WO2012152337A1

WO2012152337A1 - Segmented flow biofilm reactor

Info

Publication number: WO2012152337A1
Application number: PCT/EP2011/057724
Authority: WO
Inventors: Andreas Schmid; Katja BÜHLER; Rohan KARANDE
Original assignee: Technische Universität Dortmund
Priority date: 2011-05-12
Filing date: 2011-05-12
Publication date: 2012-11-15

Abstract

A segmented flow biofilm reactor is provided which comprises at least one capillary member, at least one reservoir for a liquid phase, and at least one reservoir for a gaseous phase, wherein the at least one reservoir for the liquid phase, and the at least one reservoir for the gaseous phase are in fluid connection with the at least one capillary member. In addition, a method for converting a substrate to a product using a biofilm as biocatalyst within at least one capillary member is provided, wherein at least one liquid phase and a gaseous phase are flowing through the at least one capillary member bearing the biofilm biocatalyst in a segmented fashion.

Description

SEGMENTED FLOW BIOFILM REACTOR

The invention concerns bioreactors, more specifically segmented flow biofilm reactors, and processes utilizing the segmented flow biofilm reactor of the present invention. In particular, the present invention concerns a segmented flow biofilm reactor wherein a biofilm can be grown and maintained for performing a biotransformation reaction or fermentation in the presence of a segmented flow. The present invention further concerns a method for transforming a substrate to a product in the presence of a segmented flow.

Biofilms are a versatile class of biocatalysts, as they are able to self- immobilize, to generate high cell density and cell mass, to exhibit resistance against many otherwise toxic chemical substances. Moreover, biofilms have the potential for long term activity ranging from days to months. Conventional reactors utilizing biofilms for the production of value added products are known and can be classified as fixed bed reactors and expanded bed reactors. In a fixed bed reactor, catalyst pellets are held in place and do not move with respect to a fixed reference frame. Fixed bed reactors suffer from excessive biofilm growth which often results in clogging of the reactor system, in limited transfer of nutrients, gaseous compounds and substrates, and in additional costs for immobilizing supports. An expanded bed reactor is a three phase reactor including a solid phase comprised of an expanded bed of a porous catalyst, a gaseous phase, and a liquid phase comprised of a feed stream. The liquid phase feedstream and the gaseous phase are introduced into the reactor flowing upward against and through the bed of catalyst so as to expand the bed of porous catalyst and maintain it in a fluidized or expanded configuration. Expanded bed reactors suffer from their size, from the high shear stress within the reactor, from low gas dissolution rates, and from high energy costs.

In said fixed bed reactors and said expanded bed reactors, the aqueous bulk phase serves as a substrate carrier and forms the natural surrounding for the biofilm, which is attached to the interphase of the liquid phase and the solid phase. However, important hydrophobic substrates have low solubility in aqueous media and/or are toxic to microorganisms. Hence, utilizing such hydrophobic substrates lead to low product yields. Usually, this problem is circumvented in biocatalysis by regulating addition of the substrate and/or by using a second, less toxic organic phase for recovering the product. To capitalize the advantages of such approach in biofilm reactors, several challenges have to be overcome, for example growth of the microorganisms in the presence of a second organic phase, detachment of the cells, and the limitation in mass transfers. In an alternative attempt, Halan and colleagues (Balan B, Schmid A and Buehler K. 2010. "Maximizing the Productivity of Catalytic Biofilms on Solid Supports in Membrane Aerated Reactors." Biotechnology and Bioengineering 106(4): 516-527) compartmentalized a biofilm reactor, wherein the organic phase is separated from the aqueous phase by a permeable polymeric membrane for the delivery of organic substrates using diffusion.

Higher extraction rates and faster chemical conversion compared to conventional bioreactors have been reported for a double liquid phase system that is operated as a segmented flow system (Johansson, PA, Karlberg B and Thelander S. 1980. "Extraction Based on the Flow- Injection Principle." Analytica Chimica Acta 114: 215-226; Burns JR and Ramshaw C. 2001. "The Intensification of Rapid Reactions in Multiphase Systems Using Slug Flow in Capillaries." Lab on a Chip 1 : 10-15). The advantages of segmented flow are enhanced mass and heat transfer rates which are achieved by the high ratio of surface area to volume, and an improved internal mixing within the segments. Additionally, segmented flow reactions can be scaled up by parallelization of segmented flow reactors.

It was an object of the present invention to provide a bioreactor integrating the characteristics of catalytic biofilms such as self immobilization, robustness and high turn-over rates with the advantages of segmented flow systems, e. g. high mass transfer rates, scalability and flexibility. It was an additional object of the present invention to provide an improved method for converting a substrate to a product having more value that said substrate, e. g . a biotransformation reaction or fermentation, by a biofilm biocatalyst. In this regard, the term "bio conversion" is used herein too.

The first object is achieved by a segmented flow biofilm reactor according to claim 1. Preferred embodiments of the segmented flow biofilm reactor are subject matters of the dependent claims 2 to 9. The additional object is obtained by the use of the segmented flow biofilm reactor as defined in claims 10 and 11, and by a method according to claim 12. Preferred embodiments of the method are subject matter of dependent claims 13 to 21.

Figure 1 illustrates an embodiment of the segmented flow biofilm reactor of the invention. Figure 2 illustrates another embodiment of the segmented flow biofilm reactor of the invention.

Figure 3 illustrates yet another embodiment of the segmented flow biofilm reactor of the invention.

Figure 4 illustrates still another embodiment of the segmented flow biofilm reactor of the invention.

Figure 5 illustrates a further embodiment of the segmented flow biofilm reactor of the present invention In a first aspect, the present invention provides a segmented flow biofilm reactor comprising at least one capillary member, at least one reservoir for at least one liquid phase, and at least one reservoir for a gaseous phase, wherein the at least one reservoir for a liquid phase, and the at least one reservoir for the gaseous phase are in interruptible fluid connection with the at least one capillary member.

The at least one capillary member provides a support for a biofilm that has to be established in the segmented flow biofilm reactor for converting a substrate to a product. Hence, the biofilm within the at least one capillary constitutes the biocatalyst for converting the substrate to the product. Said biofilm is established and thus present on the inner surface of the capillary member when the segmented flow biofilm reactor is in operating condition.

The capillary member or at least one capillary member of the segmented flow biofilm reactor of the present invention may for example be configured as a tube or as a pipe. Any suitable combination of tubes and pipes can be utilized too. The at least one capillary member of the segmented flow biofilm reactor (SFBR) may be a tube, preferably selected from the group consisting of porous tubes, non-porous tubes, photopermeable tubes, transparent tubes, non- transparent tubes, hydrophobic tubes, and hydrophilic tubes. Photopermeable tubes are permeable to photoradiation of proper wavelengths. Transparent tubes are adequately permeable to visible radiations to allow the human eye to see through it.

The at least one capillary member may consist of or may be made of any suitable material. The suitable material may be selected from the group consisting of at least one polymer, at least one metal, at least one mixture of metals, i. e. at least one alloy. Examples of suitable materials for manufacturing the at least one capillary member may be selected from the group consisting of polyvinylidene fluoride (PVDF), polyethylene (PE), poly vinyl chloride (PVC), polypropylene, polyacrylonitrile, polytetrafluoro ethylene, polysulfone, glass, steel, copper, nickel, aluminum and the like. In a preferred embodiment, the at least one capillary member is a tube which is made of a polymeric material. A particularly preferred polymeric material is polytetrafluoro ethylene (PTFE).

The at least one capillary member may have any suitable cross-section. The at least one capillary member may have a round cross-section, a square cross-section, a rectangular cross- section or a triangular cross-section. Preferably, the at least one capillary member of the segmented flow biofilm reactor has a round or circular cross-section.

The dimensions of the at least one capillary member can be varied in a wide range, depending on - for example - the peculiar needs for each species of microorganism constituting the biofilm, for each type bioconversion reaction to be carried out, for the different compositions of the different phases to be utilized, the efficiency of the bioconversion reaction to be performed, and the desired or required flow rate of the phases. The inner diameter of the at least one capillary member is preferably less than 5 mm, more preferably less than 4 mm, and most preferably less than 3 mm. The at least one capillary member is preferably at least 1 mm in inner diameter, preferably at least 2 mm. In a particularly preferred embodiment, the at least one capillary member has an inner diameter of between 1 mm and 2.5 mm. In addition, it is preferred that the at least one capillary member has a length of between 0.1 m and 2.5 m. The at least one capillary member comprises an inlet opening and an outlet opening. The inlet opening of the at least one capillary member is in fluid connection with reservoirs for each of the phases, the at least one liquid phase, and the at least one gaseous phase such that the different phases can flow through the at least one capillary member in direction from its inlet opening to its outlet opening. The fluid connection of the at least one capillary member and the reservoirs for each of the phases is interruptible such that the flow of each phase through the at least one capillary member can be interrupted separately. The phases which may flow through the at least one capillary member of the segmented flow biofilm reactor can leave the at least one capillary member at their outlet opening. The phases leaving the at least one capillary member may be collected, separated from each other and/or analyzed for their composition and/or recycled back into the system. The desired product may be separated from the phase it is contained in by suitable means.

The segmented flow biofilm reactor of the present invention comprises at least one reservoir for at least one liquid phase. At least one of said at least one liquid phases may be an aqueous phase, and/or at least one of said liquid phases may be an organic phase. Hence, the segmented flow biofilm reactor comprises at least one reservoir for at least one liquid aqueous phase, and/or at least one reservoir for at least one liquid organic phase. The at least one liquid aqueous phase may be selected from the group consisting of minimal media, complete media, waste water, and mixtures thereof. The at least one organic phase may be selected from the group consisting of include alkanes, isoparaffins, n-alkylbenzenes, isoalkylbenzenes, alicyclic hydrocarbons, ethers, aliphatic esters, silicone oils, aromatic hydrocarbons, aliphatic hydrocarbon, heterocyclic compounds, higher fatty acids, higher alcohols, phthalates or mixture of phthalates and fatty acid esters.

The segmented flow biofilm reactor of the present invention comprises at least one reservoir for at least one gaseous phase. The gaseous phase may consist of a gas or a mixture of gases. The gaseous phase is preferably selected from the group consisting of air, oxygen, noble gases, carbon dioxide, carbon monoxide, sulfur dioxide, nitrogen, hydrogen sulfide, methane, butane, volatile organic molecules and other gases. It is to be understood that each of the reservoirs for the at least one liquid phase and the at least one gaseous phase may comprise one, two or a multiple number of reservoir chambers which are in fluid connection with one another. It is also to be understood that the at least one reservoir for the gaseous phase does not necessarily have to comprise one or more reservoir chambers. Instead, ambient air can be utilized as gaseous phase. As all phases shall be supplied to the segmented flow biofilm reactor in sterile form, microbes might have to be removed from the phases or have to be destroyed before the phases enter the segmented flow biofilm before gaining access to the biofilm catalyst. Hence, the segmented flow biofilm reactor may comprise suitable means for sterilizing at least one of the phases before said phase will enter the capillary member. Such suitable means for sterilizing at least one of the phases may preferably be at least one filter. Filters are particularly preferred for sterilizing the gaseous phase, and filters are preferred above all if the gaseous phase is ambient aid. Usually, liquid phases will be supplied to their reservoirs in sterile condition, and do not necessarily require additional means for their sterilization. Means for sterilizing liquid media such as filters, means for irradiating the liquid medium and means for heat sterilization of the liquid medium or its compounds are known to the skilled.

Each reservoir comprises at least one outlet. At least one outlet of each reservoir is in fluid connection with the inlet of the at least one capillary member. Preferably, the fluid connection of the reservoirs with the at least one capillary member is mediated by conduits. The conduits may be selected from the group consisting of pipes and tubes, preferably flexible tubes. Each pipe or tube is resistant to at least the compounds constituting the phase flowing through the respective pipe or tube. Resistance of the conduit to the compounds of the phase flowing through this conduit is of particular importance for the conduit which is connects the at least one reservoir for the at least one liquid organic phase with the at least one capillary member.

The segmented flow biofilm reactor of the present invention further comprises at least one means for generating a segmented flow of the at least one liquid phase and the at least one gaseous phase from the respective reservoirs through the conduits connecting each reservoir with the at least one capillary member, and through the at least one capillary member. Said means for generating a segmented flow can be selected from the group consisting of pumps and valves. In one embodiment, each reservoir is in fluid connection with the at least one capillary member by a separate conduit, wherein each conduit is provided with a separate pump, or with a single channel of a multi-channel pump. Preferably, said conduits are tubes and are provided with a peristaltic or a piston pump. The segmented flow bio film reactor of the present invention can provide a segmented flow of the at least one liquid phase and the at least one gaseous phase through the at least one capillary member. Therefore, the segmented biofilm reactor comprises at least one means for segmenting the flow of phases, e. g. at least one means for interrupting an otherwise continuous flow of the phases. Said means for segmenting the flow of the phases can be selected from the group consisting of pumps, fittings, valves, and combinations of pumps and valves, and pumps and fittings. In a preferred embodiment, the segmented flow biofilm reactor comprises at least one pump, more preferably at least one peristaltic pump. In another or additional embodiment, the segmented flow biofilm reactor comprises at least one three way valve or at least one 4 way valve. Said three way valve and said 4 way valve are actuable/operable such that it is adjustable which reservoir provides the supply of a phase for the capillary member.

The segmented flow biofilm reactor may further comprise means for maintaining a predetermined temperature in the at least one capillary member. The means for maintaining a predetermined temperature in the at least one capillary member may for example be a basin through which the at least one capillary member runs, the basin comprising a medium having the predetermined temperature. This embodiment has the advantage that the temperature of the biofilm can be adjusted and maintained in an optimal range for growth and propagation of the biofilm, and/or for bioconversion. In a particular embodiment, wherein the at least one capillary member is a porous tube, the medium within the basin may consist of or may comprise the substrate for bioconversion, which substrate may access the biofilm biocatalyst through the porous wall of the at least one capillary member. In an embodiment, the segmented flow biofilm reactor comprises at least one capillary member, one reservoir for a liquid phase, preferably a liquid aqueous phase, and one reservoir for a gaseous phase, wherein the reservoir for the liquid phase and the reservoir for the gaseous phase are in interruptible fluid connection with the at least one capillary member.

In its operating mode, two different phases, a liquid phase and a gaseous phase, are flowing through the at least one capillary. Hence, at least the inner surface of the at least one capillary has to be resistant to the compounds that are present in the different phases. In addition, the inner surface of the at least one capillary has to be non-toxic to the microorganisms constituting the biofilm on the inner surface of the at least one capillary. Moreover, the inner surface of the at least one capillary has to permit the microorganisms to adhere to said inner surface such that a biofilm can be established thereon. Thus, in a preferred embodiment the at least one capillary consist of a material that is resistant to the compounds that are present in the different phases, that is non-toxic to the microorganisms for forming the biofilm, and that permits the microorganisms for forming the biofilm to adhere thereon. In an alternative embodiment, the at least one capillary is provided with a coating on its inner surface, wherein the coating is resistant to the compounds that are present in the different phases, is non-toxic to the microorganisms for forming the biofilm, and permits the microorganisms for forming the biofilm to adhere to the coated inner surface of the capillary. It is to be understood that a segmented flow biofilm reactor of the invention comprises at least one capillary made of a material that is resistant to the compounds that are present in the different phases, that is nontoxic to the microorganisms for forming the biofilm, and that permits the microorganisms for forming the biofilm to adhere thereon, or at least one capillary comprising a coating on its inner surface, wherein the coating is resistant to the compounds that are present in the different phases, is non-toxic to the microorganisms for forming the biofilm, and permits the microorganisms for forming the biofilm to adhere to the coated inner surface of the capillary. In another embodiment, of the present invention, the segmented flow biofilm reactor comprises at least one capillary, at least one reservoir for a liquid aqueous phase, at least one reservoir for a liquid organic phase, and at least one reservoir for a gaseous phase, wherein the at least one reservoir for a liquid aqueous phase, the at least one reservoir of the liquid organic phase, and the at least one reservoir for the gaseous phase are in fluid connection with the at least one capillary.

In its operating mode, three different phases, at least one aqueous phase, at least one organic phase, and at least one gaseous phase, flow through the at least one capillary. Hence, at least the inner surface of the at least one capillary has to be resistant to the compounds that are present in the different phases, i.e. the at least one aqueous phase, the at least one organic phase and the at least one gaseous phase. In addition, the inner surface of the at least one capillary has to be non-toxic to the microorganisms constituting the biofilm on the inner surface of the capillary. Moreover, the inner surface of the at least one capillary has to permit the microorganisms to adhere to said inner surface such that a biofilm can be established thereon. Thus, in a preferred embodiment the at least one capillary consist of a material that is resistant to the compounds that are present in the different phases, that is non-toxic to the microorganisms for forming the biofilm, and that permits the microorganisms for forming the biofilm to adhere thereon. In an alternative embodiment, the at least one capillary is provided with a coating on its inner surface, wherein the coating is resistant to the compounds that are present in the different phases, is non-toxic to the microorganisms for forming the biofilm, and permits the microorganisms for forming the biofilm to adhere to the coated inner surface of the capillary. It is to be understood that a segmented flow biofilm reactor of the invention may comprise at least one capillary made of a material that is resistant to the compounds that are present in the different phases, that is non-toxic to the microorganisms for forming the biofilm, and that permits the microorganisms for forming the biofilm to adhere thereon, and at least one capillary comprising a coating on its inner surface, wherein the coating is resistant to the compounds that are present in the different phases, is non-toxic to the microorganisms for forming the biofilm, and permits the microorganisms for forming the biofilm to adhere to the coated inner surface of the capillary.

The segmented flow biofilm reactor allows the production of value added products, for examples of hydrophobic organic compounds, in that substrates are converted to products by the microorganisms of the biofilm within the at least one capillary member of the segmented biofilm reactor. The term "substrate" refers to a chemical compound that is intended for being converted by the microorganisms of the biofilm to a different chemical compound whose value is increased compared to the value of the substrate, and which is designated "product". The substrate for the bioconversion and/or the product of the bioconversion may be selected from the group consisting of hydrophilic compounds, hydrophobic compounds, liquid compounds and gaseous compounds. In a particularly preferred use of the segmented flow biofilm reactor, the substrate for the bioconversion and/or the product of the bioconversion is a hydrophobic compound, i. e. a compound that is barely soluble in water or insoluble in water. Hence, the present invention also pertains to the use of the segmented flow biofilm reactor as described herein before in more detail for converting a substrate to a product, preferably to a value added product, by using a biofilm as biocatalyst.

In another aspect, the present invention provides a method for converting a substrate to a product by using a biofilm as biocatalyst. The method of the present invention comprises subjecting the biofilm catalyst to a segmented flow of at least one liquid phase and at least one gaseous phase. The at least one liquid phase may be a liquid aqueous phase or a liquid organic phase. The liquid phase may also comprise a combination of at least one liquid aqueous phase and at least one liquid organic phase. More specifically, the method comprises conversion of a substrate to a product by a biofilm catalyst which is present on the inner wall of at least one capillary member within a segmented flow biofilm reactor which has been described herein before. In an embodiment of the method, the biofilm is subjected to a segmented flow of at least one liquid aqueous phase and at least one gaseous phase, wherein the segments of the at least one liquid phase and the at least one gaseous phase possess a segmented flow through the at least one capillary member. In another embodiment of the method, the biofilm is subjected to a segmented flow of at least one liquid aqueous phase, at least one liquid organic phase, and at least one gaseous phase, wherein the segments of the at least one liquid aqueous phase, the at least one liquid organic phase and the at least one gaseous phase possess a segmented flow through the at least one capillary member, i.e. the different phases employed are flowing through the at least one capillary member in a segmented fashion.

Hence, the method comprises the step of establishing the biofilm on the inner surface of the at least one capillary member. The method also comprises maintaining the biofilm on the inner surface of the at least one capillary member, in particular during the bioconversion of the substrate to the product.

There is no particular limitation with respect to the microorganism that are suitable for the method of the present invention, except for the microorganism's capability of forming a biofilm. The biofilm may consist of a single species of microorganisms or may consist of two or more species of microorganisms. The microorganism may be naturally occurring microorganism or they may be genetically modified. A preferred genetic modification introduces the capability of performing a specific bioconversion reaction by the genetically modified organism. The biofilm may consist of or may comprise prokaryotic microorganisms or eukaryotic microorganisms or cells. The liquid aqueous phase is preferably a medium containing all compounds that are required by the microorganisms for growth and propagation for constituting a biofilm. The liquid aqueous phase can be selected from the group consisting of minimal media, complete media and waste water. Each species of microorganisms that may be employed as biofilm biocatalyst in the segmented flow biofilm reactor of the present invention and/or in the method according to the present invention for converting a substrate to a product, is preferably provided with the medium said microorganisms prefer. A wide variety of media are available for the skilled artisan for obtaining optimal multiplication and/or bioconversion performance. The different media may for example differ in the carbon source supplied to the biofilm biocatalyst, e.g. glucose, glycerol, citrate, etc. In a preferred embodiment, the aqueous phase consists of M9-medium. The liquid aqueous medium may contain the substrate that shall be converted to a product when the biofilm was established on the inner surface to the at least one capillary member.

The liquid organic phase may consist of or comprise an organic substrate to be converted. An organic substrate may be dissolved in an organic solvent, and the resulting organic solution may be employed as organic phase. Examples for organic solvents that may be employed for the organic phase may be selected from the group consisting of alkanes, isoparaffins, n-alkylbenzenes, isoalkylbenzenes, alicyclic hydrocarbons, ethers, aliphatic esters, silicone oils, aromatic hydrocarbons, aliphatic hydrocarbons, heterocyclic compounds, higher fatty acids, higher alcohols, phthalates and mixtures of phthalates and fatty acid esters.

The substrate to be converted is not subject of particular limitations as long as the substrate can be converted by the microbes of the biofilm biocatalyst. The substrate may be soluble in the liquid aqueous phase. Then the substrate may be incorporated into the liquid aqueous phase when the biofilm has been established and shall be used for the bioconversion of said substrate. In cases of organic substrates which are only slightly soluble or insoluble in a liquid aqueous phase, the substrate can be dissolved in an organic solvent. Specific examples of substrates for conversion by a biofilm catalyst which is subjected to a segmented flow include glucose, sucrose glycerol, lactose, hydrolyzed starch, D-alanine, whey permeate; aromatic hydrocarbons such as benzene, toluene, xylene, ethylbenzene, styrene, naphthalene, and phenanthrene; aliphatic hydrocarbons such as tridecane and tetradecane; alicyclic compounds such as cyclohexanone and cyclohexanol; heterocyclic compounds such as methyllimidazole, collidine and picoline; higher fatty acids such as lauric acid, palmitic acid,stearic acid, oleic acid and linolic acid; higher alcohols such as octyl alcohol, decyl alcohol, lauryl alcohol, cetyl alcohol and stearyl alcohol; fatty acid esters such as ethyl caprylate and ethyl caprylate; alkanes.

The gaseous phase consists of a gas or a mixture of gases. Suitable gases are selected from the group consisting of air, oxygen, noble gases, carbon dioxide, carbon monoxide, sulfur dioxide, nitrogen, hydrogen sulfide, methane, butane, volatile organic molecules and other gases. It is to be understood that the gaseous phase is chosen according to the microorganisms requirements. For example no air or oxygen-containing gaseous phase is used when anaerobic bacteria are employed for biocatalysis. The gaseous phase may consist of or comprise a gaseous substrate for the bioconversion such as, for example, carbondioxide.

The length of the segments of the different phases within the capillary may be up to 12 cm. In particularly preferred embodiments, the length of each segment is in a range of between 1 and 15 mm. The length of all segments or of the segments of two phases may be the same, of the lengths of all segments may differ from one another.

The volume of the segments of the different phases may be up to 6,000 μί. In a particularly preferred embodiment, the volume of the segments of each phase is in the range of between 1 and 15 μΐ. The volume of all segments or of the segments of two phases may be the same, of the lengths of all segments may differ from one another.

Depending on the diameter of the capillary, the volume of the segments determines the length of the segments. The flow rate of the segments/phases in the capillary should be in the range of 10 - 5,000 μΙ7ηιίη and preferably 100 - 1,000 μΙ7ηιίη (total flow rate).

The present invention provides devices and methods which are robust, convenient and reliable for converting a substrate to a value added product. The present invention allows bioconversion of gaseous substrates, hydrophilic substrates or hydrophobic and organic substrates to be converted into a product. The product may be gaseous, hydrophilic or hydrophobic. In addition, a wide variety of microorganisms can be utilized for a bioconversion. The only limitation with respect to the microorganism is the microorganisms capability of forming a bio film

Before the invention is described in detail with respect to some of its preferred embodiments, the following general definitions are provided.

The present invention as illustratively described in the following may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. The drawings as described are only schematic and non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes.

Where the term "comprising" is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term "consisting of is considered to be a preferred embodiment of the term "comprising of. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which preferably consists only of these embodiments.

Where an indefinite or definite article is used when referring to a singular noun, e.g. "a", "an" or "the", this includes a plural of that noun unless something else is specifically stated. The terms "about" or "approximately" in the context of the present invention denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value of ±10%, and preferably of ±5%.

Figure 1 displays a schematic representation of an embodiment 100 of the segmented flow biofilm reactor of the present invention. The segmented flow biofilm reactor comprises a first reservoir 2 for a liquid phase, and a second reservoir 6 for a gaseous phase. The first reservoir 2 is in fluid connection with a first inlet of a three-way fitting 21 (= T-shaped fitting) by means of a flexible tube 3. The second reservoir 6 is in fluid connection with a second inlet of the three-way fitting 21 by means of a flexible tube 7. The three-way fitting 21 comprises two inlet openings and an outlet opening, wherein the outlet opening of the three-way fitting 21 is connected to the inlet opening of the capillary member 12. A first peristaltic pump 8 mediates the transport of the liquid phase from reservoir 2 through tube 3 to the capillary member 12, and a second peristaltic pump 10 mediates the transport of the gaseous phase from reservoir 6 through tube 7 to the capillary member 12.

A segmented flow of the liquid phase and the gaseous phase is generated within the three-way fitting by coordinated operation of the first peristaltic pump 8 and the second peristaltic pump 10. The peristaltic pumps can be operated to continuously pump the different phases, wherein the segments of different phases providing the segmented flow are formed due to the different physico-chemical properties of the different phases. However, in an alternative embodiment, the pumps may be turned on and off in a coordinated manner for forming the segmented flow. For instance, one pump is turn on and pumps while the other pump is turned off. After a predetermined period the pumping pump is turned off and the other pump is turned on. The segmented flow is achieved in that only one phase at any given point of time is pumped into the three-way fitting. Thereby, an alternating sequence of phase segments is generated consisting of segments of the liquid phase 15, 15', 15" and 15"', and segments of the gaseous phase 17, 17', 17" and 17"'. The alternating order of segments migrates through the capillary member 12 providing the bio film on the inner wall of the capillary member 12 with nutrients.

During establishment of the biofilm on the inner wall of the capillary member, it may be refrained from supplying the substrate for the desired bioconversion to the microorganism of the biofilm. However, for bioconversion the substrate has to be supplied to the biofilm.

Therefore, the substrate may be added to one of the phases used for generating the segmented flow when the biofilm has been established. In an alternative embodiment, the substrate may be supplied to the biofilm in that the capillary member 12 or at least a portion thereof is placed into the substrate or a solution containing the substrate, provided that the capillary member is permeable for said substrate, i.e. porous to allow for substrate supply through membrane transfer.

In this embodiment, the segmented flow biofilm reactor comprises two pumps, one pump for each phase to be supplied to the at least one capillary member. Preferably, these pumps are peristaltic pumps. Providing each tube 3, 7 with a separate peristaltic pump has the advantage that the flow rate of each phase can be adjusted individually, thereby providing utmost flexibility in segmenting the phases for flowing through the at least one capillary member of the segmented flow biofilm reactor.

The capillary member 12 is placed with most of its length in a water bath 13 for maintaining a predetermined temperature in the capillary member 12. The medium within the water bath 13 does not have to be water, in fact any suitable medium can be utilized for transferring heat to the capillary member. A suitable medium may for example be or contain the substrate.

Figure 2 is a schematic representation of an embodiment 200 of the segmented flow biofilm reactor of the invention. The segmented flow biofilm reactor comprises a first reservoir 2 for an aqueous phase, a second reservoir 4 for an organic phase, and a third reservoir 6 for a gaseous phase. The reservoir 2 is in fluid connection with a first inlet of an X-shaped fitting 11 (= four-way fitting). The X-shaped fitting comprises three inlets, a first inlet, a second inlet and a third inlet, and an outlet. A second reservoir 4 for receiving the organic phase is in fluid connection with the second inlet of the X-shaped fitting 11 by means of a flexible tube 5. The third reservoir 6 including the gaseous phase is in fluid connection with the third inlet of the X-shaped fitting 1 1 by means of a flexible tube 7. A first peristaltic pump 8 mediates transport of the aqueous phase from the first reservoir 2 to the X-shaped fitting 11. A second peristaltic pump 9 mediates transport of the organic phase from the second reservoir 4 to the X-shaped fitting 11. And a third peristaltic pump 10 mediates transport of the gaseous phase from the third reservoir 6 to the X-shaped fitting 1 1.

The segmented flow biofilm reactor comprises a capillary 12. One end of capillary 12 is fluidly connected to the outlet of the X-shaped fitting 1 1 to receive the phases. The capillary 12 comprises an out let 14 where the phases migrating through the capillary 12 from its inlet to its outlet 14 may be collected for further analysis or processing/recirculation. The arrow indicated the direction of flowing in the capillary 12. The capillary 12 goes through a water bath 13 for maintaining a predetermined temperature in the capillary 12. Upon coordinated operating of the three peristaltic pumps 8, 9, 10 a segmented flow of at least two phases selected from the group consisting of the aqueous phase, the organic phase and the gaseous phase can be generated. A segmented flow of all three phases is shown in Figure 2, wherein a segment of the aqueous phase 15, 15', 15" and 15"' follows a segment of the organic phase 16, 16', 16" and 16"' which in turn follows a segment of the gaseous phase 17, 17', 17" and 17"' respectively. Hence, an alternating order of phase segments migrates through the capillary 12 providing the microorganisms of the biofilm on the inner surface of the capillary 12 with nutrients, the substrate for bioconversion, and suitable environmental conditions.

In this embodiment, the segmented flow biofilm reactor comprises three pumps, one pump for each phase to be supplied to the at least one capillary. Preferably, these pumps are peristaltic pumps. Providing each of the first, the second and the third tube with a separate peristaltic pump has the advantage that the flow rate of each phase can be adjusted individually, thereby providing utmost flexibility in segmenting the phases for flowing through the at least one capillary of the segmented flow biofilm reactor.

Figure 3 is a schematic representation of an alternative embodiment 300 of the segmented flow biofilm reactor of the invention, wherein the segmented flow biofilm reactor comprises an actuable four- way valve 20 instead of an X- shaped fitting as shown in Figure 2. The outlet of the four-way valve is connected to a joint supply tube 27. A single peristaltic pump 19 is positioned between the outlet of the actuable four-way valve 20 and the inlet of the capillary member 12. The pump 19 is arranged such that at least one of the three phases can be transported through the capillary member 12. The actuable four- way valve provides that only one of the three phases, the liquid aqueous phase, the liquid organic phase or the gaseous phase, is pumped by pump 19 at a given point of time. Coordinated actuating of the four- way valve 20 permits generation of an alternating order of phase segments migrating through the capillary member 12. The length of each segment of the phases can be adjusted by controlling the operating speed of the pump 19 and/or the intervals of operating the four- way valve. For generating the flow of the phases through the capillary 12, only a single peristaltic pump 19 is required. Figure 4 is a schematic representation of an embodiment 400 of the segmented flow biofilm reactor of the present invention. This embodiment comprises a set up, wherein the first reservoir 2 for the aqueous phase, and the third reservoir 6 for the gaseous phase are both fluidly connected to different inlets of T-shaped fitting 22 by tubes 3 and 7 respectively.. Coordinated operating of peristaltic pumps 8 and 10, which are positioned between the reservoirs 2 and 6 and inlets of T-shaped fitting 22, permits generating an alternating order of segments of the aqueous phase and the gaseous phase. The alternating order of phase segments is flowing through conduit 23 to a first inlet of another T-shaped fitting 21. The second inlet of T-shaped fitting 21 is fluidly connected to reservoir 4 for containing the organic phase. The organic phase may be supplied to the alternating order of segments of the aqueous phase and the gaseous phase such that an alternating order of the three phases is generated for being supplied to the capillary member 12 in that pump 9, which is arranged at tube 5 between reservoir 4 and the inlet opening of the T-shaped fitting 21 is operated in coordination with the operation of pumps 8 and 10.

In an embodiment 500 as schematically shown in Figure 5, the segmented flow biofilm reactor is provided with a three-way valve 24, wherein the first reservoir 2 for the aqueous phase, and the third reservoir 6 for the gaseous phase are both fluidly connected to different inlets of the three-way valve 24. A peristaltic pump 25 provides the flow of at least one of the phases contained in reservoir 2 and reservoir 6 through tube 26 towards t-shaped fitting 21 and through capillary member 12. The three-way valve 24 is operated in a coordinated manner for generating the segmented flow of the aqueous phase and the gaseous phase through tube 26, T-shaped fitting 21 and capillary member 12. The organic phase may be supplied to the alternating order of segments of the aqueous phase and the gaseous phase such that an alternating order of the three phases is generated for being supplied to the capillary member 12 in that pump 9, which is arranged at tube 5 between reservoir 4 and another inlet opening of the T-shaped fitting 21 is operated in coordination with the operation of pumps 9 and 25.

Examples

Example 1 - Biotransformation using a liquid-gaseous two-phase segmented flow with a membrane mediated organic substrate transport Example 1.1 - Establishing a biofilm

Establishing a biofilm in the at least one capillary member of the segmented flow is essential for efficient conversion of a substrate to a product by the biofilm catalyst. A biofilm was established in the presence of a segmented flow consisting of two phases, a liquid aqueous phase and a gaseous phase.

For establishing a biofilm in a capillary member, a culture of the respective bacterial strain (see examples 2 and 3) was grown over night. A porous silicone tube was filled with the bacterial culture, and the bacteria were allowed to settle for 2 hours and to adhere onto the inner surface of the silicone tube. Thereafter, a continuous segmented flow of nutrient medium (liquid aqueous phase) having a constant flow rate through the tube was generated to initiate biofilm formation.

After 3 to 4 days a biofilm was observed on the inner surface of the silicone tube. A segmented flow was generated in that segments of air (gaseous phase) were incorporated into the continuous segmented flow of nutrient medium. Loosely adhering bacteria were detached from the inner surface of the tube and flushed out of the tube due to the high fluidic stress caused by the segmented flow of liquid aqueous phase and gaseous phase on the tube wall. The segmented flow was continued and reestablishing of the biofilm on the inner wall of the tube was observed after another day. The reestablishment of the biofilm clearly reflects adaptation of the biofilm towards the segmented flow. The other day, part of the biofilm -containing silicone tube was placed into a closed glass bottle such that 20% of the length of the silicone tube were submerged in 20 mL of substrate for bioconversion, while the remaining portion of the silicone tube was situated in the gaseous environment above the substrate for bioconversion.

Example 1.1 - Producing octanol

For the bioconversion of octane to octanol, the Pseudomonas putida strain PpS81 pBTIO (deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ), deposition number: DSM 24710) was grown to a biofilm on the inner surface of a porous silicone tube as described in example 1.1.

After the biofilm was established, part of the porous silicone tube was submerged in octane such that the substrate octane can diffuse through the wall of the silicone tube and be converted to octanol by the biofilm biocatalyst. The product formed by the biofilm biocatalyst, i. e. octanol, diffused through the porous wall of the silicone tube into the octane bulk phase and was extracted from the octane phase at the end of the experiment.

Example 2 - Biotranformation using a liquid-liquid-gaseous three phase segmented flow

Example 2.1 - Establishing a biofilm Establishing a biofilm in the at least one capillary member of the segmented flow is essential for efficient conversion of a substrate to a product by the biofilm catalyst. A biofilm was established in the presence of a segmented flow consisting of two phases, a liquid aqueous phase and a gaseous phase.

For establishing a biofilm in a capillary member, a culture of the respective bacterial strain (see examples 2 and 3) was grown over night. A PTFE tube was filled with the bacterial culture, and the bacteria were allowed for to settle 2 hours and to adhere onto the inner surface of the PTFE tube. Thereafter, a continuous segmented flow of nutrient medium (liquid aqueous phase) having a constant flow rate through the tube was generated to initiate biofilm formation.

After 3 to 4 days a biofilm was observed on the inner surface of the PTFE tube. A segmented flow was generated in that segments of air (gaseous phase) were incorporated into the continuous flow of nutrient medium. Loosely adhering bacteria were detached from the inner surface of the tube and flushed out of the tube due to the high fluidic stress caused by the segmented flow of liquid aqueous phase and gaseous phase on the tube wall. The segmented flow was continued and reestablishing of the biofilm on the inner wall of the tube was observed after another day. The reestablishment of the biofilm clearly reflects adaptation of the biofilm towards the segmented flow.

Bioconversion of a substrate was initiated after 6 to 7 days by introducing a liquid organic phase into the segmented flow of the liquid aqueous phase and the gaseous phase such that a segmented flow of a gaseous phase, liquid aqueous phase and liquid organic phase was generated. The biofilm was maintained in the presence of the air-aqueous phase-organic phase segmented flow for another 15 to 30 days before the segmented flow biofilm reactor was actively terminated. Example 2.2 - Producing octanol

For biotransformation of octane to octanol Pseudomonas putida PpS81 pBTIO (deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ), deposition number: DSM 24710) was grown to a biofilm in a PTFE tube having an inner diameter of 2.1 mm and a length of 0.25 m according to the procedure described in example 2.1.

Pseudomonas putida PpS81 pBTI O was grown in the capillary in M9 medium as nutrient medium. The composition of M9 medium is shown in Tables 1 and 2.

Table 1 : Composition of M9-medium

Phosphate salts, sodium chloride and ammonium chlorate are dissolved in 1 L of deionized water. The pH is adjusted by 10 M sodium hydroxide (without adjustment the pH is around 7.1 when using VE water). The sterile solutions, trace elements, antibiotics and magnesium sulfate, glucose are added after sterilization. Antibiotic used in this example was kanamycin.

US* trace elements sol. 1 L

37% fuming HC1 [ml] 82.81

Table 2: Composition of US* trace elements solution

Bio film growth on the capillary wall in liquid medium took 3 to 4 days. After visualization of biomass, air segments were continuously injected, which on one hand flushed out only loosely attached cells but on the other hand dispersed the cells over the complete surface of tubing. Within the next 2 to 3 days, the growing of the biofilm in presence of air-aqueous segmented flow was detected on the complete surface of the tubing. Biotransformation was started by injecting the organic phase consisting of octane as substrate, forming an air-aqueous-organic segmented flow. Biofilm cultivation was continued for additional 15 to 30 days before the reactor was actively terminated.

Average productivity of between 2 to 2.8 g/L_aq./day was obtained for octanol synthesis for at least 15 days. Compared to non-segmented based biofilm reactor, the productivity was 4 fold higher in the air-aqueous-organic segmented flow system

Example 2.3 - Producing (S)-styrene oxide

For stereoselective production of (S)-styrene oxide from styrene, Pseudomonas sp. VLB120AC (deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ), deposition number: DSM 24711) was used. A biofilm consisting of Pseudomonas sp. strain VLB120AC was established as described in example 2.1. The medium described in example 2.1 was complemented with streptomycin and tetracycline as antibiotics. Establishing a biofilm consisting of Pseudomonas sp. strain VLB120AC was performed according to the procedures described in example 2.1 and 2.2 using M9 medium (composition disclosed in example and starting biotransformation of styrene were performed as described in Example 1, with the exception that styrene was used as substrate instead of octane.

For stereoselective production of (S)-styrene oxide from styrene, an average productivity of 15 to 20 g/La_q./day was obtained by using the air-aqueous-organic segmented flow bioreactor for 30 days.

Deposition of biological Material:

Pseudomonas putida PpS81 pBT 1 0 was deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) on April 04, 2011 under deposition number: DSM 24710.

Pseudomonas sp. VLB120AC was deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) on April 04, 2011 under deposition number: DSM 24711.

Name and address of the depositary institution:

DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH

InhoffenstraBe 7B

38124 Braunschweig

Germany

i

Claims

A segmented flow biofilm reactor comprising at least one capillary member, at least one reservoir for at least one liquid phase, and at least one reservoir for a gaseous phase, wherein the at least one reservoir for a liquid phase and the at least one reservoir for a gaseous phase are in interruptible fluid connection with the at least one capillary member.

The segmented flow biofilm reactor according to claim 1 , wherein said segmented flow biofilm reactor comprises at least one reservoir for a liquid aqueous phase and at least one reservoir for a liquid organic phase, said at least one reservoir for a liquid aqueous phase and said at least one reservoir for a liquid organic phase being in fluid connection with the at least one capillary.

The segmented flow biofilm reactor according to claim 1 or 2, wherein the capillary member is made of a material selected from the group consisting of polymers, metals and alloys.

The segmented flow biofilm reactor according to any one of claims 1 to 3, wherein the capillary member is made of a material selected from the group consisting of polyvinylidene fluoride (PVDF), polyethylene (PE), poly vinyl chloride (PVC), polypropylene, polyacrylonitrile, polytetrafluoro ethylene, polysulfone, glass, steel, copper, nickel, aluminum polyvinylidene fluoride (PVDF), polyethylene (PE), poly vinyl chloride (PVC), polypropylene, polyacrylonitrile, polytetrafluoro ethylene, polysulfone, glass, steel, copper, nickel and aluminum.

5. The segmented flow biofilm reactor according to any one of claims 1 to 4, wherein the inner diameter of the capillary member is less than 5 mm, preferably less than 4 mm, and more preferably less than 3 mm.

6. The segmented flow biofilm reactor according to any one of claims 1 to 5, wherein the inner diameter of the capillary member is at least 1 mm, preferably at least 2.5 mm.

7. The segmented flow biofilm reactor according to any one of claims 1 to 6, wherein the capillary member has a length of between 0.1 m and 2.5 m.

8. The segmented flow biofilm reactor according to any one of claims 1 to 7, wherein the segmented flow biofilm reactor further comprises at least one means for generating a segmented flow of the phases in the at least one capillary member.

9. The segmented flow biofilm reactor according to claim 8, wherein the at least one means for generating a slug flow is selected from the group consisting of pumps and valves.

10. Use of a segmented flow biofilm reactor according to any one of claim 1 to 9 for converting a substrate to a value added product by using a biofilm as biocatalyst.

11. Use according to claim 10, wherein the substrate and/or the product is selected from the group consisting of hydrophilic compounds, hydrophobic compounds, liquid compounds and gaseous compounds.

12. A method for converting a substrate to a product using a biofilm as biocatalyst, the method comprises the step of subjecting the biofilm biocatalyst to a segmented flow of at least one liquid phase and at least one gaseous phase.

13. The method according to claim 12, the method comprises subjecting the biofilm biocatalyst to a segmented flow of at least one liquid aqueous phase, at least one liquid organic phase, and at least one gaseous phase.

14. The method according to claim 12 or 13, the method comprises establishing and maintaining the biofilm biocatalyst on the inner surface of at least one capillary member.

15. The method according to any one of claims 12 to 14, wherein the liquid aqueous phase is selected from the group consisting of minimal media, complete media and waste water.

16. The method according to any one of claims 12 and 15, wherein the organic phase is selected from the group consisting of include alkanes, isoparaffins, n-alkylbenzenes, isoalkylbenzenes, alicyclic hydrocarbons, ethers, aliphatic esters, silicone oils, aromatic hydrocarbons, aliphatic hydrocarbon, heterocyclic compounds, higher fatty acids, higher alcohols, phthalates or mixture of phthalates and fatty acid esters.

17. The method according to any one of claims 12 to 16, wherein the gaseous phase consists of a gas or a mixture of gases, preferably selected from the group consisting of air, oxygen, noble gases, carbon dioxide, carbon monoxide, sulphur dioxide, nitrogen, hydrogen sulphide, methane, butane, volatile organic molecules and other gases.

18. The method according to any one of claims 12 to 17, wherein the product is contained in one of the phases selected from the group consisting of the at least one liquid aqueous phase, the at least one liquid organic phase, and the at least one gaseous phase.

The method according to any one of claims 12 to 18, wherein the segments of the different phases within each of the at least one capillary member have a length of up to 12 cm, preferably a length of between 1 and 15 mm.

The method according to any one of claims 12 to 19, wherein the segments of the different phases within the at least one capillary member have a volume of up to 6,000 μί, preferably a volume of between 1 and 15 μί.

The method according to any one of claims 12 to 20, wherein the flow rate of the segments/phases in the at least one capillary member is in the range of between 10 to 5,000 μΙ7ηιίη, preferably in the range of between 100 to 1,000 μυηιίη.