NL2011472C2 - Storage compound production by phototrophic diatoms. - Google Patents

Abstract

The invention is directed to a method for producing an enriched diatom culture, comprising subjecting a starting culture comprising one or more diatom species to selective pressure, thus giving a competitive advantage to storage compound producing species of diatoms, by subjecting said starting culture, under non-limiting bioavailable silicon concentrations, to a cycle of alternating dark phases and light phases and providing limitation of availability of at least one essential growth nutrient in one or more of said light phases.

Description

Title: Storage compound production by phototrophic diatoms

BACKGROUND OF THE INVENTION

The invention is in the field of renewables. More specifically, the invention pertains to methods of production of storage compounds by using phototrophic microorganisms.

Phototrophic microorganisms (algae) are capable of producing storage compounds such as lipids and sugars. Until recently, the majority of research has been focused on pure cultures of photosynthetic organisms. Cultivation of pure cultures is costly and unstable. WO-A-2013/012329, incorporated herein in its entirety, discloses a different approach, wherein an open phototrophic culture with improved storage compound production capacity is obtained. To this end a starting culture of a phototrophic organisms, such as algae, is subjected to selective pressure, which results in a competitive advantage to storage compound producing species, which are consequently formed in greater number. The selective pressure is applied to starting culture by subjecting it to a cycle of alternating dark phases and light phases and providing limitation of availability of essential growth nutrients in one or more of said fight phases.

It was found that the algae species used in the prior art often pose difficulties in subsequent cultivation, accumulation and/or separation steps. In particular it was found that the storage compounds produced in the prior art were mainly polysaccharides and to a lesser extent lipids, while lipid compounds are generally more desirable from a viewpoint of renewable energy. Furthermore, due to large amount of water in which algae are grown, separation can be costly since large volumes of water have to be treated and removed. Furthermore, the destruction of the cell walls of the algae used in prior art is difficult because of their tenacity; most algae species have a flexible, elastic cell wall, which is difficult to break. This makes cultivation, accumulation and/or harvesting of the phototrophic organisms and the storage compounds difficult and expensive.

It would be desirable to provide methods that result in enhanced production of lipids and easier ways of harvesting the phototrophic organisms and the storage compounds accumulated in the phototrophic organisms.

BRIEF SUMMARY OF THE INVENTION

It was found that the problems associated with the prior art may be overcome by using siliceous algae, in particular diatoms as phototrophic microorganisms. Thus, in a first aspect, the present invention is directed to a method for producing a diatom culture, comprising subjecting a starting culture comprising one or more diatom species to selective pressure, thus giving a competitive advantage to storage compound producing species of diatoms, by subjecting said starting culture, under condition favorable for diatom dominance, to a cycle of alternating dark phases and fight phases and providing limitation of availability of essential growth nutrients in one or more of said fight phases.

DETAILED DESCRIPTION OF THE INVENTION A major group of photosynthetic organisms are diatoms. One unique feature of diatoms is the silicon-based cell wall. Diatoms posses interesting characteristics for storage compound production, such as high storage compound productivity under certain conditions, easy solid / liquid separation and cell destruction. These characteristics are essential for a feasible large scale process, but are largely overlooked. By applying the procedure described in WO-A-2013/012329 under bioavailable silicon rich conditions we can obtain a culture of storage compound producing diatoms in open systems. We therefore combine the advantages of working in open systems and the advantages that diatoms posses. Another advantage is the possible market value of the silicon based cell wall remains.

Hildebrand et al. (Biofuels, 3(2012)221-240) reported that diatoms posses characteristics that allow for easier harvesting and breakdown of cell walls and can accumulate lipids under bioavailable silicon (Si) limitation.

Ulrich Sommer (Limnol. Oceanogr., 30(1985)335-346) described chemostat competition experiments with natural phytoplankton communities. Diatom presence was found to increase with increasing bioavailable silicon/phosphate ratios.

Diatoms can produce low value bulk compounds, such as precursors for biofuels or amorphous silica (also known as diatomite).

Diatoms can also be used to produce high value compounds, such as polyunsaturated fatty acids or antibiotics. Diatoms can act as, or deliver building blocks for nanotechnology, such as photonic devices, optical sensors or gas sensors, see for instance T. Fuhrmann et al., Applied Physics B 78(2004) 257-260; http://www.nanowerk.com/news/newsid=1591.php; and Parker et al., Nature Nanotechnology 2(2007) 347-353.

The present invention is based on the notion that it is much more efficient not to have to rely on sterile conditions. Using diatom cultures as a carbon and/or silicon source can reduce the production costs compared to conventional agriculture, diatomite mining or chemical technology. These diatom cultures with high storage compound content can be cultured in relatively cheap systems such as open ponds. Desirable functionality of this culture is to contain phototrophic species that have high lipid or other storage compounds producing capacity and produce a biomass which contains a substantial amount of storage compounds.

This method of the invention is based on selective enrichment and can be used to obtain desired non-axenic cultures in non-sterile environments. By subjecting a starting culture to selective pressure with the aim of improving yield of storage compounds, results in a so-called “open” culture, that has the desired improved product yield. An open culture is defined herein as a culture that needs not be confined to a single species, so generally it contains two or more diatom species. Apart from two or more different phototrophic diatom species, also other organisms may be present, such as other phototropic microorganisms, bacteria, or fungi. This means that the equipment used does not have to be sterile, which is a great advantage, in particular from an economic point of view. The culture used is thus a non-axenic culture, that may comprise more than one specie of diatoms. Preferably the open culture of the present invention is non-axenic.

In accordance with the present invention a selection step is carried out on a mixed starting population of diatoms. This selection step favors storage compound producing diatoms. This step may be followed by an accumulation step, wherein the cell division is suppressed, by stopping the addition of an essential nutrient. An example of such an essential nutrient is nitrogen (N). Because of the depletion of nitrogen, cell division stops and the diatoms accumulate sugars or lipids, because these accumulation processes can still be carried out in the absence of the essential nutrient.

However, all cellular processes dependent on nitrogen containing compounds are influenced by nitrogen limiting conditions and this may result in problems elsewhere in the diatom cells. It may for instance be no longer possible for the cell to efficiently absorb light or to sustain nonlimiting enzyme levels and as a result the metabolic pathways towards lipids and sugars may be hampered. For this reason depletion in nitrogen compounds was found to be less preferable. In accordance with the present invention the limitation can be carried out by making bioavailable silicon (Si) containing compounds limited in availability. Since silicon is specifically used to build the cell wall and has substantially no other uses for the cell, bioavailable silicon depletion results only in the cells stopping their division process, but at the same time the cells remain in relatively good condition. As a result the cells will continue to produce storage compounds in an improved rate as compared to a situation where other compounds than bioavailable silicon are limited.

The present invention is accordingly directed to a method for producing an open phototrophic diatom culture with improved storage compound production capability, comprising subjecting a starting culture to selective pressure, thus giving a competitive advantage to storage compound producing species, by subjecting said starting culture to a cycle of alternating dark phases and light phases and providing limitation of availability of essential growth nutrients in one or more of said light phases.

The open culture is obtained by selecting a starting culture, comprising one or more diatom species and subjecting them to the selective pressure by subjecting the starting culture to a cycle of alternating dark phases and light phases and providing limitation of availability of essential growth nutrients in one or more of said light phases. No special measures, such as sterilization of the equipment needs to be taken. As a result other species will also start to develop in the volume holding the original starting culture, for instance because the original culture becomes “contaminated” with these other species. Thus an open culture in accordance with the invention is obtained. Surprisingly this open culture has developed such that it provides very high production of storage compounds.

Surprisingly it was found that by using selective pressure and evolution a stable open, preferably non-axenic phototropic culture can be obtained. This open culture surprisingly produces much higher storage compound contents than observed for open cultures on which the selective pressure in accordance with the invention was not exercised. In accordance with the invention it is possible to produce diatoms wherein the total mass of storage compound is at least 50 wt.%, preferably at least 70 wt.% and more preferably 80-90 wt.% based on the weight of the organic cell mass. Apart from storage compound content also the open culture provides superior specific productivity of storage compounds. For instance, it was found possible to triple the amount of storage compounds in one light phase: the initial storage was only 22 wt.% and after 16 hours it was 64 wt.% based on the weight of the organic cell mass, which is an increase of more than a factor 3.

The term “non-axenic” culture, as used herein refers to a culture that in principle has a free exchange of biological material with its surroundings, and can constantly be invaded by all kind of new species. The specie or species that can adapt the best to the environmental conditions in the culture in terms of sustaining growth will in principle become dominant in the culture.

Selective pressure for mixed cultures it thus used as a tool to obtain the culture functionality and reactor performance of choice. In this way there is no need for expensive equipment and energy for sterilization since infections with competitive phototrophic organisms are rendered harmless or even beneficial. Moreover and importantly long term continuous operation of the reactor system becomes possible.

To obtain storage compound producing diatoms, it is important that the concentration of bioavailable silicon should not be limiting diatom growth. The concentration of bioavailable silicon in the system should therefore preferably be above 0.1 mM, more preferably above 2 mM. Other favorable conditions include a relatively high pH, a relatively low temperature and non-limiting levels of iron, boron and other essential nutrients.

Diatoms will typically dominate the system after multiple cycles, typically more than 10 cycles, in which typically 70% of the reactor volume is replaced every cycle.

Johnson et al. (Johnson, K., Jiang, Y., Kleerebezem, R., Muyzer, G. and van Loosdrecht, M.C.M. Enrichment of a mixed bacterial culture with a high polyhydroxyalkanoate storage capacity, Biomacromolecules 10 (2009) 670-676) established in their research on the production of bioplastics, such as polyhydroxyalkanoates (PHAs) from organic waste streams that these bioplastics can be produced with open mixed bacterial cultures if a suitable enrichment step based on the ecological role of PHA is used. The use of sunlight as an energy source and CO2 as carbon source for these kinds of applications, however, opens up a new field of possibilities.

The present invention relates to a selection method based on the ecological role of storage compounds, in particular compounds comprising sugars and/or polyhydroxyalkanoates and/or lipids and/or other carbon-based compounds in a day/night cycle.

Uncoupling of carbon fixation and growth in a phototrophic community can be established by feeding the phototrophic community with a hmited amount of nutrients in the absence of autotrophic carbon fixation (in the dark phase). By providing nutrients in the dark period the species capable of growing on storage compounds have a competitive advantage over species that depend directly on fight for growth. By full (or almost full, e.g. more than 95 wt.%) consumption of one or more essential growth nutrients during growth on storage compounds in the dark phase, no significant active biomass growth is possible in the subsequent light period. Therefore, in the light phase, substantially all carbon dioxide fixation that occurs leads to the formation of storage compound molecules in the microorganism and not to any substantial growth. Application of these conditions of repeating fight phases without nutrients and dark phases with nutrients circumstances over many cycles (typically more than 5, more typically more than 10 cycles) was found to lead to a phototrophic community that is enriched with species with a superior storage compound producing capacity and the capacity to grow on storage compounds in the dark.

The method of the present invention is conceptually different from the method used by Johnson et al. mentioned above. This is because the approach of Johnson et al. is based on an overflow metabolism in which the difference between the maximum growth rate and carbon uptake of the bacteria is used as strategy for application of selective pressure. The method of the present invention, on the other hand, comprises the separation of growth and carbon uptake of phototrophic diatoms under bioavailable silicon sufficient conditions. The advantage of the method of the present invention is that it provides for a relatively simple operation and a high productivity, resulting in cost reduction compared to existing options, in particular because it is not necessary to work under sterile conditions.

The method of the invention may be followed by conventional process steps, including: - accumulation of extra storage compounds; - collecting the biomass; - extracting the storage compounds from the biomass; and - converting the storage compounds into a valuable product, preferably biofuel.

Without wishing to be bound by theory it is believed that organisms that somehow can perform enhanced storage compound accumulation in response to inducing time varying environmental conditions such as combined temporary nutrient depletion and day/night cycling have a competitive advantage over other species and will therefore become dominant in an open culture, essentially defining therewith the properties of this culture.

The dark phase is typically 2-72 hours, preferably 4-48 hours and more preferably 6-18 hours. There is essentially no light source present during the dark phase. The fight phase is typically from 2-72 hours, preferably 4-48 hours and more preferably 6-18 hours. A light source is present during the fight phase. A suitable fight source may be sunlight or an artificial fight source. Preferably the fight source is sunlight. Preferably the method of the invention is carried out in an open pond, subjecting the open culture to sunlight during daytime while limiting nutrients and feeding nutrients during night time.

The amount of bioavailable sibcon containing compounds present should never limit, the growth of diatoms. A growth limiting amount of an essential growth nutrient should be supplied in the dark phase. In this case growth limiting nutrients for phototrophic organisms may include compounds containing nitrogen, phosphorus, sulfur, molybdenum, magnesium, cobalt, nickel, iron, zinc, copper, potassium, calcium, boron, chlorine, sodium, selenium, specific vitamins and any other compounds that may be essential for biomass assimilation of phototrophic species.

The typical amount of nutrients present in a phototrophic fresh water growth medium may be according to the composition of the COMBO medium developed by Kilham, S.S., Kreeger, D.A., Lynn, S.G., Goulden, C.E. and Herrera, L., Hydrobiologia (1998) 377, 147-159. However, other phototrophic growth media suitable for diatoms known to those skilled in the art may also be suitably used. In accordance with the invention a modified version of these know media may be used, specifically with regard to the one or more omitted essential nutrients. The amount of bioavailable silicon containing compounds present in the medium should always be enough to sustain growth of diatom species. A preferred nutrient that can be supplied in growth limiting amounts is nitrogen. While nutrient deficiency (i.e. nitrogen deficiency) prevents growth and production of most cellular components, the production of storage compound synthesis remains possible.

Repetition of the abovementioned dynamic pattern over many day/night cycles leads to the selection of a phototrophic community with a superior storage compound production capacity.

Other preferred nutrients which may be depleted include iron, phosphorus and magnesium.

The cultivation method of the present invention is different from the prior art in that it is open and thus does not rely on pure cultures, in which no selective pressure is present.

The method of the present invention also differs from methods wherein cultures that are depleted for nutrients in order to induce storage compound production, but in which however no selective pressure in applied.

The terms “phototroph” and “phototrophic” are defined as properties of organisms that use photons as an energy source. In principle all phototrophic diatomic life forms can be used in accordance with the present invention, in particular species from the genus of Nitzschia. “Diatom” is defined herein as a phototrophic microorganism with an absolute requirement for bioavailable silicon.

In accordance with the invention the open culture generally contains a mix of a number of species, which may include many more species than those mentioned above. In fact it is not even necessary to know which species are present in the open culture, since the desirable species are selected by applying the selective pressure, as explained above.

The terms “storage compound” and “microbial storage compound” include lipids, polysaccharides or other carbon-based compounds like for example polyhydroxyalkanoates. Preferably the storage compound is a lipid.

The term “lipids” includes naturally occurring fats, waxes, sterols, phospholipids, free fatty acids, monoglycerides, diglycerides and triglycerides and other hydrophobic or amphiphilic carbon based biological molecules. Free fatty acids typically have a carbon chain length from 14 to 22, with varying degrees of unsaturation. A variety of lipid derived compounds can also be useful as biofuel and may be extracted from phototrophs. These include isoprenoids, straight chain alkanes, and long and short chain alcohols, with short chain alcohols including glycerol, ethanol, butanol, and isopropanol. Preferably, the lipids are triacylglycerides (TAGs) and are synthesized in phototrophs through a biochemical process involving various enzymes such as trans-enoyl-acyl carrier protein (ACP), 3 -hydroxy acyl- ACP, 3-ketoacyl-ACP, and acyl-ACO or other enzymes.

The term “polysaccharides” includes glycogen, starches, chrysolaminarin and other carbohydrate polymers. Preferably the polysaccharide is chrysolaminarin.

Typically the phototrophic community is grown in an open system. An open system is defined as a system that may comprise more than one species. Preferably the culture making up the phototrophic community is non-axenic. No sterilization is required and all sorts of microorganisms can enter the system. In a preferred embodiment the method of the invention is carried out under non-sterile conditions. Thus the equipment used for carrying out the invention is preferably not sterilized prior to use, which saves tremendously in costs. Open systems may be open reactors, open tanks, natural water bodies, (raceway) ponds and artificial reservoirs or other water containing open spaces. The open systems are typically fairly shallow (typically less then 1 m deep, preferably from 20 cm to 50 cm) so as to allow light to reach the majority of the phototrophs within the systems, and typically have a consistent depth to provide the maximum area for growth within the zone that is accessible to fight. Preferably the open system used is an open reactor. A typical open reactor design for phototrophic organisms is a raceway pond.

The advantage of growing the phototrophs in an open system is that it is cheaper to operate than closed systems since there is no need for sterilization equipment, and the investment and maintenance costs are generally lower due to cheaper construction.

Alternatively the phototrophic community may be grown in a closed system. Suitable closed systems may include flat-panel reactors, tubular reactors and any other closed reactor design. The systems may be supplied with natural or artificial light as an energy source.

The basis for the medium is typically a water source. Suitable water sources include natural water sources such as lakes, rivers and oceans; and waste water sources such as municipal and industrial waste water. The temperature of the medium is typically 10-40 °C, preferably 15-25 °C. The pH of the medium is about 4-10, preferably about 6-9. A further advantage of the methods of the present invention is that it may be coupled with flue gas CO2 mitigation produced by power stations and waste water treatment. Waste water (e.g. sewage) may be pretreated for removal of organic carbon, while maintaining the essential nutrient concentrations required for cultivation of phototrophs. The pretreated waste water and the carbon dioxide may be used in the methods of the present invention. CO2 produced from power stations or incineration installations may be used as a source of CO2 in the methods of the present invention. Also CO2 from the atmosphere may be used.

The phototrophic diatomic biomass may be cohected by using conventional methods, such as microscreens, centrifugation, flocculation, broth flotation, ultrasound and combinations thereof.

The storage compound may be extracted from the biomass by existing technology, either destructive or non-destructive means. Such extraction processes may include physical extraction methods including crushing, pressing, osmotic shock and ultrasonication; and chemical extraction methods including solvent, enzymatic and supercritical carbon dioxide extraction.

There are numerous methods of converting the storage compound into a biofuel which are dependent on the type of storage compounds produced and the desired biofuel to be produced. Biofuels may include biodiesel, bio-ethanol, biogas, bio-hydrogen, bio-oil and bio-syngas. Preferably the biofuel is biodiesel or bio-ethanol. Biodiesel production utilizes a transesterification process, wherein the storage compounds, preferably lipids, undergo an alkali or acid catalyzed transesterification reaction. Glycerol is released as a byproduct of transesterification and fatty acid methyl esters are produced. This process may be run in either continuous or batch mode.

Bio-ethanol is naturally produced by some phototrophs and may be collected by non-destructive means without killing the microorganisms. The ethanol can be evaporated and subsequently condensed and collected. Alternatively, bio-ethanol may be produced by the action of microorganisms and enzymes through the fermentation of storage compounds, preferably polysaccharides such as glycogen or starch.

The methods of the present invention may be operated as a continuous process, but also as a sequenced batch.

The present invention is now elucidated on the basis of some examples.

Examples

Example 1 A reactor was run with light/dark cycles with nitrogen only present during the dark phase and a bioavailable silicon concentration of 8 mM at pH 7.5. The inoculum consisted for 1 % of diatoms. After 11 cycles in which 70% was removed every cycle diatoms accounted for 54% of the culture.

Example 2 A reactor was run with light/dark cycles with nitrogen only present during the dark phase and a bioavailable silicon concentration of 8 mM at pH 8. The inoculum consisted for 1 % of diatoms. After 10 cycles in which 60% was removed every cycle, diatoms accounted for more than 90% of the culture. This culture was, under bioavailable silicon limiting condition, able to accumulate 50% of lipids on organic weight in 48 hours of fight.

Example 3

The culture obtained in example 2 was subjected to a continuous light phase with nitrogen and bioavailable silicon limitation. After 48 hours of bioavailable silicon limitation 50 % of lipids on organic weight was produced, where under nitrogen limitation sugars were the main storage product.

Claims (6)

A method for producing a diatom culture, comprising subjecting a starting culture comprising one or more diatom species to selective pressure, so that a competitive advantage is given to storage compound-producing diatom species, by subjecting said starting culture, to non-limiting biologically available silicon concentrations, to a cycle of alternating dark phases and light phases and providing the availability limitation of essential growth nutrients in one or more of said light phases. A method for producing a diatom culture according to claim 1 which is performed for 5 or more cycles. The method of any one of the preceding claims, wherein 60% or more of the reactor volume is replaced each cycle. A method for producing storage compounds, preferably lipids, by subjecting the culture obtained according to claim 2 to biologically available silicon restriction. A method for producing storage compounds, preferably sugars, by subjecting the culture obtained according to claim 2 to nitrogen limitation. A method for producing storage compounds by subjecting the culture obtained according to claim 2 to it does not indicate which essential nutrient nutrient restriction other than nitrogen or biologically available sihcium.