportion of the world's microorganisms are capable of growing on laboratory media. However, these microorganisms represent a potential source of novel antibiotics, the demand for which is growing increasingly urgent. The difficulty of culturing many microorganisms on standard laboratory media therefore represents an obstacle to the identification and isolation of new antibiotics. The present invention provides devices and methods for culturing such microorganisms and for identifying factors that promote their growth.

A method of the invention may comprise a step of isolating a microbial strain that has been selected, the isolation step is performed after the selection gradient has been applied in order to select a microbial strain. The method may further comprise a step of producing bioethanol using the microbial strain that has been isolated.

The microbial cells used in the device and methods of the invention in connection with bioethanol production are preferably Geobacil!us cells, and preferably LDH^~ Geobacillus cells. Preferably the Geobacillus cells are Geobacillus thermoglucosidasius, and more preferably Geobacillus thermoglucosidasius strain LLD-R. However the device and methods described herein are suitable for the selection of many types of microbial cell.

Terminology

A "microbe" or "microbial cell", also known as a microorganism, is a microscopic organism consisting of a single cell. These include eukaryotes such as yeasts, amoebae or some algae, all prokaryotes and all archeae. In the context of the present invention, a microbe is preferably a prokaryote, and preferably a Geobacillus.

The term "Geobacillus" used herein refers to the group of thermophilic bacteria, commonly referred to in the literature as a thermophilic Bacillus. It includes Geobacillus

stearothermophilus, which was formerly known as Bacillus stearothermophilus, and Geobacillus thermoglucosidasius strain LLD-R, formerly known as Bacillus

stearothermophilus strain NCIMB 12403.

An "LDH- Geobacillus" is a Geobacillus lacking L-lactate dehydrogenase (LDH) activity. An LDH- Geobacillus may lack lactate dehydrogenase genes, or have one or more mutations in lactate dehydrogenase genes such that they lack functional lactate dehydrogenase enzyme. One such LDH- Geobacillus strain is LLD-15, formerly known as Bacillus stearothermophilus strain LLD-15 (NCIMB 12428).

The term "culture medium ", or "medium", is used herein to refer to liquid culture medium, which is an aqueous solution of various nutrients suitable for the growth or maintenance of a cell culture. Many types of microbial culture medium are known in the art.

Fresh and/or sterile culture medium refers to culture medium free from microbial cells. In the devices and methods of the invention, the culture medium is preferably sterile culture medium. The term "culture broth", or "broth", is used herein to refer to culture medium containing microbial cells. The term "effluent broth" is used to refer to culture medium that exits a fermenter compartment via an outlet. For example effluent broth may be culture medium that is transferred from a fermenter compartment to the next (adjacent) fermenter compartment in the series. Effluent broth may be culture medium that exits a fermenter compartment for waste disposal and or collection and analysis. For example, in uses of the device for the culture of environmental microorganisms effluent broth may be collected for determination of antibiotic activity.

An "aerobic culture" in the context of the present invention may mean a culture containing an excess of dissolved oxygen, so that growth by the PDH pathway is sufficient to oxidise all sugars to CO₂. An aerobic culture may be aerated with air (20% oxygen). The dissolved oxygen concentration varies with medium temperature and composition.

The term "semi-aerobic" may mean a culture containing insufficient dissolved oxygen so that growth by the PDH pathway is insufficient to oxidise all sugars to CO2, and some anaerobic products such as lactate or ethanol are produced. A semi-aerobic culture may be aerated with a mixture of air and nitrogen and/or CO2 containing 10% oxygen, or 15% oxygen. The term "micro-aerobic" may mean a culture supplied with very little oxygen, so that growth is by the PDH pathway and controlled by the rate of oxygen supply. All other sugars are converted to anaerobic products. A micro-aerobic culture may be aerated with a mixture of air and nitrogen and/or CO2 containing less than 5% oxygen

The term "anaerobic" means a culture supplied with no oxygen. In anaerobic Geobacillus cultures growth is by the PFL pathway. The term "anaerobic" may refer to conditions under which the dissolved oxygen concentration is too low for a microorganism to use oxygen as a terminal electron acceptor.

The term "feedstock" in the present context may refer to the raw material (such as biomass) used in the production of a desired product (such as bioethanol). The term feedstock may also be used to refer to a particular nutrient (such as glucose or xylose) or group of nutrients, (such as biomass sugars, hemicellulose sugars, C5 or C6 sugars).

The term "C5 sugars" refers to pentose sugars, which have five carbon atoms, such as arabinose, ribose or xylose.

The term "C6 sugars" refers to hexose sugars, which have six carbon atoms, such as glucose, fructose or galactose.

The term "redox death" refers to a phenomenon observed in LDH^{^} Geobacillus cultured in conditions that are both non-aerobic (i.e. low oxygen conditions: anaerobic, micro-aerobic, or semi-aerobic conditions) and relatively high sugar concentration. This phenomenon occurs because unregulated sugars uptake eventually exceeds the maximum possible flux through the PDH pathway. In this situation pyruvate then accumulates and is excreted as illustrated in Figure 1. That creates metabolic imbalance, causing the NAD/NAD H ratio to drop. At a critical point, there is insufficient NAD to maintain adequate ATP production from glycolysis. This leads to a collapse in membrane potential and a catastrophic 'redox death" (Aristou et a/. 2012). Since high sugar concentrations and low oxygen conditions are desirable for commercial ethanol production, the sensitivity of LDH- Geobacilli to "redox death" represents a significant obstacle to the commercial exploitation of these microbial strains in bioethanol production. Brief description of the drawings

Figure 1. Anaerobic pathways in Geobacilli that lack lactate dehydrogenase activity (LDH- Geobacilli). PFL = the pyruvate-formate lyase growth pathway which produces formate and acetate, which are excreted. PDH = the pyruvate dehydrogenase overflow pathway. The broken arrow illustrates that excess pyruvate is secreted when the PDH flux is saturated

Figure 2. Micro-aerobic pathways in LDH^~~ Geobacillus strains. The PDH pathway on the left operates in micro-aerobic conditions to produce ATP for cell growth. Oxygen suppresses the anaerobic PFL growth pathway so growth is controlled by the oxygen supply to the electron transport chain. The PDH pathway on the right operates as an anaerobic overflow pathway to produce ethanol

Figure 3. Pathways in anaerobic utilisation of glucose plus glycerol. Sugars are converted to acetyl-Co by glycolysis and the PFL-pathway on the left. Glycerol uptake is linked to glycerol-kinase activity (GK), which is repressed by glucose uptake, and enters the glycolytic pathway via glycerol phosphate dehydrogenase (GPD) which yields additional NADH. The resulting pyruvate is converted to acetyl CoA by the anaerobic PDG-pathway, which also produces NADH. To achieve redox balance, all of the acetyl-CoA produced by both pathways is reduced to ethanol.

Figure 4. The novel anaerobic PFL-FDH growth pathway, resulting from the expression of a foreign formate dehydrogenase (FDH) gene, yields only ethanol + C0₂.

Figure 5. An autoselector device in accordance with an embodiment of the invention. The upper figure shows a plan of the device, having five fermenter compartments (beakers) set in a plastic sheet. The viewing cap comprises around its periphery an aerator (1 ), a gas exit (2), a sampling port (3), a port for medium and broth feed (4), a broth exit (5) and a sampling port (6). Each beaker has a perspex viewing cap. The device also comprises a number of thermometer pockets. The central A to B cross section of the device, shows the sintered glass spargers used for aeration and the medium feed and broth exit ports. The beakers rest snugly within the cylindrical holes in the metal block, which has a thermostated heating element at each end to enable a constant temperature or a gradient temperatures in the series of fermenters. The fermenter array is set in a heat-lagged plastic box, designed to sit on a base containing five magnetic stirrers which rotate stirrer bars resting on the floor of each beaker.

Figure 6. Pump connection configurations. The thick lines indicate flexible connections to peristaltic pump tubing (indicated by an arrows within a circle for controlled supply of medium or other additives, gas sparging or broth transfer at rates Indicated as V, 2V, etc. C1 configuration: Increasing dilution rates (= growth rates) is used to study microbial physiology and to select mutant strains that can grow even more rapidly. In the embodiment shown, the fermenter compartments are linked such that all of the effluent broth removed from a fermenter compartment is transferred to the next fermenter compartment in the series That is, the fermenter compartments (apart from the final compartment in the series) have no waste effluent outlet. The final fermenter compartment in the series has an outlet for effluent broth waste disposal and/or collection.

C2 configuration: Increasing aeration. This is achieved by mixing air and nitrogen to study the switch in metabolic pathways between anaerobic (no air;), micro-aerobic ( 5% Cte), semi-aerobic (10% Cte) and (15% Cte) to fully aerobic (no N2 ; 20% Ct ) or vice versa. Mutant strains can then be select at the preferred aeration rate..

C3 configuration: Increasing feedstock concentration, e.g. a stepwise gradient of 4% w/v sugars to 2% w/v sugars, provides a selection gradient for mutants that are resistant to

'redox death'. Or that consume unutilised nutrients such as pectin hydrolysates.

C4 configuration: Increasing toxin resistance, such as higher ethanol tolerance, is provided by a selection gradient of increasing toxin mixed into the medium inlet line.

C5 configuration: Increasing growth temperature, provides a selection gradient for mutants that express higher amounts of, or more thermostable varieties of, a growth-limiting enzyme.

A decreasing temperature gradient will select mutant strains that grow better at low temperatures

Figure 7a. A device in accordance with an embodiment of a development of the invention. Each fermenter compartment is a helical glass coil contained within a cylindrical thermostatted container, with inlet and outlet ports connecting to a multichannel peristaltic pump. The inlet port supplies a segmented flow of feedstock plus medium or fermentation broth from a previous compartment. A gas mixture such as nitrogen / air is introduced, so as to divide the liquid flow into a series of elongated droplets or segments, separated by gas bubbles that provide a regulated supply of oxygen. This 'segmented flow' traverses the coil and exits, after removing the gas bubbles, through a quartz tube in which the optical density is continuously monitored by a UV detector. The broth then flows through a section of tubing in which it can be irradiated sporadically by a powerful UV lamp in order to cause in situ mutagenesis. A portion of the exit stream is passed to the next compartment, while the rest is collected for product analysis or waste disposal.

Figure 7b. Coupled helical coil fermenter compartments. This figure shows how two or more fermenter compartments comprising helical coils can conveniently be coupled to form a single selection compartment with increased retention time.

Figure 7c. An array of five coupled helical coil fermenter compartments. This is a plan of one of many possible arrays of fermenter compartments, showing five pairs of fermenter compartments coupled as shown in Figure 7b, wherein each coupled pair is arranged to form a train of five fermenter compartments analogous in function to the embodiment shown in Figure 5. Figure 8. A 'Thermophilic Yeast'. LDH^" Geobacilli expressing a thermostable pyruvate decarboxylase (PDC) use pathways similar to those used by yeasts. The micro-aerobic growth pathway uses pyruvate dehydrogenase (PDH) plus the electron transport chain, and the overflow pathway uses PDC to restore redox balance.

Figure 9a. A fermenter compartment of a segmented flow device (i.e. a miniselector or microselector), in accordance with an embodiment of a second development of the invention.

Figure 9b. A pair of coupled fermenter compartments of a miniselector device, in accordance with an embodiment of a second development of the invention.

Figure 9c. A circular array of fermenter compartments arranged around a UV lamp.

Figure 10 An example of a coupled segmented flow device: a microselector coil (a) is coupled to a miniselector coil (b).

Detailed description of the invention

The present invention will now be described in detail, with reference to the accompanying drawings.

In a first aspect, the present invention provides a device for the efficient selection of microbial strains in continuous culture, and provides methods and uses of the device for selection of microbial strains having particular characteristics. The present invention also provides a device for the efficient selection of environmental microorganisms, and uses of the device for culturing microorganisms having particular characteristics, such as the ability to produce antibiotics.

The device comprises at least two fermenter compartments, it may comprise at least three, at least four or at least five fermenter compartments. The device may comprise two, three, four, five, six, seven, eight, nine or ten fermenter compartments. The device may comprise two to ten fermenter compartments. Preferably the device comprises three to seven fermenter compartments. Preferably the device comprises five fermenter compartments. The device may comprise fermenter compartments, which fermenter compartments consist of two, three, four, five, six, seven, eight, nine or ten fermenter compartments, that is, the device may have only two, three, four, five, six, seven, eight, nine or ten fermenter compartments. Preferably the fermenter compartments consist of five fermenter compartments. Fermenter compartments may also be referred to herein as fermenters, or as compartments. In one development of the invention, a fermenter compartment may comprise one or more coils of tubing, thus fermenter compartments may also be referred to as coils of tubing, coils, helical coils, coupled helical coils, spiral coils, or coupled spiral coils. Practical issues may influence the number of compartments in a device. Two are simplest but provide only a single selection step that must be repeated manually. Three allows two consecutive selection steps per experiment. The number of peristaltic pump channels may increase the overall cost of the device. With relatively fewer fermenter compartments sterilisation and assembly is easier and risk of accidents is lower. Five compartments is a conveniently small number of compartments that allows simultaneous comparison of several dilution rates, pH or temperatures, or use of an exponential selection gradient to select stepwise improvements arising from multiple takeovers. One does not have to use all compartments in every experiment. Each fermenter compartment provides a vessel suitable for the continuous culture of microbial cells.

In some embodiments of the invention, particularly in relation to "autoselector" developments of the invention, the volume of a, or of each, fermenter compartment may be less than about 1 litre less than about 500 ml, less than about 400 ml, less than about 300 ml, less than about 200 ml, or less than about 100ml. The volume of a, or of each, fermenter compartment may be about 10 - 1000 ml, about 10 - 500 ml, about 10 - 400 ml, about 10 - 300 ml, about 10 - 200 ml, or about 40 - 200ml, about 50 - 200 ml, about 50 - 500 ml, about 40 - 120 ml, about 10 - 100 ml, or about 10 - 100 ml. Preferably the volume of each fermenter compartment is from about 50 to about 200 millilitres. Preferably the volume of each fermenter compartment is from about 40 to about 100 millilitres. Relatively low volume compartments conserve medium and nutrient supply and simplify stirring and aeration. However large inoculum transfers may be used to increase mutant cell residence time, and hence a volume of about 50 to about 200 millilitres may be preferred. The term volume here refers to the usable volume for culturing microbial cells.

In another development of the invention, a fermenter compartment contains a coil suitable for segmented flow. The fermenter compartment comprises tubing that is suitable for segmented flow of culture broth through the fermenter compartment. The tubing may be arranged in a helical coil. Each fermenter compartment may have an internal volume of about 0.2ml to 10 ml. Each fermenter compartment may have an internal volume of about 50 pi to 50 ml, about 100 μΙ to 50 ml, about 100 μΙ to 20 ml, about 100 μΙ to 10 ml, about 100 μΙ to 5 ml, about 200 μΙ to 5 ml, about 50 μΙ to 500 μΙ, about 100 μΙ to 500 μΙ, about 200 μΙ to 500 μΙ. Each fermenter compartment may have an internal volume of about 5 μΙ, about 50 μΙ, about 100 μΙ, about 200 μΙ, about 500 μΙ, about 5 ml, or about 50 ml. A miniselector preferably has fermenter compartments having a volume of about 5ml to about 50ml. A microselector preferably has fermenter compartments having a volume of about 5 μΙ to about 1000 μΙ. The volume may be referred to herein as the working volume, which is the usable volume of the fermentation compartment or coil.

Relatively low volume fermenter compartments conserve medium and nutrient supply and simplify stirring and aeration. However large inoculum transfers may be used to increase mutant cell residence time and so higher volume fermenter compartments may be preferred. In embodiments of an autoselector device of the invention, lower limits of about 10 ml, about 20 ml, about 40 ml, or about 50 ml may be preferred. Upper limits of about 100 ml, about 150 ml, about 200 ml, about 500 ml or about 1000 ml may be preferred. Any of these recited lower limits may be combined with any of the recited upper limits to define a volume range for a fermenter compartment in an autoselector device of the invention. In embodiments of the miniselector device of the invention, lower limits of about 5 ml, or about 10 may be used. Upper limits of about 10 ml, about 20 ml, about 40 ml or about 50ml may be used. Any of these recited lower limits may be combined with any of the recited upper limits to define a volume range for a fermenter compartment in a miniselector device of the invention. In embodiments of the microselector device of the invention, lower limits of about 5 μΙ, about 10 μΙ, about 20 μΙ or about 50 μΙ may be used. Upper limits of about 100 μΙ, about 500 μΙ, or about 1000 μΙ may be used. Any of these recited lower limits may be combined with any of the recited upper limits to define a volume range for a fermenter compartment in a microselector device of the invention

In some embodiments, a device may comprise one or more fermentation compartments in accordance with the microselector developments described herein and may additionally comprise one or more fermentation compartments in accordance with the miniselector developments described herein. Such devices may be referred to herein as coupled segmented pulse flow devices, coupled segmented flow devices, or coupled devices.

Preferably such devices comprise a microselector fermentation compartment coupled to a miniselector fermentation compartment, such that broth from a segment in the microselector fermentation compartment is transferable to a segment in a miniselector fermentation compartment. Such an arrangement is depicted in Figure 10.

In developments of the invention using segmented flow, segments of broth separated by gas bubbles flow through a coil. Each segment acts as a tiny batch fermenter, so the residence time of each segment within the coil is inversely equivalent to the dilution rate in a conventional continuous culture. The volume of each segment and the number of segments will depend on the internal diameter (ID) and length of the coil, which can be chosen from within a wide range for a variety of specific purposes, as discussed below.

For example, a miniselector may be a glass or plastic coil with an ID from 4 -10mm so the volume of a 1 cm segment will be from 126pL to 785pL. A im. long coil of this ID hold 50 such segments plus 55 gas bubbles, so the total working volume of the fermenter compartment will be from 5.3 ml to about 40 ml. Each coil may be supplied via a multichannel peristaltic pump immersed in a water bath equipped with a temperature heating element, as illustrated inn Figure 7A. In this way, the device may be used to select improved microbial strains in a variety of selection gradients.

For example, a microselector differs may use less than 1 mm ID tubing in which the volume of each segment will be below 10 μΙ. The consequent back-pressure means that syringe pumps or diaphragm pressure pumps should be used. Embodiments of the invention in accordance with microselector developments make use of uses the novel concept of 'pulsed segmented flow fermentations' to create suitably regulated retention times. In this way a series of 1 cm segments of < 10μΙ_ volume are created which can each be inoculated with a mixture of 'uncultivatable' microorganism, diluted such that there is less than one cell per segment on average. Each segment then acts as a micro batch fermentation, which can be retained within the coil until it reaches stationary phase. In this embodiment, the microselector acts as an automated alternative to the iChip

Moreover, exit broth samples from may be diverted into another series of miniselector coils that are configured to screen the toxicity of broth from environmental microorganisms against a choses pathogen. In this way the search for new antibiotics may be automated i.e. effected with minimum human intervention.

For use in selection of novel soil microorganisms, the iChip has about 100 x 1 μΙ holes, each designed to deigned to hold, on average, a single cell; so the volume of a segment in a microselector used for an analogous purpose will preferably be about 1-2 μΙ. A microselector compartment preferably holds about 10 segments, and on exit each will be injected into a bigger segment (e.g. 250-500 μΙ) in a shorter and fatter miniselector coil. Hence each coupled microselector / miniselector coil will may provide 0.25 - 0.5 ml droplets of up to ten different soil microorganisms. These may be collected in Falcon tubes and/or screened automatically for antibiotic activity.

A fermenter compartment is suitable for the continuous culture of microbial cells. A fermenter compartment that is suitable for the continuous culture of microbial cells allows the culture of microorganisms in an open flow system of essentially constant volume. A fermenter compartment that is suitable for the continuous culture of microbial cells allows the culture of microorganisms to reach a steady state of growth. The fermenter compartment may have a lid. The fermenter compartment may be built in to a heating block or may be a detachable vessel such as a beaker, which has a mouth, and a reversibly attachable lid or stopper. Preferably a beaker has a flat bottom to allow stirring with magnetic bars. The vessel and/or lid or stopper comprise glass or a transparent polymer such as poly(methyl methacrylate) (Perspex®).

The compartment is suitable for an "open flow" system but, as is understood in the art, this does not mean that the compartment is physically open to contaminants. A compartment is preferably sterile (microorganism-free), apart from the cell population of interest, i.e. the cell population under selection, which is derived from the inoculum. Accordingly, the methods and uses of the invention may comprise a step of sterilising the device before the inoculum is added, for example using ethanol or UV. In use, the device preferably only contains cells derived from the initial inoculum. In use, the device may contain a population of cells that is substantially homogenous, that is, the cells of the population are of the same species, cell type or cell line. In use, the device contains a population of cells that is derived from the inoculum (i.e. free of contaminating cells).

The fermenter compartments are linkable to form a linked series of fermenters. The links are conduits for the transfer of culture broth from one fermenter to another. In this way the linked fermenter compartments are in fluid connection, which allows the transfer of microbial cells from one fermenter compartment to another.

In the device of the invention, the fermenter compartments are linked to form a series of fermenter compartments. The fermenter compartments may be referred to as being linked in series. A series of fermenter compartments refers to a train, chain, serial array, or cascade of fermenter compartments. The fermenter compartments of the series are in fluid connection, such that in use culture broth is transferred successively from the first fermenter compartment in the series to the second fermenter compartment in the series, and then (if there are more than two fermenter compartments in the device) from the second fermenter compartment to the third fermenter compartment in the series, and so on through each fermenter compartment in the series. A "first fermenter compartment" or "first fermenter" herein may refer to the first fermenter compartment in a series of fermenter compartments. The next fermenter compartment in the series is the "second fermenter compartment" or "second fermenter" and so on. The next fermenter compartment in the series may be referred to as the adjacent fermenter compartment. The identity of the first fermenter compartment may be determined structurally, by the configuration of links between fermenter compartments, and on configuration of pumps which determine the direction of transfer of culture medium between fermenter compartments. The identity of the first fermenter compartment may be determined functionally, the first fermenter compartment being the fermenter compartment to which an inoculum of cells is added.

A link may comprise any kind of conduit, such as a tube or a pipe. A link may comprise a flexible conduit, such as a flexible tube, preferably a flexible plastic tube. A link may comprise a rigid conduit such as a rigid pipe, preferably a glass or metal pipe. A link may be glass or plastic tubing.

Each fermenter compartment of the device has one or more liquid inlets, for the addition of culture medium and/or culture broth to the fermenter compartment, and has one or more liquid outlets for the removal of culture broth from the fermenter compartment.

A liquid outlet of a first fermenter compartment is linkable to a liquid inlet of a second fermenter compartment, such that culture broth is transferrable from the first fermenter compartment to the second fermenter compartment. Similarly, a liquid outlet of the second fermenter compartment is linkable to a liquid inlet of a third fermenter compartment, such that culture broth is transferrable from the second fermenter compartment to the third fermenter compartment, and so on, such that each fermenter compartment in the device is linkable to at least one other fermenter compartment to form a linked series of fermenter compartments in fluid connection.

A liquid outlet of a fermenter compartment may be linkable to a waste disposal facility, and/or to a collection vessel for storage and/or analysis. A fermenter compartment may have a liquid outlet that is linked to a liquid inlet or another fermenter compartment and a liquid outlet that is linked to a waste disposal facility. In this configuration not all of the effluent broth from the fermenter compartment is transferred to the next fermenter compartment in the series. A fermenter compartment may have a liquid outlet that is linked to a liquid inlet of another fermenter compartment and may have no liquid outlet that is linked to a waste disposal facility. In this configuration all of the effluent broth from the fermenter compartment is transferred to the next fermenter compartment in the series.

A liquid inlet of a fermenter compartment is linkable to one or more reservoirs for holding fresh culture medium. Optionally, the device includes one or more reservoirs for holding fresh culture medium. Optionally, the device includes other inlets linkable to other reservoirs for supplying increasing feedstock concentration, pH control, or other additives such as suspected toxins or growth factors. Addition of fresh culture medium to a fermenter compartment may be referred to herein as diluting, and hence the rate of addition of fresh culture medium to a fermenter compartment is the dilution rate. A link between a liquid inlet of a fermenter compartment and a culture medium reservoir may be referred to herein as a feed line.

Optionally, a liquid inlet of a fermenter compartment is linkable to both a reservoir of fresh (i.e. sterile) culture medium and to the liquid outlet of another fermenter compartment, such that culture broth diluted with fresh culture medium may be added to a fermenter compartment via the inlet. Culture broth diluted with fresh culture medium may be added directly to a fermenter compartment via the same inlet.

A device of the invention may comprise liquid inlets having one or more pumps for the delivery of feedstock, culture medium, gas, or cells (including a cell inoculum). Segmented flow devices may comprise a plurality of pumps arranged to inject culture medium and/or cells into a segment, and/or to inject gases into the bubbles that separate segments, or to inject gases to create a bubble in a segment and thereby separate it into two segments.

In use, each fermenter compartment in the device is linked to another fermenter compartment to form series of fermenter compartments in fluid connection. The links between fermenter compartments are conduits through which culture broth is transferable. In use, culture broth may be regularly or continually transferred from one fermenter compartment to the next fermenter compartment in the series. A portion of culture broth (effluent broth) that is transferred from a fermenter compartment to an adjacent fermenter compartment acts as a growth inoculum in the adjacent fermenter compartment. Preferably culture broth is continuously transferred from one fermenter compartment to the next in the series. Preferably, similar amounts of culture broth are transferred between each fermenter compartment in the series. Self-evidently, the first fermenter compartment in the series does not receive culture broth from another fermenter compartment in the series and the final fermenter compartment in the series does not transfer culture broth to another fermenter compartment in the series (overflow of culture broth from the final fermenter in the series may be discarded). The first fermenter compartment in the series receives the original cell inoculum.

In use, the proportion of culture broth transfer from a fermenter compartment to the next fermenter compartment in the series will depend on the maximum growth rate (p_max ) of the cells in the chosen growth conditions. It may be relatively low, e.g. 5 -10% for rapidly growing microorganisms such as Geobacilli, that will quickly reach steady state, or much higher, e.g. 60-80% for slowly growing species such as yeasts or algae.

In continuous cultures, the dilution rate, (D) = the total feed rate (ml / h.) per ml. of culture volume, and in steady state, D = the cell growth rate (μ). Accordingly the dilution rate used in methods of the invention will be below Mmax in the first fermentation compartment, so as to supply a continuous inoculum of fresh cells to the next compartment. Moreover in the final compartments, D = > p_max so that residual sugars are available to allow takeovers by faster growing mutant strains. That is, culture conditions in the final compartments may be adjusted so that D - > _η3χ to allow takeovers by faster growing mutant strains

Accordingly high dilution rates in the range 0.2 to 0.8 lr¹ may be used with rapidly growing Geobacilli, whereas a range of 0.02 to 0. 10 h^'1 may be used may be more suitable for slowly growing algae, or even lower rates below of 0.005 lr¹ for single cell animal or plant tissue cultures.

The 'Miniselector' or 'Microselector' device of this invention may be preferred for use at low dilution rates; for example, optimising growth medium, selecting faster growing strains and optimising CO2 fixation and/or biofuels production by photosynthetic marine microalgae, Each fermenter compartment of the device may be readily adapted to supply constant light illumination.

The 'Miniselector' or 'Microselector' is particularly suitable for single cell selection of animal or plant cells. Segmented flow of culture broth offers prolonged residence times under sterile conditions in each fermenter compartment, and sterile transfer between compartments with minimal mechanical disturbance.

The flow of liquids in to, out of, and between fermenters may be driven by one or more pumps, which are optionally peristaltic pumps, and optionally multichannel peristaltic pumps. The pumping rate of the pumps and/or the internal diameters of the fluid connections may be varied to vary the liquid flow rates. Syringe pumps and diaphragm pumps may also be used.

The configuration of the device may be easily changed by varying the pumping rates and/or links between the fermenter compartments.

Accordingly, the present invention provides a method of connecting the ports of a device of the invention in which a similar portion of the culture broth is transferred from each compartment to the adjacent compartment (the next fermenter compartment in the series) so as to form a connected series. In the method microbial cells may be grown in identical conditions in each fermenter compartment except that one condition is varied in a progressive, or step-wise, fashion so as to apply as selection gradient across the series of fermenter compartments. Preferably only one condition is varied, i.e. a single condition is varied.

The addition of culture medium dilutes the microbial culture, thereby reducing the cell density of the culture. The culture medium is preferably sterile culture medium. The rate of addition of culture medium to a fermenter compartment (the dilution rate) may be set to a predetermined value to control the rate of growth of the microbial culture, by restricting the amount of a limiting nutrient in the culture. Such a continuous culture fermenter is known as a chemostat. At steady state the growth rate of the microbial culture is equal to the dilution rate. The dilution rate can be adjusted as desired up to the maximum growth rate of the organism under the chosen culture conditions. Thereafter the cells will all wash out unless a mutant strain with a higher growth rate arises. For GeobaciHi dilution rates of 0.01 to 0.8 hr¹ may be used. Transfer rates from one fermenter compartment to another in the series are preferably similar rates across the series of fermenter compartments.

In particular, the addition of sterile culture medium to a compartment dilutes the resident microbial culture, thereby reducing the cell density and provides fresh feedstock to allow growth to a new steady state. The dilution rate can be adjusted as desired up to the maximum growth rate of the organism under the chosen culture conditions. Thereafter the resident cells will reach stationary state and feedstock uptake will decline unless a mutant strain with a higher growth rate arises. The faster growing mutant cells will use all of the available feedstock and cause gradual 'wash out' of the resident cells until a new higher steady state population of mutant cells is established. Such an event is called a 'takeover". In this way, dilution rates of up to 0. 6 tv¹ may be used, for example to select fast-growing GeobaciHi.

Each fermenter compartment has a plurality of ports. A port may be an inlet and/or outlet for the addition and/or removal of liquids and/or gasses from fermenter. Alternatively or additionally, a port may be a hole for the introduction of a device, such as a probe, to the fermenter compartment. Ports may be reversibly sealable.

A fermenter compartment may have a gas inlet for the introduction of gasses such as air, oxygen, or nitrogen or mixtures thereof, and a gas outlet for the exit of gasses such as ethanol vapour. The gas outlet may allow exit of residual gasses and products such as C0₂ or ethanol vapour. A gas inlet may be connected to a sintered glass disc for gas sparging. Gas sparging creates bubbles, in particular a cloud of tiny bubbles, that may help to stir and/or agitate the culture broth, and may help remove vapours produced by the culture. A gas outlet may be connected to a chamber for condensation of vapours, or analysis.

Analysis may use an infra-red monitor or mass spectrometer for gas analysis.

Optionally, a fermenter compartment has one or more ports for sampling, insertion of a sensor such as a pH meter, addition of growth supplements or inhibitors, addition of toxins, or addition of substances to adjust the pH of the culture. Ports may be capped when not in use. Ports may be located in a lid.

A fermenter compartment may comprise means for stirring or agitating a culture of microbial cells therein. Such means may comprise a rotatable stirrer bar, or a sintered glass element through which gasses can be passed. The device optionally comprises a frame in which a plurality of fermenter compartments are mountable. The frame may comprise a block, which may have a plurality of holes or recesses in each of which a fermenter compartment is mountable. A hole or recess In the block may comprise a lip at its base for holding a fermenter compartment in the block. Preferably the holes or recesses are cylindrical and the fermenter compartments are cylindrical. The block may be formed from a thermally conductive material, such as a metal. The block may comprise one or more cavities or pockets for receiving a thermometer.

The device may comprise one or more elements which are heating elements or cooling elements for maintaining the same temperature in each fermenter compartment and/or maintaining a temperature gradient across the series of fermenter compartments.

A cooling element may be referred to as a "cold heater", and a heating element may be referred to as a "hot heater". Preferably the device comprises elements which are heating elements. The heating element may be a cartridge heater, and may be a thermostated cartridge heater. The fermenter compartment may be contained within a temperature controlled liquid jacket. The elements may be arranged to maintain the same temperature in each fermenter compartment. Preferably the heating elements and/or cooling elements are thermostatically controlled. The elements may be arranged to maintain a temperature gradient across the series of fermenter compartments. The device may comprise a block formed from a thermally conductive material, such as a metal, in which the one or elements are fitted.

The device may comprise one or more stirrer motors. A stirrer motor may comprise a magnet, or a magnetic induction motor, for rotating a magnetic stirrer bar located in a fermenter compartment.

The device may comprise a lifting means for lifting the plurality of fermenter compartments from the frame, thereby allowing convenient removal of the plurality of fermenter compartments from the frame for sterilisation or inspection. The lifting means may comprise a rigid sheet or support, which is engageable with the fermenter compartments mounted in the frame such that they can be lifted out of the frame by lifting the lifting means. A lifting means may comprise a plastic sheet having a plurality of holes, wherein the edge of each hole is engageable with a lip or protrusion on a fermenter compartment.

Preferably a fermenter compartment has one or more translucent or transparent parts to allow light to pass through fermenter compartment, such that in use light is allowed to pass through a culture of microbial cells in the fermenter compartment. Translucent or transparent parts may comprise glass or a polymer such as poly(methyl methacrylate) (Perspex®). Preferably the device comprises a frame in which a plurality of fermenter compartments are mountable, and the frame has one or more transparent, translucent, or open portions that are alignable with a transparent or translucent parts of a fermenter compartment to allow light to pass through a microbial culture inside a fermenter compartment mounted in the device. Preferably the ports and/or links are arranged to allow light to pass through a microbial culture inside the fermenter. Preferably the ports and/or links are arranged around the periphery of the top and bottom of the fermenter compartment to provide central viewing window.

In this way the optical density, or fluorescence, of a culture in a fermenter compartment can be observed, which provides an indication of the microbial cell density in the culture. In this context, observed means viewed by eye, inspected, or quantitatively measured e.g. using a spectrometer. Preferably a fermenter compartment, or the whole device, can be placed on or next to a light box in order to observe the optical density of a culture within a fermenter compartment. Preferably the device allows overhead inspection of one or more cultures in a fermenter compartment, to detect cell takeovers or to view fluorescence.

In this way also the cell culture can be exposed to UV light, in order to mutagenise cells in the culture. Preferably a fermenter compartment, or the whole device, can be placed under a UV lamp to mutagenise cells within one or more fermenter compartments.

Thus, a fermenter compartment may comprise transparent parts arranged for visual inspection or UV irradiation of resident cells.

Preferably an autoselector fermenter compartment is constructed from transparent glass and/or plastic so that a culture of microbial cells in the fermenter compartment can be visually inspected or UV irradiated as described above. If the fermentation compartment has a lid it should be transparent. If the lid holds ports and/or links, these are arranged, to allow light to pass vertically through a microbial culture inside the fermenter. Preferably the ports and/or links are arranged around the periphery of the top and/or bottom of the fermenter compartment to provide a central viewing window.

A plurality of fermenter compartments may be arranged in a circular array. The array may be around a UV lamp.

In one development of the invention, a fermenter compartment comprises tubing through which culture broth may be passed In segmented flow. The tubing may be arranged as a coil. The tubing may be arranged as a helical coil or spiralled coil. In this way a relatively long tubing can be accommodated in a relatively small volume, which enables the construction of a relatively compact and portable device. The tubing may be any appropriate material, including glass, plastic or metal. The tubing may be microbore tubing. The coils may be disposable, which is particularly useful for working with pathogens. A fermenter compartment may comprise two coupled helical coils, or may comprise three or more coupled helical coils. A coil may be of glass, quartz or water-repellent plastic, wound around a cylindrical heating element and immersed in a temperature-controlled heating bath. The coil is fed with buffered medium containing an appropriate concentration of feedstock via a multi-channel peristaltic pump, as illustrated in Figure 7a. The culture medium is segmented before it enters the coil with a mixture of nitrogen and oxygen sufficient to maintain cell growth in the adjacent segment

The internal diameter (ID) of the coil will dictate the length and volume of each segment and the surface area of its meniscus with the gas bubbles. The latter controls the diffusion rate of oxygen into the segment, so small segments are favoured, but longer segments are convenient for injection of inoculum, toxins or growth factors. A suitable range may lie between 4 mm and 10mm; the volume of a 1 cm segment will then be from 126μΙ to 785μΙ. A 1 m long coil of this ID hold 50 segments plus 50 gas bubbles, so the total working volume of the fermenter compartment will be from 6.3 ml to about 40 ml which overlaps the working volume of an autoselector compartment.

This development the invention may be referred to as a "miniselector" or "microselector", because of the small, or extremely small, volumes of culture broth required. As disclosed herein, in the miniselector or microselector successive fermentation compartments are spiral coils contained within a thermostatted compartment. An embodiment of a miniselector is illustrated in Figure 7. The fermentation compartments are supplied via a pump with medium, feedstock, growth factors or inhibitors, and a mixture of gases which can be used to create selection gradients as described above. However the liquid mixing is here done outside the spiral coil, and the gas is injected so as to separate the fermentation broth into a series of droplets which traverse the spiral as fermentation proceeds. The liquid components of the medium, i.e. feedstock, growth factors or inhibitors and acids or alkalis for pH control, are added by peristaltic pumps, jointly or separately, via T-tube connectors. Finally, gas mixtures, e.g. air and nitrogen, are added, jointly or separately, in order to segment the liquid medium immediately before cells inoculum or cells transfer

A microselector device of the invention may comprise a series of tubes, for example bore plastic tubes. The tubes may be coiled around a thermostatted heating element. As in the miniselector, each coil may form a fermentation compartment. The tubes may be formed from microbore tubing designed to contain 1 μΙ_ segments of culture broth. Relatively high back-pressures will limit the use of the device for fermentations requiring long residence time, but we have seen that a solution is to use "pulsed injection segmented flow" as recommended by Francis et al. (2002) chemical analyses.

A miniselector device of the invention may comprise fermenter compartments which are coils of glass or plastic tubing designed for segmented flow, through which flow tiny segments of culture broth separated by bubbles of air, oxygen or nitrogen (Figure 9a) . Each segment acts as a tiny batch fermenter, so the residence time of each segment within the coil is inversely equivalent to the dilution rate in a conventional continuous culture. The volume of each segment depends on the internal diameter (ID) of the tubing and the number of segments, the length of the coil and the size of the gas bubbles. For aerobic

fermentations, each bubble must contain sufficient oxygen to supply each segment. For example, the volume of a 1 cm long segment in a 2 mm ID coil will be 31.5 μΙ_, so a 50 cm. long coil holding 20 such segments, with hold a total broth volume of about 0.6 ml. The residence time of each segment will depend on the flow rate and the length and ID of the coil. For some selection procedures, e.g. increasing feedstock utilisation, it is desirable that this should almost complete, so longer tubes with larger ID may be preferred. A working range may be from 0.5 ml to 5ml. For glass coils, this may be achieved conveniently by coupling adjacent compartments as illustrated in Figure 9b. These may be arranged in a circular array around a powerful UV lamp for sporadic in situ mutagenesis, as shown in Figure 9c. In embodiments using plastic coils (particularly relatively long thin plastic coils) back-pressure may mean that syringe or diaphragm pumps are preferred over peristaltic pumps. Advantageously, the high pressures associated with use of syringe or diaphragm pumps may increase dissolved oxygen concentration in aerobic fermentations.

In embodiments of the autoselector device of the invention, relatively low volume fermenter compartments conserve medium and nutrient supply and simplify stirring and aeration. However large inoculum transfers may be used to increase mutant cell residence time and so higher volume fermenter compartments may be preferred hence the working volume may be between 40 ml and 200 ml. A miniselector device of the invention may comprise fermentation compartments of about 5 ml to 40 ml, as described above, but different constraints apply to the microselector device of this invention, which may use tubing of less than 1mm or less than 2mm internal diameter. Except at very low flow rates or very short tubing, very high back-pressure makes peristaltic pumps are less suitable, so syringe pumps or diaphragm pressure pumps are preferred. Even then, low flow rates and short coil are needed to supply adequate retention times, so this embodiment of the invention uses the novel concept of computer-controlled 'pulsed segmented flow fermentations' to allow suitably regulated retention times. In this way a series of 1cm segments of 1-10 μΙ_ volume, separated by 1 cm bubbles, can be created. There is no theoretical limit to the length of such a coil, so the broth volume from a 1 m coil carrying 50 such segments, will be from 50- 500 pi, whereas a 4m coil will hold 0.2- 1ml.

Segmented flow within the tubing means that a plurality of droplets of culture broth (i.e. segments of culture broth) flow through the tubing. Segmented flow is a technology that has been in use for many years. Applications of segmented flow are known for example in the field of "AutoAnalyser" devices, which are automated analysers using segmented flow in a technique called continuous flow analysis (Skeggs, 1957). The apparatus and equipment used in such "AutoAnalyser" devices is readily adaptable to the autoselector devices of the present invention. In segmented flow each droplet of culture broth is separated from an adjacent droplet of culture broth by a gas bubble. Because the each droplet of culture broth is a relatively small volume, and because gases may diffuse between a droplet of culture broth and a contacting gas bubble, there is no need for sparging or stirring in order to maintain a desired gas tension or concentration in the culture broth. The relatively small volume of each droplet also facilitates maintenance of a constant temperature within the droplet.

Typically each droplet of culture broth in developments that use segmented flow is about 1 μΙ to 10 μΙ, about 1 μΙ to 5 μΙ, about 1 μΙ to 2 μΙ, about 5 μΙ to 200 μΙ, about 5 μΙ to 100 μΙ, about 5 μΙ to 50 μΙ, about 5 μΙ to 20 μΙ, about 10 μΙ to 100 μΙ, or about 10 μΙ to 50 μΙ. Each droplet may have a volume of about 1 μΙ, about 5 μΙ, about 10 μΙ, about 20 μΙ, about 50 μΙ, about 100 μΙ, about 200 μΙ, or about 500 μΙ.

The droplet size, or segment volume, and residence time will vary according to the internal diameter (ID) of the tubing and the desired residence time. Thus a relatively short glass coil of 2mm ID x 200 mm = 628μΙ (i.e. volume 628μΙ) may contain thirty 10 μΙ droplets and thirty 10 μΙ bubbles, or three 100 μΙ droplets and three 100 μΙ bubbles (such AutoAnalyzer coils are commercially available). Alternatively the volume of a 1 cm segment in a relatively narrow glass coil of 4mm ID will be about 12.5μΙ, so a relatively short 50 cm coil may contain about 25 droplets and 25 bubbles (such AutoAnalyzer coils are commercially available). A droplet volume range for a fast growing Geobacillus may be about 5 to 50μΙ. Similar glass coils of 0.4 mm ID would accommodate droplets of about 1 - 10 μΙ. This may be suitable for growth of photosynthetic marine algae for example. The lower range of the ID may be limited in practice by the back pressure on the peristaltic pump. Back pressure constraints will be minimal at the very slow flow rates required for tissue culture selection, where disposable plastic tubing may be preferred.

In the devices of the invention each fermenter compartment in the device is linked to another fermenter compartment to form series of fermenter compartments in fluid connection. In the developments of the invention that uses segmented flow, each fermenter compartment comprises an outlet for transfer of a portion of culture broth from a droplet in that fermenter compartment, for transfer to a droplet in the next fermenter compartment in the series via an inlet in that next fermenter compartment. Each fermenter compartment also comprises an outlet for transfer of a portion of culture broth from a droplet in that fermenter compartment for storage and / or analysis.

In this development, in use each fermenter compartment comprises a plurality of droplets of culture broth. Each droplet can be regarded as a batch culture, and each fermenter compartment can be regarded overall as a continuous culture, because microbial cells are regularly added to, and removed from, each fermenter compartment. In this way, each fermenter compartment provides an open culture system of substantially constant volume (i.e. a continuous culture).

The present invention provides a device for the selection of cells, the device comprising a plurality of fermenter compartments for the culture of cells, wherein the fermenter compartments are linked to provide a series of fermenter compartments in fluid connection, such that cells are successively transferable through each fermenter compartment in the series. The device may have any or all of the features associated with the "miniselector" development discussed above. The device may have any or all of the features associated with the "microselector" development discussed above. The device may be suitable for the selection of animal cells or plant cells. Animal cells may be mammalian cells, and may be mammalian tumour cells. In this context, plant cells may be algal cells. The device may be suitable for the selection of cells in single cell culture.

The "miniselector" or "microselector" is a development of this invention which uses the concept of segmented flow in spiral tubes contained within a thermostatted compartment as illustrated in Fig. 7a. Such fermenter compartments may have working volumes of about 0.2 millilitres to about 10 millilitres. This reduces time and cost or medium preparation and is particularly desirable for selection of microbial strains that require expensive culture media, or of pathogens which have to be handled in small volumes with care. The retention time in such configurations is the inverse of the dilution rate in conventional continuous cultures and can conveniently be increased by reducing the input rate and / or by lengthening the spiral or by coupling two spirals together as illustrated in Fig.7b.

As in other embodiments of the invention, miniselector and microselector compartments form a linked series. Miniselector and microselector compartments could be used in the embodiment illustrated in Figure 5, but many alternative configurations are possible, since the liquid and gas mixing, broth analysis and UV irradiation is all done online.

Figure 7c illustrates an example which comprises five fermenter compartments, each composed of a pair of linked spirals.

The microselector and miniselector developments of the present invention lends themselves to complete automation with feedback control, signalling only when a significant strain improvement has been detected.

Segmented flow is illustrated in Figure 10. Figure 10a represents segmented flow in a microselector and Figure 10b represents segmented flow in a miniselector. More particularly Figure 10 illustrates "pulsed injection segmented flow". Each fermentation compartment comprises a coil, such as a microbore plastic coil, equipped with pumps, such as syringe or diaphragm pumps. The pumps may be computer regulated to inject defined volumes of reagents or gas into the coil in sequential timed pulses, as follows: • Pump 1 may inject a defined volume of feedstock medium into the entrance of a microselector coil, which may be the first coil of a series of interconnected fermentation coils. The volume injected will normally be constant, but may increase in each subsequent compartment in order=to be able to study the effects of increasing dilution rate (equals growth rate).

• Pump 2 may inject a pulse of gas to separate the medium into the defined segment.

For anaerobic fermentations, the gas may be nitrogen, For aerobic fermentations the gas may be air or oxygen, sufficient to oxidize all of the feedstock in the subsequent segment.

• Pump 3 may inject an appropriate cell inoculum into the resulting feedstock segment which will transit the coil in a series of timed pulses sufficient to ensure growth to stationary phase. The effluent segments may then be directed into the entrance of a similar coil which is the first in a series designed to provide a variety of selection gradients such as are described above.

Alternatively the inoculum may be a suspension of mixed environmental microbial cells, diluted such that on average, less than a single cell is injected into a feedstock segment. As before, this segment transits the coil in timed pulses, but in this case the effluent cells may be amplified in a coupled miniselector coil, as shown in Figure 10.

• Pump 4 is programmed to inject the major part of the exit segment into the feedstock segment of a much larger ID (internal diameter) miniselector coil, fed with identical feedstock and operated under similar conditions. The final segment emerges via a UV spectrophotometer, which operates a two-way valve designed to direct only high OD segments into a chilled fraction collector for further analysis, thereby automating the whole process that is manually operated by use of an Chip.

Moreover there are other uses for the devices of the present invention, in particular the coupled segmented flow devices, for example:-

• To identify microorganism that produce novel antibiotics.

Increasing samples of effluent broth from a coupled Micro/Miniselector containing novel microorganisms may be injected into cultures of a chosen pathogen growing in a series of similar Micro/ Mini selector coils. A significant drop in optical density indicates the LD₅o of antibiotics produced by the novel microorganisms. • To identify and analyse growth factors for "uncultivatable" soil microorganisms.

The cell broth emerging from a Micro/Miniselector growing on soil dialysate will be depleted in specific growth-limiting nutrients, e.g. small peptides that are not present in conventional minimal media. These may be revealed by peptide mapping and/or mass spectrometry and analysed so as to supply synthetic growth factors for large scale production of antibiotic- producing microorganisms.

The invention provides methods for the selection of microorganisms that are difficult to grow in standard laboratory media, for example environmental microorganisms. The invention thus provides methods for the cultivation of microorganisms that are difficult to grow in standard laboratory media. The invention provides methods for identifying one or more growth factors or nutrients necessary for cultivating a microorganism or cell type.

A device in accordance with the microselector development of the invention is particularly useful for the selection of microorganisms that are difficult to culture in standard laboratory media. Pulsed flow injection allows long retention times of very small volumes in relatively long coils of low internal diameter. For example in the coupled coil arrangement shown in Figure 9b, the ID of the coil may be chosen so that the length of a segment is at least 10mm, so the volume of such segments in a coil of 0.4mm ID will be at least 1 ,26μΙ. The temperature of both coils may be maintained within the range 15 to 25^°C and the pH of the soil dialysate is in the range 6.5 to 7.5, so as to mimic the soil environment. The dialysate may be concentrated and supplemented to ensure that the feedstock segments contain sufficient total nitrogen, phosphate and added glucose, xylose, and arabinose to maintain cell growth to stationary phase. The length of the coil and the pulse rate will be chosen so that the residence time of each segment is sufficient to ensure growth to stationary phase and utilization of almost all of the feedstock.

As shown in Figure 10 a series of pumps may be arranged to add, remove and manipulate the culture medium and culture broth in the fermenter compartments. For example Pump 1 may inject pulses of cell-free feedstock into the coil entrance and Pump 2 may segment this with sufficient oxygen to maintain aerobic growth. Pump 3 may inoculate the feedstock segment with a mixture of cells obtained by washing and filtering the soil and centrifuging the cell therein. The optical density allows cell dilution with soil dialysate until on average only one cell is injected into the feedstock segment.

The segments then proceed through the coil in pulses until they exit, and are automatically injected into bigger segments of fresh medium as shown in Figure 10. The fermentation conditions remain identical and the length of the coil is sufficient to allow growth to stationary phase. The exit broth is passed through a UV detector, which directs segments with high optical density into a chilled computer-controlled mini fraction collector (not shown) or another array for determination of antibiotic activity against a pathogen, as described above.

The device of the invention may comprise cells, such as microbial cells. That is, the device may comprise a population, or culture, of cells, such as microbial cells, for selection. In use, the device of the invention comprises microbial cells. In use, the microbial cells are present in one or more of the fermenter compartments, and may be referred to herein as "resident cells". The device as sold may be sterile, that is, the fermenter compartments may be sterile. The fermenter compartments before use may be sterile. In use, and in the methods of the invention, the device may be sterilised before an inoculum is added to a fermenter compartment. In use, the device preferably only contains cells derived from the inoculum. The device may comprise a population of homogenous or substantially homogeneous cells, that is the device may comprise a population of cells of the same species, same organism, or same cell line. The device may be free of, or substantially free of contaminants. A contaminant in this context may be any microorganism that is not the microorganism of interest (i.e. is not the microorganism derived from the inoculum). The "device" in this context may be internal surfaces, the interiors of the fermenter compartments and links. The device may be free, or substantially free of contaminating microorganisms. For example, where the device is used to select for a Geobacillus having certain characteristics, the device may be free of non-Geobacillius cells.

The present invention provides methods, which methods comprise providing a device of the invention and using it as disclosed herein. The present invention provides a method of selecting a cell, which cell is preferably a microorganism. The invention provides methods of selecting a microbial strain and methods of culturing microorganisms. Methods comprise providing a device of the invention, adding cells to a fermenter compartment, and applying a selection gradient across the series of fermenter compartments. The present invention also provides uses of the device of the invention, in accordance with the methods disclosed herein.

Methods may comprise a step of sterilising the device before adding cell to a fermenter compartment. Methods may comprise a step of picking out an inoculum of cells, before adding the inoculum of cells to a fermenter compartment. The methods may comprise adding culture medium to each fermenter compartment. The methods may comprise transferring culture broth from the first fermenter compartment through each fermenter compartment in the series. The methods of the invention may comprise applying a selection gradient across the series of fermenter compartments. The methods of the invention may comprise maintaining a substantially constant temperature in the fermenter compartments. The temperature may be maintained at a substantially constant temperature that is in the range of about 15°C to 70°C.

During use of the device of the invention, and in the methods of the invention, a cell take over occurs in a fermenter compartment when a mutant cell in that fermenter compartment is able to grow faster than the other cells in that compartment. The mutant cell population takes over the cell culture in that fermenter compartment (I.e. it out-competes the other cells in that fermenter compartment), and this may be observable as an increase in cell density in that compartment. For example, in a method for selecting an increased maximum growth temperature, a temperature gradient may be applied across the fermenter compartments of the device. A fermenter compartment in the series may provide a temperature at, or close to, the maximum temperature at which the cells of the inoculum (initial culture) are capable of growing. Thus, the rate of cell growth will be low in that compartment and in subsequent compartments in the series, and therefore the optical density of the culture in that fermenter compartment will be relatively low. That fermenter compartment will continuously be repopulated with cells from the previous fermenter compartment in the series, and when it is populated with a mutant cell having improved ability to grow at the higher temperature in that compartment, then that mutant cell will form a population that takes over the compartment. This cell take over may be observable as an increase in optical density of the culture in that fermenter compartment.

Figure 5 illustrates a device in accordance with an embodiment of the invention. It comprises a metal block with 5 cylindrical holes, which is heated at each end by thermostatted cartridge heaters to create an exponential temperature gradient along the block. The temperatures can conveniently be measured by placing thermometers in the pockets provided in the metal block.

The block is contained in a plastic box lined with polystyrene foam, which is normally seated on a stirrer block containing a series of motors. Magnets attached to the motors are used to rotate stirrer bars within the beakers, as shown in Figure 6. Magnetic induction motors will be a satisfactory alternative. The fermenter block can easily be moved onto a light box to allow viewing from above, or on to a UV lamp for in situ mutagenesis. Alternatively a gradient of chemical mutagens such as nitrosoguanidine can be supplied intermittently to each fermenter.

The beakers sit snugly into holes within a thermostatted heating block, and their lips fit into holes in the lid of the block so that the whole array can be easily removed for cleaning. Each vessel has a circular Perspex cap, held tightly in place by silicone rubber bands, with 5 ports through which glass or steel tubes are inserted. These are arranged around the periphery, so as leave a transparent centre to view cell density when the block is placed on a light-box. The ports are conveniently connected by plastic tubes to a peristaltic pump which allows transfer of a portion of the effluent broth to the adjacent compartment to act as a continuous inoculum. Each fermenter is grown under identical conditions except for a single variable. That may be, for example, successive increases in temperature. Growth rates will be expected to increase successively, but eventually they will decline because some critical component, e.g. an enzyme, has reached the limit of its thermostability. Residual feedstock will accumulate in that compartment, so that any mutant cell with increased thermostability will use this to grow faster than the wild type and will eventually take over in that compartment. This takeover will be signalled by a rise in resident cell density, which may often be recognised by visual inspection and samples of the mutant cell can be withdrawn for analysis. However selection for increased thermostability will continue, so the autoselector will do its job when left alone.

The ports have the following functions:

^■ Port 1 is for gas sparging via a sintered glass disc (e.g. Dixon Glass, cat.no.525) which creates bubbles that help to stir the broth, aerate it if necessary, and to sparge the vapour that is produced.

^■ Port 2 is the gas exit. The vapour can be condensed or trapped in wash bottles for analysis and / or passed through a gas analyser for 0₂and C0₂ analysis.

■ Port 3 is for feed medium + broth from the preceding vessel via a multi- channel peristaltic pump (e.g. Watson-Marlow 205S/CA)

■ Port 4 is the broth exit, which can be controlled by the pump and optionally sampled in a fraction collector.

■ Ports 5 and 6 are capped spare ports that can be used as necessary for sampling, pH control, addition of growth supplements or of toxins, etc.

Feed and aeration rates are readily varied by changing the pump rate and/or pump tubing diameter, and differing configurations of the pump connections allow a variety of different methods of use for the equipment, as set out below.

As explained above, when the device of the present invention is in use, one or more culture conditions is varied across the series of fermenter compartments in a progressive manner, such that one or more culture conditions varies in a gradient across the series. Preferably, only one culture condition is varied across the series. Such a gradient of culture conditions is a selection gradient. Thus in use the device provides a series of continuous culture fermenter compartments, wherein a selection gradient is applied across the series. For example, the temperature of the culture may be varied in a gradient across the series such that the temperature is progressively higher in each fermenter compartment in the series, and/or the concentration of a nutrient such as glucose is progressively lower in each fermenter in the series.

The methods of the present invention comprise providing a device of the present invention, adding one or more microbial cells to a first fermenter compartment in the series of fermenter compartments, and applying a selection gradient across the series of fermenter compartments. A selection gradient may be, for example, a gradient of increasing or decreasing: pH, temperature, feedstock concentration or composition, or rates supply of feedstock (dilution rate), air, growth supplement or toxin. Different selection gradients may be applied by configuring the device in different ways.

The methods of the invention are preferably methods of selecting an LDH^" Geobacillus strain that is resistant to "redox death". Such a strain may have improved characteristics for the production of bioethanol.

The methods of the invention are preferably methods of selecting a microorganism that is incapable of growing on, or difficult to grow on, standard laboratory medium. Such microorganisms are sometimes termed "uncultivatable" microorganisms, and many environmental microorganisms fall into this category. The invention thus provides methods of selecting (that is cultivating, culturing, or isolating) environmental microorganisms in laboratory media. Sources of such environmental cells include environmental water, such as sea water, and soil.

In the methods of selecting environmental microorganisms, a microselector coil is supplied with a minimal medium containing sugars and essential fatty acids salts and vitamins. The unknown growth-limiting nutrient may be provided by adding the appropriate environmental water (e.g. soil dialysate). The feedstock segments are inoculated with, on average, a less than single cell from a diluted suspension of washed environmental cells, and traverses the coil in pulsed flow designed to allow growth to stationary phase.

The major portion of the effluent broth may be the injected into a much larger feedstock segment in a miniselector coil operated under similar pulsed flow conditions, so that an amplified number of cell emerge in the exit segment. This is led through a UV spectrometer which diverts high density segments via a two-way valve into a chilled fraction collector.

Alternatively, or additionally, the effluent broth may be collected and assayed for antibiotic activity, for example by determining the effect of the effluent broth on the growth of a chosen pathogen or standard laboratory microorganism (e.g. E. coli). Accordingly, the present invention provides a method of selecting a microbial strain, the method comprising providing a device of the invention (such as a coupled microselector / miniselector device), picking an inoculum of microbial cells, and adding the inoculum of microbial cells to a first fermenter compartment of the series of fermenter compartments. The method may also comprise applying a selection gradient across the series of fermenter compartments.

The method may comprise a step of sterilising the device before adding the inoculum to the first fermenter compartment. The method preferably uses a segmented flow device of the invention, that is, the method preferably uses a miniselector or a microselector. The method preferably comprises adding sterile culture medium to the fermenter compartments. The sterile culture medium is preferably minimal media. The method may comprise transferring a portion of culture broth from the first fermenter compartment through each fermenter compartment in the series. The method may comprise taking an effluent sample for analysis. The effluent sample may be analysed for antibiotic activity. The effluent sample may be analysed by comparing its composition to that of an environmental dialysate, and identifying a growth factor by its relative depletion from the effluent.

The device of the invention can be configured in a variety of ways in order to apply different selection gradients to microbial cells cultured in the device, as described below. Different configurations can easily be achieved by changing the links between the fermenters and/or reservoirs of culture medium, and/or by adding fermenter compartments to the device or removing fermenter compartments from the device.

Figure 6 illustrates a variety of configurations (configurations C1 to C5) of an autoselector device in accordance with embodiments of the invention, which configurations are suitable for use in the methods and uses of the device according to embodiments of the present invention.

The various selection gradients described below in connection with an autoselector development of the invention, are applicable to miniselector and microselector

developments.

Configuration C1 provides a selection gradient of increasing dilution rates.

In this configuration, there is a stepwise increase in dilution rate in successive

compartments. For example aeration and broth transfer rates (V) may be substantially identical in each fermenter compartment, but there is a stepwise increase in medium feed rate, and hence dilution rate in successive compartments. For example there may be a stepwise increase in broth transfer rates between fermenter compartments and medium feed rates are substantially constant, such that dilution rates increase in successive compartments. Dilution rates equal growth rates in continuous chemostat cultures (Herbert et al. 1956), so such simultaneous comparisons of steady state cell density and product formation will be of particular value to compare specific sugars uptake and product yield with growth rate, and thereby calculate maintenance energy requirements under various growth conditions. Hence this configuration will be widely useful in microbiology research, and also an inexpensive tool for teaching microbial physiology.

Accordingly, the present invention provides a method of selecting a microbial strain, such as a Geobacillus strain, wherein the method comprises providing a device of the invention and adding microbial cells to the first fermenter compartment in the series of fermenter compartments, and applying a selection gradient which is an dilution rate gradient. In this method the device is configured to provide a series of fermenter compartments in fluid connection each containing culture medium, wherein the rate of dilution of the culture broth is progressively higher in each successive fermenter compartment.

A dilution rate gradient may vary for example from about 0.01 h-1 to about 0.9 h^"1, from about 0.01 to 0.8 hr¹, from about 0.01 to about 0.5 h ¹, from about 0.01 to about 0.2 h^"\ from about 0.01 to about 0.1 IT¹, or from about 0.05 to about 0.2 f¹.

Configuration C2 provides a selection gradient of increasing aeration:

This can be used to regulate and study the switch between anaerobic and aerobic metabolism in facultative anaerobes, and thereby find optimal aeration conditions. In this configuration the culture conditions in each fermenter compartment are identical, but the intracellular redox potential is regulated by injecting an increasing proportion of air or oxygen into the nitrogen gas sparging line of each vessel (e.g. via a T-junction in the gas sparging line). Different genes are regulated at different levels of aeration, so this configuration will be useful to find suitable microaerobic conditions to select strains optimised for production of specific products, such as ethanol.

A selection gradient which is an aeration gradient may be used to select for microbial strains capable of growing in conditions of relatively low oxygen, such as semi-aerobic, microaerobic or anaerobic conditions. Such a selection gradient may be used to determine optimised aeration conditions for grown of particular strains, as well as to select for strains optimised for microaerobic fermentation.

Accordingly, the invention provides a method of selecting a microbial strain, such as a Geobacillus strain, wherein the method comprises providing a device of the invention and adding microbial cells to the first fermenter compartment in the series of fermenter compartments, and applying a selection gradient which is an aeration gradient. In this method the device is configured to provide a series of fermenter compartments in fluid connection each containing culture medium, wherein the aeration rate or the composition of the aeration gas varies progressively between each successive fermenter compartment. For example, the aeration gradient may be a gradient of oxygen concentration in the gas used to aerate each fermenter compartment. The gas may also contain nitrogen and/or carbon dioxide. The aeration gradient may be from about zero to about 100% oxygen, from about zero to about 50% oxygen, from about zero to about 20% oxygen, or from about 10% to about 1 %, from about 5% to about 1%, or from about 2% to about 0.1 %.

Configuration C3 provides a selection gradient of increasing feedstock concentration

Configuration C3, which is suitable for selecting for strains capable of increased feedstock utilisation. In this configuration the dilution, aeration and transfer rates are identical, but feed concentrations are progressively higher in successive fermenter compartments. This can provide a variety of improved Geobacillus strains individually adapted for maximum bioethanol production from a particular feedstock. For example :- a). Strains that grow in high sugar concentrations - increased resistance to "redox death". As discussed above, high sugar concentrations can kill LDH ^" Geobacillus cells in a process known as "redox death".

A selection gradient which is a feedstock gradient may therefore be used to select for microbial strains that are resistant to "redox death". A feedstock gradient may be used to select Geobacillus strains that are capable of growing, or capable of producing ethanol (fermenting), using relatively high concentrations of hemicellulosic sugars, C5 sugars, or specific C5 sugars such as xylose or arabinose, or reduced sugars such as arabitol, ribitol, sorbitol or xylitol. Such Geobacillus strains will utilise higher concentrations of sugars to yield more concentrated ethanol, this will increase volumetric productivity of the process and reduce distillation energy costs.

Hartley and Javed (2007) proposed a system in which the culture growth rate of LDH^{^} Geobacillus is regulated by sparging with a minimum amount of air, which restores redox balance by oxidising the excess pyruvate to carbon dioxide and water via the Electron Transport Chain (Figure 2), thereby avoiding "redox death". Inhibition of culture growth by ethanol is avoided by continuous removal of ethanol vapour under mild vacuum or sparging with recycled carbon dioxide. Although this minimum amount of oxygen allows sufficient production of ATP for growth, ethanol yields are reduced. Fortunately however flux through the PFL pathway is inactivated by oxygen, so ethanol yields can still be > 80% of theoretical.

Nevertheless "redox death" remains a drawback in this system because PDH flux becomes saturated above 6% w/v sugars feed in this system, so this process can only produce less than 3% v/v ethanol. However, the autoselector device of the present invention may be used to select Geobacillus mutants that grow on relatively high sugar concentrations, as described herein, (for example due to increased PDH activity) and thereby increase volumetric ethanol productivity. b). Strains that utilise pectin hvdrolvsates and minimise effluent treatment

Most wild-type Geobacillus strains utilise the more abundant hemicellulosic sugars, xylose and arabinose, but not galactose or galacturonic acid derived from pectin. Pectins are abundant together with hemicelluloses in residual biomass from food processing plants such as from cider production, fruit juice production, potato crisp manufacture or sugar beet processing. Pectins have negligible animal feed value but are easily hydrolysed by enzymes, so galactose and galacturonic acid would emerge in the effluent from a bioethanol plant using such feedstocks.

An "autoselector" in Configuration C3 will readily select wild-type Geobacillus strains that grow well on such effluents so could provide an efficient effluent disposal process providing additional valuable feed.

Alternatively, deletion of the LDH gene in pectin-utilising Geobacillus strains would provide a continuous fermentation process with negligible effluent that yields even more ethanol. Moreover the rest of the pectin hydrolysate is converted to high protein Geobacillus cells that have high animal feed value.

Accordingly, the present invention provides a method of selecting a microbial strain, such as a Geobacillus strain, wherein the method comprises providing a device of the invention and adding microbial cells to the first fermenter compartment in the series of fermenter compartments, and applying a selection gradient which is an increasing feedstock gradient or an increasing sugar gradient. In such methods the device is configured to provide a series of fermenter compartments in fluid connection each containing culture medium, wherein the concentration of feedstock or sugar in the culture medium is higher in each successive fermenter compartment.

A sugar gradient may vary for example from about 2% to about 20% w/v sugars, from about 2% to about 20% w/v sugars, from about 2% to about 10% w/v sugars, from about 3% to about 10% w/v sugars, from about 4% to about 10% w/v sugars, or from about 4% to about 8% w/v sugars. For example the first fermenter compartment may contain, and be diluted with, culture medium comprising about 4% w/v sugars, the second and successive compartments may contain, and be diluted with, culture medium comprising, about 6%, 8%, 10% and 12% w/v sugars respectively. For example the first fermenter compartment may contain, and be diluted with, culture medium comprising about 4% w/v sugars, the second compartment may contain, and be diluted with, culture medium comprising, about 5% w/v sugars, the third compartment may contain, and be diluted with, culture medium comprising about 6% w/v sugars, the fourth compartment may contain, and be diluted with, culture medium comprising about 7% w/v sugars, the fifth compartment may contain, and be diluted with, culture medium comprising about 8% w/v sugars, and so on.

A method of selecting a microbial strain which method comprises applying a selection gradient which is an increasing feedstock gradient, wherein the feedstock is sugar, may be a method of selecting a strain of LDH^~Geobacil!us capable of growing micro-aerobically or semi-aerobically in a culture medium containing at least about 6% w/v sugars, at least about 7% w/v sugars, at least about 8% w/v sugars, at least about 9% w/v sugars, at least about 10% w/v sugars, at least about 1 1 % w/v sugars, or at least about 12% w/v sugars.

The sugar may be glucose, for example in a method for selecting an LDH^~ Geobacillus having improved resistance to redox death. The sugar may be galactose or galacturonic acid or a mixture thereof, for example in a method for selecting a Geobacillus for improved effluent disposal methods.

A method of selecting for resistance to redox death is preferably performed under semi-aerobic or micro-aerobic conditions.

The above described methods using the autoselector in Configuration C3 may be used for selecting for strains of LDH^~Geooac///us capable of using substantially all sugars derived from biomass. The strain selected using this method may then be selected for growth at relatively high temperatures using the device in Configuration C5 as described below, thereby providing a method for selecting for a strain of LD ^~ Geobacillus capable of using substantially all sugars derived from biomass at relatively high temperatures. Alternatively, the selection for growth at higher temperature using Configuration C5 may be applied first, followed by the selection for use of substantially all sugars derived from biomass. In this context, a relatively high temperature may be about 50^°C or higher, about 55^'C or higher, about 60^°C or higher, about 65^°C or higher, or about 70^° C or higher.

Configuration C4 provides a selection gradient of increasing toxin concentration.

This is suitable for selecting for strains having resistance, or improved resistance, to growth inhibitors (toxins) such as a fermentation product (e.g. ethanol), or more commonly, toxic products arising during feedstock preparation, such as furfural from breakdown of pentoses.

In Configuration C4, culture conditions are identical in each fermenter compartment, but there is a stepwise increase in toxin concentration in successive compartments either through spare ports or to the feed lines through a T-tube. For example:- a) Ethanol tolerant strains

External ethanol concentration up to 4% v/v has little effect on the growth rate of LDH- Geobacilli at 60^"C - 70^°C, either aerobically or anaerobically, or in rich or defined medium (Amartey et al. 1997). This natural ethanol tolerance is sufficient for most fermentation processes considered herein. However increased ethanol tolerance is a desirable feature for improved strains growing on higher sugar concentrations, or at higher temperatures, such as are here envisaged.

Ethanol tolerant strains growing in these new extreme conditions can readily be selected by stepwise increase in ethanol additions to successive compartments. Cell death and residual sugars concentration will successively increase. After in situ mutagenesis, ethanol tolerant mutant strains with increased growth in successive compartments will be selected. b) Strains resistant to toxins in industrial or agricultural effluents

Configuration C4 is useful to select strains that are resistant to toxins such as pesticides, that will naturally proliferate in dumps or compost heaps that contain such toxins. Resistant strains in samples from such sources will be selected by the autoselector in configuration C4 growing aerobically or semi-aerobically on glucose or sucrose with a gradient of increasing effluent concentration. Their pesticide resistance can be enhanced by sporadic

mutagenesis, and in many cases this will arise from enzymes that hydrolyse or otherwise modify the toxin. Analysis of toxin concentrations in the effluent broth after significant takeovers will identify strains that produce such enzymes, such strains will be invaluable for effluent treatment.

A selection gradient which is a toxin gradient may be used to select for a microbial strain from a microbial culture which strain is are capable of growing in relatively high

concentrations of substances which are normally inhibitory to the growth of that microbial culture. A toxin may also be referred to as a growth inhibitor. A toxin may be

2-deoxyglucose, ethanol, furfural, or a pesticide.

Accordingly, the present invention provides a method of selecting a microbial strain, such as a Geobacillus strain, wherein the method comprises providing a device of the invention and adding microbial cells to the first fermenter compartment in the series of fermenter compartments, and applying a selection gradient which is an increasing toxin concentration. In this method the device is configured to provide a series of fermenter compartments in fluid connection each containing culture medium, wherein the concentration of a toxin in the culture medium is higher in each successive fermenter compartment.

The toxin gradient may be an ethanol gradient. An ethanol gradient may vary for example from about 1% to about 10% v/v ethanol, from about 1 % to about 6% v/v ethanol, from about 1 % to about 5% v/v ethanol, from about 2% to about 10% v/v ethanol, from about 2% to about 6% v/v ethanol, or from about 2% to about 5% v/v ethanol.

For example the first fermenter compartment may contain, and be diluted with, culture medium comprising about 4% v/v ethanol, the second compartment may contain, and be diluted with, culture medium comprising, about 4.5% v/v ethanol, the third compartment may contain, and be diluted with, culture medium comprising about 5% v/v ethanol, the fourth compartment may contain, and be diluted with, culture medium comprising about 5.5% v/v ethanol, the fifth compartment may contain, and be diluted with, culture medium comprising about 6% v/v ethanol, and so on. For example the first fermenter compartment may contain, and be diluted with, culture medium comprising about 4% v/v ethanol, the second compartment may contain, and be diluted with, culture medium comprising, about 6% v/v ethanol, the third compartment may contain, and be diluted with, culture medium comprising about 8% v/v ethanol, the fourth compartment may contain, and be diluted with, culture medium comprising about 10% v/v ethanol, the fifth compartment may contain, and be diluted with, culture medium comprising about 12% v/v ethanol, and so on.

A method of selecting a microbial strain which method comprises applying a selection gradient which is an increasing toxin gradient, wherein the toxin is ethanol, may be a method of selecting a strain of LD ^~Geobacillus capable of producing, and/or growing in a culture medium containing, at least 4% v/v ethanol, at least 6% v/v ethanol, at least 8% v/v ethanol, or at least 10% v/v ethanol in anaerobic or microaerobic conditions. c). Strains for production of bioethanol from a mixture of biomass sugars and glycerol,

LDH- Geobacilli cells grow well on glycerol aerobically, but anaerobic growth produces excess NADH, causing redox death. Another solution to the "redox death" problem that has been suggested is to grow LDH- Geobacillus anaerobically on a mixture of sugars and glycerol, which would restore redox balance by allowing the excess NADH from the glycerol to be used to reduce the excess acetyl-CoA arising from the PFL-pathway (Baghaei-Yazdi ef a/., 2009). As shown in Figure 3, such a mixed fermentation process would produce more ethanol and half as much CO2 as a yeast ethanol fermentation. The other half is removed in the effluent as formate and retained in the stillage, thereby reducing atmospheric CO2 Since glycerol is a by-product of biodiesei production with low commercial value, this strategy offers the prospect of producing both biofuels (bioethanol and biodiesei) from waste biomass. Ethanol yields will be much higher, and carbon dioxide emissions lower, than those from conventional yeast fermentations of food sugars.

However attempts to demonstrate this process have failed, because glycerol uptake by Geobacilli is repressed during growth on sugars, as in many other microorganisms; a phenomenon known as 'catabolite repression".

The autoselector of the present invention can be configured and used in methods to select mutants that will utilise both sugars and glycerol simultaneously. Mutant microbial strains selected for resistance to 2-deoxyglucose no longer repress glycerol uptake, and take up glycerol even when growing on glucose (Hodgson 1982). Configuration C4 of the device, growing aerobically on low glucose and excess glycerol with a selection gradient of 2-deoxyglucose, will select catabolite repression-resistant strains of LDH- Geobacilli, (if necessary after UV mutagenesis), that lack a functional glycerol-uptake repressor. These will grow anaerobically on mixtures of glycerol and biomass sugars, so will be suitable production strains for the envisaged bioethanol process.

Accordingly the present invention provides a method of selecting a strain of LDH^~

Geobacillus capable of utilising glucose and glycerol simultaneously in anaerobic culture (i.e. capable of growing using a mixture of glucose and glycerol), the method comprising performing a first selection step comprising providing a device of the invention and inoculating the first fermenter compartment in the series with LDH- Geobacilli, and applying a selection gradient which is an increasing gradient of 2-deoxyglucose. In this method the autoselector is configured to provide a series of fermenter compartments containing a culture medium containing a mixture of glucose and glycerol, and culture medium in successive compartments contains 2-deoxyglucose in increasing concentrations. Glucose uptake and steady state cell density will decrease in successive compartments. Mutants resistant to catabolite repression will take over, and be capable of utilising both sugars and glycerol aerobically and, preferably, capable of growing anaerobically on a mixture of sugars and glycerol.

Accordingly, the above described method may further comprise a second selection step, comprising isolating the strain selected in the first selection step and selecting for anaerobic growth, by inoculating the first a fermenter compartment with the strain selected in the first selection step, and applying a selection gradient which is a decreasing gradient of oxygen. In this method the autoselector is configured to provide a series of fermenter compartments containing a culture medium containing a mixture of glycerol and glucose as feed stock, wherein the feedstock concentration is the same in each fermenter compartment, and wherein the oxygen content decreases in successive fermenter compartments.

Configuration C5 provides a selection gradient of increasing or decreasing temperature

In this configuration, culture conditions are identical in each fermenter compartment, except that there is a stepwise temperature increase or decrease. Such temperature gradients may be combined with any of the other configurations described above, for example to determine optimum growth temperature at increasing dilution rates or aeration rates. The device in these configurations will have the following uses:- a). Determination of optimum growth temperature.

This is useful for process optimisation, since the optimum temperature for any particular strain will vary according changes in, for example, pH, aeration, feedstock composition or concentration. For this purpose, the C2 configuration can be used with an identical inoculum of medium and cell broth from a batch culture to each compartment. To determine the growth temperature range of an unknown isolate, a broad temperature gradient can be chosen, say 20^°C to 90^°C. Alternatively a narrow temperature range such as 65^°C to 75^°C can be used to optimise growth temperature for a particular feedstock. b) . Selection for increased maximum growth temperature.

It will be of particular advantage to select more thermostable Geobacilli for ethanoi production since the ethanoi vapour can then be more easily removed from the fermentation broth by mild vacuum. The C5 configuration with a temperature range of 65^°C to 80^°C can be used, preferably with optimised culture conditions in each compartment. Sporadic mutagenesis will create more thermostable mutants which will be readily selected in the hotter compartments. c) . Selection for decreased minimum growth temperature.

Unexpectedly, the invention in the C4 configuration used to select for decreased minimum growth temperature may be important for selection of improved Geobacillus strains for bioethanol production, as illustrated in Example 1.

A selection gradient which is an increasing temperature gradient may be used to select microbial strains capable of growing at relatively high temperatures, that is, to select thermostable strains. The temperature at the lower end of the gradient is applied to the first fermenter in the series and the temperature at the upper end of the gradient is applied to the final fermenter in the series.

A temperature gradient for an unknown mixture of natural isolates may vary for example from around 40°C to around 90°C, whereas a narrow range of from around 65°C to around 80°C, may be suitable to select thermophile strains capable of growing at relatively high temperatures.

The Configuration C5 shown in Figure 6 is suitable for selecting thermostable strains. In this configuration culture conditions are identical in each fermenter compartment, except that there is a stepwise temperature increase (or decrease) in successive compartments.

Sporadic mutagenesis will select mutants of whatever genes regulate the range of growth temperature. It will be of particular advantage to select more thermostable Geobacilli for ethanoi production since the ethanoi vapour can then be more easily removed from the fermentation broth by mild vacuum.

The present invention provides methods, and uses of the autoselector device, for selection of improved strains of Geobacilli for bioethanol production. As mentioned above, "redox death" of LDH~ mutant Geobacilli in anaerobic conditions in relatively high sugar culture medium is an obstacle to the efficient commercial exploitation of Geobacilli for bioethanol production. Methods of the invention, and uses of the device of the invention, offer routes to strain improvement by selecting for Geobacilli strains which are resistant to "redox death".

Strategies for selecting for Geobacilli strains that avoid the "redox death" phenomenon have previously been suggested (but so far none of these selection strategies has led to the isolation of a strain that is commercially useful for the production of bioethanol). The autoselector device of the present invention allows for very efficient selection of microbial strains having characteristics useful for bioethanol production, such as characteristics associated with resistance to "redox death". It is therefore expected that implementation of selection strategies using the autoselector device, as set out herein, will provide new and commercially useful Geobacillus strains for the production of bioethanol.

Strategies for selecting for Geobacilli strains that avoid the "redox death" phenomenon include selecting for strains that grow on high glucose concentrations, and selecting for strains capable of fermenting sugars plus glycerol, as explained above. A further two strategies for selecting for Geobacilli strains that avoid the "redox death" phenomenon include introducing a foreign formate dehydrogenase (fdh) gene, and introducing and expressing a foreign pyruvate decarboxylase gene (pdc), as explained below. a) Selecting a microbial strain having a thermostable formate dehydrogenase (fdh) gene. This strategy for avoiding "redox death" was designed to create a novel PFL-FDH anaerobic growth pathway which maintains redox balance and produces only ethanol and carbon dioxide (Figure 4). Since the PDH overflow pathway also produces only ethanol and carbon dioxide, such strains should produce ethanol of almost 100% of theoretical maximum yields anaerobically at neutral pH where cell viability is highest.

Javed er at. (2007) constructed a synthetic fdh gene using Geobacillus preferred codons to encode the amino acid sequence of Pseudomonas 101 FDH, the most thermostable of known formate dehydrogenases (Tishkov & Popov, 2006). The gene was transformed into a Geobacillus strain under control of the powerful lactate dehydrogenase LDH promoter, but no formate dehydrogenase activity could be detected in cells grown at 52^*C although the holoenzyme is stable up to 60^°C in vitro. This may be because the apoenzyme (lacking bound NAD) is less stable than the holoenzyme (with bound NAD^*). Therefore a Geobacillus host with a much lower growth temperature is required to allow the thermostable holoenzyme time to assemble.

Use of the autoselector device of the present invention offers a route to select a Geobacillus that expresses a more thermostable formate dehydrogenase. The formate dehydrogenase is preferably a heterologous, or foreign, formate dehydrogenase.

Accordingly, the present invention provides a method of selecting a microbial strain, such as a Geobacillus strain, having a thermostable heterologous (foreign) formate dehydrogenase (FDH). The method comprises transforming microbial cells with a heterologous gene encoding FDH, or providing microbial cells that comprise a heterologous gene encoding FDH. The method further comprises providing a device of the invention and adding the cells to a first fermenter compartment in the series. The method has two or three selection steps: (1 ) selecting for decreased minimum temperature growth by applying a decreasing temperature gradient across the series of fermenter compartments; taking the strain selected by the first selection step and (2) selecting for growth on high sugar concentrations by applying a sugar concentration gradient; and optionally (3) taking the strain selected by the second selection step and selecting for growth at relatively high temperatures by applying an increasing temperature gradient. Preferably in this method the culture medium used in the device may be supplemented with nicotinamide so as to increase the intracellular concentrations of both NAD and NADH. b) Selecting a microbial strain having a thermostable pyruvate decarboxylase (pdc) gene. This strategy may solve the "redox death" problem by creating a yeast-like overflow pathway to convert the excess pyruvate directly to ethanol and carbon dioxide. Consequently Green et al, (2001 ) introduced the Zymomonas pdc gene into an LDH- Geobacillus strain under control of the powerful LDH promoter, to yield strain TN-T9-P1 . Unfortunately the pyruvate decarboxylase proved to be unstable at the minimum temperature required for growth of the mutant Geobacilli (Thompson et al. 2008) so the project was abandoned.

However the autoselector provides a convenient route to select mutants that express a more thermostable heterologous, (foreign) pyruvate decarboxylase.

Accordingly the present invention provides a method of selecting a Geobacillus strain expressing a thermostable heterologous pyruvate decarboxylase. The method comprises transforming microbial cells with a heterologous gene encoding pyruvate decarboxylase, or providing Geobacillus cells that already express a heterologous gene encoding pyruvate decarboxylase. The method further comprises providing a device of the invention and adding the transformed cells to the first fermenter compartment in the series of fermenter compartments. The method has two or three selection steps:

(1 ) selecting for decreased minimum temperature growth by applying a decreasing temperature gradient across the series of fermenter compartments. Growth will decrease as the temperature drops and residual sugars will rise. Mutant cells that grow more rapidly will take over in these compartments, taking the strain or strains thus selected and;

(2) selecting for growth on high sugar concentrations at the minimum growth temperature by applying a sugar concentration gradient, this will select strains that are resistant to redox death by virtue of expressing PDC activity, The strain selected by this second selection step with have improved resistance to redox death and a thermostable PDC, such that they are particularly suitable for bioethanol production. The method may optionally include a third step, which comprises taking the strain selected by this second selection step; and

(3) selecting for growth at higher temperatures by applying an increasing temperature gradient. This step selects for strains with an even more thermostable PDC. Preferably in this method the culture medium used in the device may be supplemented with thiamine to increase the intracellular concentration of the co-enzyme, thiamine pyrophosphate. Both the above methods for selecting a strain of LDH- Geobacillus having a thermostable heterologous formate dehydrogenase or having a thermostable heterologous pyruvate decarboxylase, each comprise (1 ) a step of selecting for decreased minimum temperature growth by applying a decreasing temperature gradient; and (2) a step of selecting for growth on high sugar concentrations by applying an increasing sugar concentration gradient; and optionally (3) a step of selecting for growth at increased temperatures by applying an increasing temperature gradient.

In the above methods, the first step of selecting for decreased minimum growth temperature may comprise applying a decreasing temperature gradient that varies from about 65^°C to about 40^"C. The second step of selecting for growth on high sugar concentrations may be as described above for methods of using the device in Configuration C3. Preferably the sugar gradient varies from about 2% w/v to about 10% w/v. The third step of selecting for growth at relatively high temperatures may be as described above for methods of using the device in Configuration C5. Preferably the temperature gradient is from about 55°C to about 70°C.

Other selection gradients that may be applied using the device of the invention in methods of the invention include a pH gradient and a composition gradient. In a pH gradient the pH may increase or decrease progressively across the series of fermenter compartments. For example a pH gradient may vary from about pH 7 to about pH 9, or from about pH 7 to about pH 5. The latter may be useful to increase growth below pH 6, wherein expression of the PFL pathway is suppressed.

A feedstock composition gradient is a gradient of concentration of any growth-limiting component in a culture medium composition, For example, the effluent broth from fermentations of a biomass hydrolysates will contain a mixture of unfermentable sugars, such as those derived from pectins. Hence a culture medium rich in these components may be obtained by centrifuging such an effluent. The supernatant may then be used in a device of the invention as an increasing feedstock gradient, so as to select strains that grow more rapidly on these unfermentable components

The autoselector device of this invention is admirably suited to the task of identifying further desirable growth factors to add to this feedstock. The growth conditions and feedstock will be the same as used in the process, except that stepwise additions of suspected growth- limiting factors will be made to successive compartments. The latter, notably essential amino acids, can be identified as components of the minimal defined medium described for anaerobic growth of Geobacilli (San Martin er a/. 1992). Desirable additives will be easily identified by stepwise increases in steady state cell density.

Novel thermostable strains can be selected for use with a specific feedstock, such as a specific biomass hydrolysate, (for example enzymic hydrolysates of beet pulp), by inoculating cells (for example Geobacilli taken from their native environment, such as a compost heap or beet pulp) into an autoselector device supplied with that feedstock in an increasing temperature gradient. Strains selected under these conditions will be useful hosts for conversion to ethanol producers by metabolic engineering.

Moreover the autoselector device may be used as a tool to select mutants of many other enzymes, inducers, repressors or permeases where these are growth limiting for the host strain. Example 1 illustrates its use to select a more thermostable formate dehydrogenase to allow growth by the PFL-FDH pathway shown in Figure 4.

The selection gradient will vary across the series of fermenter compartments in a step-wise manner. The size of the steps between each fermenter compartment may be constant, so as to create a liner gradient. For example the dilution rate (growth rate) of the cultures may be maintained at 0.1 h^~1 in a first fermenter, 0.2 h ¹ in a second fermenter, 0.3 h^"1 in a third fermenter and so on. Alternatively, the size of the steps between each fermenter may be variable. For example an increasing or decreasing exponential gradient of growth temperature may be applied. This can be achieved by embedding a series of fermenters at regular intervals within a metal block, which is differentially heated at each end.

Thus methods of the invention may comprise applying a selection gradient, wherein applying the selection gradient comprises a step of varying a culture condition variable in a progressive manner across the series of fermenter compartments. The progressive manner of varying the culture condition variable may be (a) an increase in the culture condition variable or (b) a decrease in the culture condition variable. A culture condition variable is a culture condition that can be progressively varied e.g. temperature, feedstock concentration. The progressive variation in a culture condition may be a step-wise progressive variation. In use, the device of the invention may be arranged to provide a series of fermenter compartments in which a culture condition varies in a step-wise manner across the series of fermenter compartments. Preferably, the culture conditions in each fermenter are identical, except for the culture condition that is varied to from the selection gradient.

In this way, the device in use maintains a continuous culture of microbial cells at least in the first fermenter, which are subject to a series of progressively changing selection pressures in successive fermenters in the series. The transfer of culture broth from the first fermenter to the second fermenter in the series provides a regular input of populations of microbial cells capable of growing in the culture conditions of the first fermenter, many or most of which may be incapable of growing in the culture conditions of the second fermenter or may grow at a slower rate in the culture conditions of the second fermenter, owing to less favourable culture conditions. In each fermenter compartment, the microbial cells able to grow, or able to grow fastest, will outcompete the other cells, and thereby "take over" the fermenter compartment. Strains of microbial cells having improved characteristics may arise as a result of spontaneous or sporadic mutations. To facilitate genesis of mutant cells, the methods of the present invention may optionally comprise a mutagenesis step.

Microbial cells may be mutagenised in the device, in order to facilitate the genesis of mutant cells having improved traits. The mutagen may be UV light or a chemical mutagen. For example at least part of the device, or at least part of a fermenter compartment, may be placed under a UV lamp for mutagenesis by UV light. Alternatively or additionally, a chemical mutagen may be added to one or more fermenter compartments. Microbial cells may be exposed to mutagens intermittently. Accordingly, a method or use of the invention may comprise a step of mutagenising the microbial cell culture. The mutagenizing step is preferably performed in situ.

Examples

Example 1 - Selection for strains expressing a thermostable formate dehydrogenase

Javed & Baghaei-Yazdi (2007) describe a Geobacillus strain containing a synthetic

Pseudomonas 101 formate dehydrogenase gene under control of the LDH promoter. Figure 4 shows that this should provide a strain resistant to redox death that grows anaerobically by a novel PFL-FDH pathway to produce only ethanol and C(¾. However although the enzyme is stable up to 60^°C In vitro, it did not express FDH activity in vivo when grown very slowly on sugars at 52^'C. This can be explained if the apoenzyme (lacking bound NAD) is less stable than the holoenzyme (with bound NAD*). Therefore a Geobacillus host with a much lower growth temperature is required to allow the thermostable holoenzyme time to assemble.

A stepwise procedure may be used to select such strain:-

(i) A recombinant strain with decreased minimum growth temperature.

Lower growth temperature of the FDH transformant strain is selected by using the autoselector in C5 configuration at constant 2% w/v glucose feed rate with a decreasing temperature gradient of 65^°C to 40^°C. Growth rates will decrease as temperature drops, and residual sugars accumulate in successive compartments. Sporadic mutagenesis creates mutants that grow better at lower temperatures such as 50^°C to 45^°C, which take over in the colder compartments. These strains are then selected for expression of FDH activity as follows:-

(ii) . Selection for resistance to redox death.

If the recombinant apo-FDH, or a mutant thereof, is thermostable at the lower growth temperature, it will bind NAD to form the more stable hoio-FDH. Recombinant strains that express formate dehydrogenase activity at the lower temperature can be selected by the device in Configuration 3 growing anaerobically at that temperature at pH 7, with a sugar gradient of 2% to 10% w/v. To encourage holoenzyme formation, each compartment is supplemented with nicotinamide, (a precursor of NAD), to increase the intracellular NAD concentrations. Redox death will kill the cells at higher sugar concentrations, but strains expressing FDH activity will be more resistant to redox death and will take over in compartments fed with high sugar concentrations (e.g. these strains will probably be resistant to redox death at 60^°C, since the purified holoenzyme is stable at this temperature.) Even more thermostable mutants of these can be selected as follows:-

(iii) . Selection for strains expressing a more thermostable formate dehydrogenase

These will be selected by the device in configuration C5, growing anaerobically at pH 7 on high sugars (e.g. 6% w/v glucose, supplemented with nicotinamide), with a temperature selection gradient of 55 o 70^*C. The improved strains will grow rapidly at pH 7 on high concentrations of biomass to give close to theoretical yields of ethanol, at temperatures in which it can be continuously removed as vapour by mild vacuum. Example 2 - Selection for strains expressing a thermostable pyruvate decarboxylase (PDC). Figure 8 shows that such an LDH~ Geobacillus would provide a thermophile strain that could make ethanol from biomass sugars by the same pathway as a thermophilic yeast. For this reason, Green ef a/. (2001 ) transformed the Zymomonas PDC gene into a Geobacillus under control of the LDH promoter. The gene in the transformant, strain TN-T3-P1 , was transcribed and translated when grown at 52 ^°C, which is the minimum growth temperature of its host. Unfortunately, although the transformed strain appeared to be slightly more resistant to redox death, it showed negligible PDC activity in cell extracts, even though the isolated enzyme is stable up to 64^'C (Thompson et al. 2011 ). This can be explained if the newly synthesized apoenzyme is much less thermostable until it has bound, its coenzyme, thiamine pyrophosphate (TPP). Therefore host strains able to grow below 50^*C are needed.

A stepwise procedure like that described for Example 1 above may be used in to select such strains, as follows:-

(i) A recombinant strain with decreased minimum growth temperature

As in Example 1 at step (i) above, strain TN-T3-P1 ( or an analogous LDH- strain expressing a PDC gene under the LDH promoter) will be selected for anaerobic growth at lower temperatures.

(ii) Selection for resistance to redox death.

As in Example 1 at step (ii) above, recombinant strains that express PDC activity at the lower temperature can be selected by the device in Configuration 3 growing anaerobically at that temperature at pH 7, with a gradient of 2% to 10% w/v sugar concentration, To encourage holoenzyme formation, each compartment will be supplemented with thiamine, (a precursor of its coenzyme, thiamine pyrophospate, TPP). Strains expressing PDC activity will be more resistant to redox death and will take over in compartments fed with high sugar concentrations.

(Hi). Selection for strains expressing a more thermostable pyruvate decarboxylase.

Here the active enzyme is stable up to 64^*C, so the device will be used as in Example 1 step

(iii) in configuration C5, growing anaerobically on high sugars ( e.g. 6% w/v glucose supplemented with thiamine), using a temperature selection gradient of 55^°to 70^°C, The improved strains will grow rapidly at over 65^°C, pH 7, on high concentrations of biomass sugars in a batch, fed-batch or continuous microaerobic process, which will give close to theoretical yields of ethanol. This can be continuously removed as a > 30% v/v vapour by mild vacuum and distilled directly into 95% v/v bioethanol, thereby halving distillation costs.

Example 3 - Selection of novel soil microorganisms

A suitable finely ground soil sample is filtered through a fine mesh, steeped in water overnight, and then centrifuged to remove soil particles. The supernatant is dialysed to provide cell-free dialysate and the residue is ultracentrifuged to provide a mixed cell inoculum

In order to determine a range of mixed-cell growth rates samples of this are grown up at 15°C in shake-flasks containing a suitable minimal medium. A suitable minimal medium may be mineral salts, trace elements and vitamins, supplemented with glucose to provide the energy source and soil dialysate to act as the growth controlling nitrogen source. The optical density of samples from tis is taken from these at intervals, to determine the range of varying growth rates so as to select appropriate residence times for the pulsed rate segmented culture, which will be performed a follows:-

A coupled micro/ miniselector device of the invention (Figure 10) is fed with the above culture medium and segmented with oxygen, as described above The internal diameter of the microselector coil is chosen so that the volume of a 2cm segment is 5 μΙ_. The length of the coil and the pulse rate are chosen to fit the range of growth rates determined above The culture medium provided by Pump 1 is segmented by Pump 2 at a pulse rate, chose to provide 2cm bubbles of oxygen, adequate to utilise all of the glucose the culture medium therein, Pump 3 then inoculate the first segment with a mixed cell suspension, diluted such that each segment contains about one cell.

The effluent segments therefore emerge in stationary phase, and each is transferred by Pump 4 into a 250-500 μΙ_ segment of culture broth provided by Pumps 1 and 2 of an adjacent miniselector coil, whose ID provides such segments. The pulse rate and number of segments is identical, so discrete clones of different cells will emerge successively from the two-way valve. Each of these may be collected in a fraction collector holding Falcon tubes containing 5 ml of culture medium at 15°C, so that growth continues during the course of the experiment.

Example 4 - Screening for novel antibiotic production

The Falcon tubes described above may be ultracentrifuged, and samples of the supernatant mat be manually screened for antibiotic activity by conventional techniques, e.g. streaking on to plates of a target pathogen growing on conventional rich medium.

Alternatively, potential antibiotics may be automatically screened against a target pathogen in a series of plastic Miniselector coils, used to provide a toxin selection gradient for the pathogen growing on rich medium as described above. The suspected antibiotic may be injected in increasing quantity into the second and subsequent fermentation compartments, until a drop in growth rate is detected in the effluent broth. The successive drop in subsequent compartment will allow calculation of the LD₅o for that antibiotic.

Preferably however, the whole screening process may be carried out automatically by fist amplifying the number of novel cells emerging from the two-way valve by directing then into an even larger miniselector coil and growing them up to stationary phase in the same culture medium.

A significant part of the effluent broth may then be diverted directly into the series of coils described above that housie the target pathogen, before it is collected in Falcon tubes. A decline in pathogen growth rate caused by the rising toxin gradient will automatically signal to the attendant devices operator, so that a novel antibiotic will have been discovered with minimum human intervention.

The following numbered clauses describe aspects of the invention, and form part of the description.

1. A device for selection of microbial strains, comprising:

a plurality of fermenter compartments for the continuous culture of microbial cells, wherein the fermenter compartments are linked to provide a series of fermenter compartments in fluid connection, such that microbial cells are successively transferable through each fermenter compartment in the series.

2. A kit of parts, comprising:

a plurality of fermenter compartments for the continuous culture of microbial cells; and,

a plurality of conduits for linking the fermenter compartments to provide a series of fermenter compartments in fluid connection, such that microbial cells are successively transferable through each fermenter compartment in the series.

3. The device according to clause 1 , or kit according to clause 2, which comprises two, three, four, or five fermenter compartments.

4. The device or kit according any one of the preceding clauses, wherein a fermenter compartment has a volume of about 40 to about 200 millilitres.

5. The device or kit according to any one of clauses 1 to 3, wherein a fermenter compartment has a volume of about 0.2 to about 10 millilitres.

6. The device or kit according to clause 5, wherein each fermenter compartment comprises tubing for segmented flow of culture broth.

7. The device or kit according to clause 6, wherein each fermenter compartment comprises tubing arranged in a helical formation.

8. The device or kit according to any one of the preceding clauses, further comprising one or more heating elements and/or cooling elements for: (a) maintaining the same temperature in each fermenter compartment; or

(b) maintaining a temperature gradient across the series of fermenter compartments.

9. The device or kit according to any one of the preceding clauses wherein the fermenter compartments are identical.

10. The device or kit according to any one of the preceding clauses, wherein each fermenter compartment has a removable cap supplied with multiple adjustable ports. 1. The device or kit according to clause 7 in which the ports are connected to a multichannel pump, such as a peristaltic pump, to allow regulated inlet and outlet of liquids or gas mixtures.

12. The device or kit according to any one of the preceding clauses, wherein the fermenter compartments comprise inlets for regulated supply of growth medium, buffer for pH control, gas mixtures, other additives such as growth factors or toxins, or gas mixtures for oxygen supply and gas sparging.

13. The device or kit according to any one of the preceding clauses, wherein the fermenter compartments comprise outlets for removal or continuous sampling of fermentation broth, or gases and vapour, or for transfer of a portion of effluent broth to an adjacent fermenter compartment to act as a growth inoculum.

14. The device or kit according to any one of the preceding clauses, wherein each fermenter compartment comprises transparent parts arranged for visual inspection or U V irradiation of resident cells.

15. A method of selecting a microbial strain, the method comprising:

providing a device according to any one of the preceding clauses;

adding microbial cells to a first fermenter compartment of the series of fermenter compartments; and

applying a selection gradient across the series of fermenter compartments.

16. The method according to clause 15, wherein the selection gradient is:

(a) a temperature gradient;

(b) an aeration gradient;

(c) a feedstock concentration gradient;

(d) a dilution rate gradient

(e) a pH gradient

(f) a composition gradient; or

(d) a toxin or growth inhibitor concentration gradient. 17. The method according to clause 16, wherein applying the selection gradient comprises a step of varying a culture condition variable in a progressive manner across the series of fermenter compartments, wherein the progressive manner of varying the culture condition variable is (a) an increase in the culture condition variable or (b) a decrease in the culture condition variable.

18. The method according to any one of clauses 15 to 17, wherein the method is for selecting a strain of Geobacillus.

19. The method according to clause 18, wherein the method is a method of selecting a strain of LDH- Geobacillus having improved characteristics for the production of bioethanoi.

20. The method according to clause 18 or clause 19, which is a method for selecting:

(a) a strain of LDH- Geobacillus capable of growing microaerobically in a culture medium containing at least about 6% w/v sugars;

(b) a strain of LDH- Geobacillus capable of producing at least 4% v/v ethanol in microaerobic fermentations;

(c) a strain of LDH- Geobacillus capable of utilising substantially all sugars derived from hydrolysis of biomass,

(d) a strain of LDH- Geobacillus capable of utilising substantially all sugars derived from hydrolysis of biomass, at temperatures higher than about 60^"C.

(e) a strain of LDH- Geobacillus capable of growing anaerobically utilising a mixture of sugars and glycerol;

(f) a strain of LDH- Geobacillus expressing a thermostable heterologous formate dehydrogenase.

(g) a strain of LDH- Geobacillus expressing a thermostable heterologous pyruvate decarboxylase.

21. The method according to clause 20, which is a method for selecting a strain of LDH^~ Geobacillus having a thermostable heterologous formate dehydrogenase or having a thermostable heterologous pyruvate decarboxylase, wherein the method comprises:

(i) a step of selecting for decreased minimum temperature growth by applying a decreasing temperature gradient; and

(ii) a step of selecting for growth on high sugar concentrations by applying an increasing sugar concentration gradient; and optionally

(iii) a step of selecting for growth at increased temperatures by applying an increasing temperature gradient.

22. The method according to any one of clauses 15 to 21 , further comprising a step of mutagenising microbial cells. 23. The method according to any one of clauses 15 to 22, wherein after the step of applying a selection gradient the method comprises a step of isolating a microbial strain from a fermenter compartment.

24. The method according to clause 23, further comprising using the isolated microbial strain for the production of ethanol.

25. Use of the device according to any one of clauses 1 to 11 , in a method for selecting a microbial strain.

26. A microbial cell or a microbial strain selected using the device, or method or use of the device according to any one of the preceding clauses.

27. Use of a microbial cell or a microbial strain selected according to clause 26 for bioethanol production.

28. A device for selection of microbial strains, comprising a series of identical fermenter compartments for the continuous culture of microbial cells, wherein the compartments may or may not be linked to allow transfer of a portion of the microbial cells successively through each fermenter compartment.

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Claims

58 CMms

1. A device for selection of microbial strains, comprising:

a plurality of fermenter compartments for the culture of microbial cells,

wherein the fermenter compartments are linked to provide a series; of fermenter compartments in fluid connection, such that microbial cells are successively transferable through each ferm enter compartment in the series.

and wherein a fermenter compartment has a volume of about 5 μί to 200 ml

2. The device according to claim 1 which comprises two, three, four, or five fermenter compartments.

3. The device or kit according fo any one of the preceding claims* wherein each fermenter compartment comprises tubing for segmented flow of culture broth.

4. The device or kit according to claim 5, wherein each fermenter compartment comprises tubing arranged in a helical formation.

5. The device or kit according to any one of the preceding claims, wherein the fermenter compartments are suitable for continuous culture of microbial cells.

6. A method of selecting a microbial strain, the method comprising:

providing a device according to any one of the preceding claims;

picking an Inoculum of microbial ceils, adding the inoculum of microbial ceils to a first fermenter compartment of the series of fermenter compartments.

7. The method of claim 8, wherein the microbial cells are environmental

microorganisms, optionally soil microorganisms.

8. The method of claim 8 or claim 7 wherein effluent broth is collected from a fermenter compartment and assayed for antibiotic activity.

9. The method according to any one of claims 8 to 8, wherein the selection gradient is:

(a) a temperature gradient;

(b) an aeration gradient;

(c) a feedstock concentration gradient;

(d) a dilution rate gradient

(e) a pH gradient

(f) a composition gradient; or

(d) a toxin or growth inhibitor concentration gradient.

10. The method according to any one of claims 6 to 9, wherein applying the selection gradient comprises a step of varying a culture condition variable in a progressive manner across the series of term enter compartments, wherein the progressive manner of varying the culture condition variable Is (a) an increase in the culture condition Yariable or (b) a decrease in the culture condition variable.

11. The method according to any one of claims 8 to 10, or the deylce of any one of claims 1 to 5, wherein the device further comprises one or more heating elements and/or cooling elements for:

(a) maintaining the same temperature in each fermenter compartment; or

12. The method according to any one of claims 6 to 11 , or the device of any one of claims 1 to 5 or 11 , wherein the fermenter compartments are identical.

13. A kit of parts, suitable for providing a device according to any one of claims 1 to 5, comprising:

a plurality of fermenter compartments for the culture of microbial ceils; and, a plurality of conduits for linking the fermenter compartments to provide a series of fermenter compartments in fluid connection, such that microbial cells are successivel transferable through each fermenter compartment in the series.