Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/54—Acetic acid
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/56—Lactic acid
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
  • C12P7/6409—Fatty acids

Definitions

• the present invention relates to methods for the microbial production of lipids and fatty acid, in particular short chain fatty acids (SOFA) by fermenting organic feedstock and to a reactor system for carrying out such a fermentation method.
• SOFA short chain fatty acids
• Lignocellulosic biomass is the most abundant source of fixed, renewable carbon and is considered as promising alternative to fossil oil.
• Modern biotechnology faces the challenge to convert this complex, heterogeneous, low-cost feedstock cost-competitive and selective to a variety of biochemicals.
• interdisciplinary approaches with the highest possible degree of process integration need to be developed to reduce the capital costs.
• Consolidated bioprocessing integrates all biotechnological process steps necessary for the conversion of polymeric carbohydrate from lignocellulosic biomass to a target product in a single bioreactor, including (i) production of cellulolytic and hemicellulolytic enzymes, (ii) enzymatic hydrolysis to fermentable carbohydrates and (iii) fermentation of the released saccharides to the target product.
• the classic CBP approach is based on genetic modification to merge all required metabolic functionalities into a single strain.
• biochemically difficult tasks are catalyzed according to the principle of metabolic compartmentalization by multiple species whereby each member of the community is specialized in a subtask and occupies its ecological niche.
• cooperative features such as the provision of cellulolytic activities enable the community the access to release carbon sources as public goods.
• SCFAs also referred to as volatile fatty acids (VFAs)
• VFAs volatile fatty acids
• a method for the microbial production of fatty acids, such as SCFAs or VFAs from organic feedstock, in particular from lignocellulosic biomass is provided, wherein a consortium of at least three different strains of microorganisms is provided, wherein the consortium comprises
• At least one strain of an aerobic, micro-aerophilic or facultative anaerobic lactic acid and/or acetic acid producing microorganism and - at least one strain of an obligate anaerobic microorganism for fermenting lactic acid and/or acetic acid to at least one short chain alkanoic acid.
• a multi-functional synthetic cross-kingdom CBP consortium for the selective production of VFAs (or SCFAs) from lignocellulose is provided.
• Lactic acid was selected as primary metabolite due to its central role in the metabolism of various VFA producers (‘Lactate Platform’).
• Lactic acid bacteria (LAB) were selected to compensate the missing metabolic flexibility of VFA producers and metabolically funnel the major monomeric sugars of lignocellulose to lactic and acetic acid.
• lactic acid as primary metabolite also acetic acid can be used as a primary metabolite which is either released from the plant biomass, or is produced by microorganisms.
• all sugars from the lignocellulosic biomass are tunneled via the central intermediates lactic acid and acetic acid which are subsequently converted to the target products, such as SCFAs or lipids.
• the aerobic microorganism for example the fungus Trichoderma reesei
• the aerobic microorganism is able to produce external cellulolytic enzymes which allow the enzymatic degradation of the organic feedstock to fermentable sugars.
• the aerobic microorganism e.g. a fungus
• the aerobic microorganism essentially requires oxygen, while the presence of oxygen is highly toxic for the VFA-producing anaerobic bacteria.
• the present invention provides a platform technology for the design of an aerobic - anaerobic microbial consortium to utilize heterogeneous feedstocks for the selective yield-optimized production of valuable biofuels, chemicals and food/feed stuff, in particular SCFAs.
• the consortium of microorganism synthesizes at least one the following SCFAs: formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid (2-methylpropanoic acid), valeric acid (pentanoic acid), isovaleric acid (3- methylbutanoic acid), hexanoic acid, heptanoic acid, octanoic acid, wherein acetic acid, propionic acid and butyric acid are the preferred short chain acids.
• SCFAs formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid (2-methylpropanoic acid), valeric acid (pentanoic acid), isovaleric acid (3- methylbutanoic acid), hexanoic acid, heptanoic acid, octanoic acid, wherein acetic acid, propionic acid and butyric acid are the preferred short chain acids.
• the at least one strain of an aerobic microorganism produces and may secret cellulolytic and hemicellulolytic enzymes, in particular endoglucanases (EG), cellobiohydrolases (CBH) and xylanases (XLN) beta-glucosidases (BG) and/or lignin modifying enzymes (LME).
• cellulolytic enzymes can be produced as cell wall bound enzymes (cellulosome) or can be externally secreted enzymes.
• the at least one strain of an aerobic microorganism providing (ligno)cellulolytic enzymes is a fungus, such as of the genus Trichoderma, Aspergillus, Penicillium or Coprinopsis.
• Further aerobic strains can be Cellulomonas uda, Chaetomium globosum, Myceliophthora thermophila (Synonym; Sporotrichum thermophile), Myrothecium verrucaria, Phanerochaete chrysosporium, Sporotrichum pulverulentum, Thermoascus aurantiacus,, Alternaria solani, Rhizopus oryzae, , Microbacterium barkeri, Bretanomyces clausenii (Synonym: Dekkera anomala), Thermoascus aurantiacus, Gloeophyllum trabeum, Lysobacter enzymogenes subsp.
• the at last one strain of an aerobic, micro- aerophilic lactic acid and/or acetic acid producing microorganism is selected from fungi of the order Mucorales, in particular of the genus Rhizopus such as R. oryzae or R. arrhizus ; or of the order Eurotiales, in particular of the genus Aspergillus such as A. niger, A. itaconicus, A. terreus, or A. brasi lien sis.
• At least one aerobic strain of the family Bacillaceae in particular of the genus Bacillus, such as Bacillus coagulans or Bacillus subtilis is used for producing lactic acid and/or acetic acid.
• the at least one strain of a facultative anaerobic lactic acid and/or acetic acid producing microorganism is from the order Lactobacillales, in particular of the families Lactobacillaceae, Aerococcaceae, Carnobacteriaceae, Enterococcaceae, Leuconostocaceae, Streptococcaceae.
• a strain from one of the following genus: Aerococcus, Vagococcus, Lactobacillus, Carnobacterium, Leuconostoc, Oenococcus, Weissella.
• Lactobacillus is a genus of Gram-positive, facultative anaerobic or microaerophilic, rod shaped, non-spore-forming bacteria. They are a major part of the lactic acid bacteria group (i.e. they convert sugars to lactic acid). In humans, they constitute a significant component of the microbiota at a number of body sites, such as the digestive system, urinary system, and genital system.
• Lactobacillus species can be divided into three groups: obligately homofermentative (group I) including L. acidophilus, L. delbrueckii, L. helveticus, L. salivarius ⁇ , facultatively heterofermentative (group II) including L. casei, L. curvatus, L. plantarum, L. sakei, L.pentosus and obligately heterofermentative (group III) including L. brevis, L. buchneri, L. fermentum, L. reuteri.
• group I including L. acidophilus
• L. delbrueckii L. helveticus
• facultatively heterofermentative group II
• group II including L. casei, L. curvatus, L. plantarum, L. sakei, L.pentosus
• group III obligately heterofermentative
• Facultatively heterofermentative Lactobacillae are characterized by their ability to ferment hexoses through the Embden-Meyerhof-Parnas (EMP) pathway like homofermenters, and use the phosphoketolase (PK) pathway for pentose assimilation (hexoses to lactate, pentoses to lactate and acetate or ethanol).
• EMP Embden-Meyerhof-Parnas
• PK phosphoketolase
• Obligate heterofermentative Lactobacillae use the PK pathway for hexoses and pentoses (see also Fig. 15).
• the at least one strain of a facultative anaerobic lactic acid producing microorganism is selected from a group containing L. pentosus, L. brevis, L. buchneri, L. fermentum, L. reuteri, L. casei, L. curvatus, L. plantarum, L. sakei, L. acidophilus, L. delbrueckii, L. helveticus, L. salivarius, in particular L. pentosus.
• At least two strains of a facultative anaerobic lactic acid producing microorganism are provided, in particular at least two Lactobacillus strains, such as L. pentosus and L. brevis.
• the metabolic flux is tuned.
• the tuning of the metabolic flux from carbohydrates to acetic acid is based on the extension of the microbial consortium with the obligate heterofermentative LAB Lactobacillus brevis which produces lactic and acetic acid from both hexoses and pentoses.
• L. pentosus alone the extended microbial consortium resulted in an increased production of acetic acid. This allows the fine tuning of the ratio of lactic to acetic acid.
• the consortium comprises at least one strain of an acetate producing microorganism.
• the at least one strain of an acetate producing microorganism can be of the genus Acetobacterium, in particular Acetobacterium woodii.
• Acetobacterium is a genus of anaerobic, Gram-positive bacteria that belong to the Eubacteriaceae family.
• the type species of this genus is Acetobacterium woodii.
• the name, Acetobacterium has originated because they are acetogens, predominantly making acetic acid as a endproduct of anaerobic metabolism. Most of the species reported in this genus are homoacetogens, i.e. solely producing acetic acid as their metabolic end product.
• Acetobacterium strain to the CBP consortium (i) omits the carbon loss in heterofermentative lactic acid fermentation of hexoses by L. brevis and (ii) compensates an unfavorable glucan/xylan/acetic acid ratio by producing acetate from lactate.
• homoacetogens provides the possibility to provide the required amount of lactic acid and acetic acid and to obtain a complete conversion of the available carbohydrates also if no further pentose sources such as xylan or mannan for the heterofermentative production of acetate and only minor acetic acid from the plant biomass are available.
• At least one strain of an obligate anaerobic or strict anaerobic microorganism, which preferably ferments lactate or co-ferments lactate and acetate which are released by the at least one lactic acid / acetic acid producing microorganism is selected from the genus Clostridium, in particular Clostridium tyrobutyricum, Clostridium aceticum, C. formiaceticum, or the family Veillonellaceae such as Veillonella criceti or Megasphaera elsdenii or Acetobacterium woodii.
• Clostridium tyrobutyricum is a rod-shape, gram-positive bacteria that grows under anaerobic condition and typically produces butyric acid, acetic acid and hydrogen gas as their major fermentation products from glucose and xylose.
• Clostridium tyrobutyricum (which is typically unable to utilize lactic acid as sole carbon source) is now forced to use lactic and acetic acid as carbon sources and not sugars.
• the present method allows the creation of an ecological niche for the obligate anaerobic microorganism due to the metabolic activity of the facultative anaerobic strains providing lactic acid.
• the present method uses the fact that C.
• tyrobutyricum is also able to co-consume lactic acid (provided by the lactic acid producing strain) and acetic acid (provided as external source e.g. from plant biomass or by the acetic acid producing strain) to butyric acid where no unwanted side products are formed. If carbohydrates are used as substrates acetic acid is co produced which increases the downstream costs and reduces the yields. Another advantage is the utilization of acetic acid as carbon source which is released from acetyl side chains by enzymatic hydrolysis of the xylopuranose backbone (from hemicellulose).
• the co-cultivation of all microorganisms reduces the metabolic flux of carbohydrates to C. tyrobutyricum due to the intra-consortium competition.
• the product selectivity will be increased since butyric acid is the only short chain acid which is a product from C. tyrobutyricum from lactic acid and acetic acid.
• Veillonella criceti is used as a lactate fermenting obligate or strictly anaerobic microorganism producing acetic acid and propionic acid. It is important to note that V. criceti is not able to use any sugar as carbon source, but rather requires the product of facultative anaerobic strain (i.e. lactic acid).
• acetic acid for producing short chain fatty acids
• propionic acid for producing short chain fatty acids
• butyric acid a) aerobic microorganism for producing cellulolytic enzymes for converting biomass to sugar molecules (e.g. T. reesei, A. niger, etc.) -> facultative anaerobic microorganism for converting sugar molecules to lactic acid and/or acetic acid (e.g. Lactobacillus) -> obligate anaerobic microorganism for strictly anaerobic conversion of lactic acid to short chain fatty acid (e.g. V.
• acetic acid e.g. T. reesei, A. niger, etc.
• facultative anaerobic microorganism for converting sugar molecules to lactic acid and/or acetic acid
• acetic acid e.g. Lactobacillus
• criceti for producing propionic acid
• b) aerobic microorganism for producing cellulolytic enzymes for converting biomass to sugar molecules e.g. T. reesei, A. niger, etc.
• facultative anaerobic microorganism for converting sugar molecules to lactic acid and/or acetic acid e.g. Lactobacillus
• acetic acid e.g. Lactobacillus
• obligate anaerobic microorganism for strictly anaerobic conversion of lactic acid to short chain fatty acid e.g. C. tyrobacetrium for producing butyric acid
• aerobic microorganism for producing cellulolytic enzymes for converting biomass to sugar molecules (e.g. T. reesei, A.
• niger, etc. facultative anaerobic microorganism for converting sugar molecules to lactic acid and/or acetic acid (e.g. Lactobacillus) -> obligate anaerobic microorganism for strictly anaerobic conversion of lactic acid to acetic acid (e.g. A. woodii, C. formiaceticum, C. aceticum etc.).
• facultative anaerobic microorganism for converting sugar molecules to lactic acid and/or acetic acid e.g. Lactobacillus
• obligate anaerobic microorganism for strictly anaerobic conversion of lactic acid to acetic acid e.g. A. woodii, C. formiaceticum, C. aceticum etc.
• At least one microorganism from the genus Megasphaera may also be used which may utilize lactate among other carbon sources. Megasphaera are anaerobic, Gram-negative cocci.
• Megasphera may be used for producing carboxylic acids with up to eight carbon atoms.
• Megasphaera elsdenii produces acetic, butyric, isobutyric, iso-valeric, valeric acid, hexanoic acid and in smaller quantities formic acid, heptanoic acid and octanoic acid.
• a modular‘Lactate Platform’ is provided by implementing a microbial consortium for the direct production of butyric acid or propionic and acetic acid from lignocellulosic biomass.
• the Lactate Platform compensates the missing metabolic flexibility of obligate anaerobic bacteria such as e.g. C. tyrobutyricum (which forms acetic acid as undesired side product when using carbohydrates as carbon source) or V. criceti (which is not able to utilize carbohydrates) since both C. tyrobutyricum and V. criceti convert lactic acid as intermediate to butyric acid and propionic and acetic acid, respectively.
• C. tyrobutyricum which forms acetic acid as undesired side product when using carbohydrates as carbon source
• V. criceti which is not able to utilize carbohydrates
• polyhydroxybutyrate and lipids can be produced through the lactate platform.
• the lactate platform boosts the carbon flux from lignocellulose to the target product compared to the alternative direct sugar fermentation routes for acetic acid, polyhydroxybutyrate, ethanol and butanol.
• Such a switch to another target product requires the exchange of the lactic acid fermenting strain(s) and with that, different abiotic requirements such as pH, temperature or presence of co-substrates will likely have to be fulfilled.
• the consortium of the microorganisms according to the invention is cultivated and grown at temperatures between 15 and 60‘O, preferably between 26 and 37 °C, in particular preferably between 28 and 30 °C.
• the pH-value of the culture medium is between 3.0 and 10.0, preferably between 4.0 and 8.0, in particular preferably at 6.0.
• the method according to the invention allows also the use of lignocellulosic biomass, such as wood or straw, for the production of short chain fatty acids, in particular butyric acid or propionic acid / acetic acid.
• beech wood may be used.
• the beech wood is pretreated in a two-stage steam pretreatment procedure wherein temperature sensitive xylooliogomers are valorized and acetic acid is recovered in a moderate inhibitory process stream. This stream also contains solubilized lignin (see also Fig. 16).
• a method for microbial production of lipids from organic feed stock, in particular lignocellulosic biomass is provided, wherein a consortium of at least three different strains of organisms is provided, wherein the consortium comprises
• At least one strain of a lipid producing microorganism in particular an algae, for fermenting lactate and/ or acetate to at least one lipid.
• lipid producing method More specific examples of the different embodiments of the lipid producing method are: a) aerobic microorganism for producing cellulolytic enzymes for converting biomass to sugar molecules (e.g. T. reesei, A. niger, etc.) -> facultative anaerobic microorganism for converting sugar molecules to lactic acid and/or acetic acid (e.g. Lactobacillus pentosus,) -> lipid producing microorganism such as algae for the conversion of lactic acid and/or acetic acid to lipids; b) aerobic microorganism for producing cellulolytic enzymes for converting biomass to sugar molecules (e.g. T. reesei, A.
• a) aerobic microorganism for producing cellulolytic enzymes for converting biomass to sugar molecules e.g. T. reesei, A.
• facultative anaerobic microorganism for converting sugar molecules to acetic acid (e.g. Acetobacterium woodii, Clostridium formiaceticum, etc.) -> lipid producing microorganism such as algae for conversion of acetic acid to lipids; c) aerobic microorganism for producing cellulolytic enzymes for converting biomass to sugar molecules (e.g. T. reesei, A. niger, etc.) -> facultative anaerobic microorganism for converting sugar molecules to lactic acid and/or acetic acid (e.g.
• acetic acid e.g. Acetobacterium woodii, Clostridium formiaceticum, etc.
• lipid producing microorganism such as algae for conversion of acetic acid to lipids
• aerobic microorganism for producing cellulolytic enzymes for converting biomass to sugar molecules (e.g. T. reesei, A. niger, etc.)
• Lactobacillus pentosus, etc. -> anaerobic conversion of lactic acid and/or acetic acid to acetic acid (e.g. Acetobactierum woodii, etc.) -> lipid producing microorganism such as algae for conversion of acetic acid to lipids; d) aerobic microorganism for producing cellulolytic enzymes for converting biomass to sugar molecules (e.g. T. reesei, A. niger, etc.) -> facultative anaerobic microorganism for converting sugar molecules to lactic acid and/or acetic acid (e.g.
• Lactobacillus pentosus, etc. -> anaerobic conversion of lactic acid and/or acetic acid to short chain fatty acids (e.g. butyric acid, propionic acid, acetic acid etc.) as descibed in detail above-> lipid producing microorganism such as algae for conversion of short chain fatty acids to lipids.
• short chain fatty acids e.g. butyric acid, propionic acid, acetic acid etc.
• the consortium synthesizes triglycerides comprising fatty acids of different chain lengths, in particular C8-C22 saturated and/or unsaturated fatty acids. Furthermore, aliphatic hydrocarbons 0 3 7H 64, C 3 6H 6 2, C 36 H 6 2 , C 34 H 58, Botryococcene, Isobotryococcene may be produced.
• the algae may be selected from a group of microalgae comprising Volvocales, such as Chlamydomonas, Chlorellales or Trebouxiophyceae, such as Botryococcaceae, Botryococcus (e.g. Botryococcus braunii ), Bacillariophyceae or Diatomophyceae.
• Volvocales such as Chlamydomonas, Chlorellales or Trebouxiophyceae
• Botryococcaceae such as Botryococcaceae, Botryococcus (e.g. Botryococcus braunii ), Bacillariophyceae or Diatomophyceae.
• a preferred algae is Chlamydomonas reinhardtii.
• Microalgae are photosynthetic and so can grow on C0 2 in the presence of light (autotrophic growth). Many also are able to utilize carbon sources such as acetate or glucose together with oxygen and grow heterotrophically in the absence of light. More common is mixotrophic growth in the light - the combination of autotrophic and heterotrophic growth. This offers many advantages such as higher growth rates, higher biomass density and higher lipid content and an immobilization of the microalgae in a biofilm would facilitate the harvest of intracellular microalgal products. To enable the mixotrophic growth of a microalgae in combination with T. reesei, an ecological niche has to be created that provides oxygen, organic carbon, light and carbon dioxide.
• microalgae C. reinhardtii is able to grow next to T. reesei in a two-layered biofilm on top of the aerated membrane with the illuminated fiber, despite potential competition for space, oxygen and organic carbon with the other microbe.
• the present methods may be carried out in a reactor containing an oxygen permeable membrane separating an oxygen supply compartment from a fermentation compartment, and oxygen is transported from the oxygen supply compartment to the fermentation compartment through the membrane and an oxygen zone is formed in the fermentation compartment adjacent to the membrane, wherein the oxygen permeable membrane contains a surface, which is facing the fermentation compartment and on which a biofilm is located, and the biofilm contains at least one strain of an aerobic microorganism for producing external cellulolytic enzymes in an aerobic zone of the fermentation compartment, and wherein the at least one strain of aerobic microorganism is supplied with oxygen through the membrane.
• the main advantages of merging enzymatic hydrolysis and fermentation in one reactor are the enhanced rate of hydrolysis of lignocellulosic biomass due to removal of end-product which reduces end- product inhibition and reduction of investment costs.
• An oxygen gradient with decreasing oxygen content with increasing distance from the membrane is established within the biofilm located on the membrane.
• the oxygen may be transferred at a rate of 0.3 - 80 1 /h, preferably 1 - 50 1 /h, more preferably 4 - 20 1/h (Volumetric oxygen transfer coefficient).
• the pressure may be in a range between 0.05 bar - 20 bar, preferably between 0.1 - 10 bar, more preferably between 1 -1 5 bar, such as 1 .1 bar.
• the reactor engineering allows for a stabilization and control of the microbial consortium. Since oxygen harms cultures of anaerobes either in planktonic or biofilm phenotype, it is preferred to cultivate the at least one strain of an aerobic microorganism for producing external cellulolytic enzymes, such as T. reesei, immobilized as biofilm directly on the surface of an oxygen membrane.
• the strains were selected in order to synthesize the final products with high yield and high selectivity. The strains grow in specific metabolic niches for this purpose.
• a reactor system for carrying out the method according to the invention wherein the reactor system comprises at least one reactor, wherein the at least one reactor contains:
• an oxygen supply compartment for accommodating a fluid containing oxygen
• the oxygen permeable membrane is a tubular body and the oxygen supply compartment lies in the tube-like space within the tubular membrane and the fermentation compartment lies outside the tubular membrane or vice versa.
• the membrane which separates the oxygen supply compartment from the fermentation compartment is preferably dense but oxygen permeable.
• the dense membrane is preferably made of or contains silicone, preferably polydimethylsiloxane. Further the membrane can contain or consist of fluorocarbon compounds (e.g. polytetrafluorethylene), hydrocarbon compounds (e.g. polyethylene, polypropylene), polysulphone or polyalkylsulphone.
• the fresh fluid is flushed through the membrane.
• the oxygen carrying fluid is recirculated. Instead of pumping always fresh fluid through the membrane, the fluid is recirculated.
• the fluid is a gas.
• the oxygen content in the gas is measured and oxygen / air is metered into the fluid when required (i.e. oxygen content in the fluid is too low).
• the fluid is a liquid which can be recirculated. In this case the oxygen containing liquid is transported into the reactor and is metered as required.
• a major obstacle of co-cultivation of different microorganisms is often the different temperature requirements of the microorganisms.
• the optimal temperature for the enzymatic hydrolysis is regularly above the tolerated growth temperature range of the enzyme producer and/or the product forming strain(s).
• the temperature is usually lowered in simultaneous saccharification and fermentation processes and consolidated bioprocesses to enable growth of the microorganism(s). Since temperature influences the rate of enzyme-catalysed reactions the hydrolysis is performed at temperatures below the optimum which prolongs the process and reduces the productivity.
• the selection of product forming microorganisms is limited, since e.g. thermophilic microorganisms cannot be co-cultivated with other microorganisms if the tolerated temperature range do not overlap.
• Another challenge is the integration of product-forming microorganisms which require lower temperatures in comparison to the tolerance ranges of the enzyme producer. Furthermore, the product formation is often influenced by the temperature.
• One example process is the production of unsaturated fatty acids, where the increase in the degree of fatty acid unsaturation in response to a decrease in growth temperature is an almost universal phenomenon known from blue-green algae, eukaryotic algae, yeasts and fungi.
• the content of fatty acids and triglycerides is affected by the temperature (e.g. in Nannochloropsis) and the temperature is an important abiotic parameter for the fine tuning of the proportion of the fatty acids.
• one challenge is that the temperature optimum of enzymes secreted by mesophilic microorganisms is above the temperature tolerated by producing microorganisms. This causes the enzymatic hydrolysis to be slower under suboptimal conditions and the process has a lower productivity.
• the selection of microorganisms for product formation is limited since thermophilic microorganisms cannot be co-cultivated if they do not have overlapping tolerance ranges.
• a temperature gradient or temperature profile is established by cooling the circulating fluid. This enables the co-cultivation of microorganisms with different temperature requirements and the creation of a niche for microorganisms, which tolerate only lower temperatures. Furthermore, the temperature gradient may accelerate the enzymatic hydrolysis. The temperature gradient may be provided by cooling via the membrane or via heating via the membrane. It is also conceivable to provide different temperature levels with multiple membranes.
• a reactor and a method for operating said reactor which comprises sub-compartments allowing the formation of temperature gradient.
• a fluid carrying the oxygen into the reactor is cooled or heated for example in a suitable cooling unit or heating unit, respectively.
• the cooled oxygen carrying fluid passes the membrane not only an oxygen gradient is formed but also a temperature gradient is formed within the reactor.
• the temperature in the biofilm on the membrane may be lower than in the fermentation slurry or broth. This allows for a higher temperature in the fermentation broth and the enzymatic hydrolysis may be carried out at higher temperatures.
• a heated oxygen carrying fluid passes the membrane the temperature in the biofilm on the membrane may be higher than the fermentation slurry or broth. This allows the creation of an ecological niche for at least one microorganism which requires or benefits from elevated temperatures. Thus, microorganisms with different temperature optimums may be co-cultivated.
• the fluid entering the reactor is used to cool the membrane and the adjacent biofilm.
• a temperature controlled fluid is fed to the reactor wherein the fluid contains oxygen to form a temperature and oxygen gradient in the reactor.
• the temperature in the fermentation slurry is increased compared to the zone located close to the membrane and the enzymatic hydrolysis is performed at increased temperature although the microorganism itself is unable to grow under these elevated temperatures.
• the co-cultivation of at least two microorganisms is achieved by growing the microorganisms in compartments with different temperatures.
• the microorganisms are characterized by missing intersection of the tolerated temperature range their co-cultivation is enabled in one reactor.
• at last one aerobic (ligno)cellulolytic enzyme producing microorganism is cultivated in the compartment located close to the membrane.
• At least one microaerophilic, facultative anaerobic or anaerobic microorganism is cultivated in the compartment with increased temperature.
• gradients for bioavailable nitrogen or pH value are provided in addition to the temperature gradient.
• bioavailable nitrogen such as urea, ammonia, ammonium salts
• a pH-gradient may be provided via the membrane by metering substances affecting the pH (i.e. substances that form acid or base in the presence of water).
• the pH gradient can be also combined with a temperature and oxygen gradient.
• a pH-gradient is provided in an electrochemical fashion by splitting water.
• Another option to alter the pH locally in the reactor is based on the principle of electrolysis of water. Electric energy can be used to split water whereby the following reactions take place at the electrodes. Two water molecules react at the anode and form four protons, one oxygen molecule and four electrons. At the cathode four water molecules react with four electrons and form two hydrogen molecules and four hydroxide ions (see Equation below). The formed protons and hydroxide ions affect the pH in the vicinity of the anode and the cathode, respectively.
• a bioreactor may be equipped with two electrodes and an electrical current is applied. Details are described in the experimental section.
• there are two approaches to locally alter the pH in the bioreactor either by flushing the membrane with fluids such as C0 2 or NH 3 solution or by using electricity to split water. In this way local pH niches can be created and with that, the microbial community can be widened and the product range can be extended.
• an aerobic fungus secreting (ligno-) cellulolytic enzymes a facultative anaerobic microorganism for producing lactic acid and/or acetic acid (which reduces the redox potential and provides a niche for an anaerobic microorganism) and an anaerobic microorganism for fermenting lactate and/or acetate (which contains a cellulosome) are co cultivated.
• This allows the beneficial co-digestion of lignocellulosic biomass by the combined action of (ligno-) cellulolytic enzymes which are secreted by the fungus and cellulosomes bound to the bacterial cells despite different tolerated temperature ranges of the producing microorganisms.
• thermophilic strains which produce cellulosomes are from the genus Clostridium, e.g. C. thermocellum with a growth range from 58-68 ' ⁇ or from the genus Thermoanaerobacterium, e.g. Thermoanaerobacterium saccharolyticum with a growth range of 30-66 °C.
• the (ligno-) celluloytic enzymes and the cellulosome allow for the rapidhydrolysis of the polymeric carbohydrates.
• the enzymatic hydrolysis rate of secreted enzymes produced by e.g. mesophilic microorganisms is increased by increasing the temperature in the reactor above the tolerated temperature range of the mesophilic microorganisms (e.g. > 32 q C for T. reesei) and by simultaneously creating a stable local ecological niche for the enzymes producer in a compartment with decreased temperature (e.g. in the vicinity of the membrane cooled by the oxygen transfer fluid).
• a reactor and a method for operating said reactor allow for the co-cultivation of at least three microorganisms whereby e.g. a psychrophilic microorganism is co-cultivated along with two microorganisms which require increased temperatures.
• the three microorganisms have no overlapping tolerated temperature ranges.
• the temperature gradient enables the formation of a two-layered biofilm, which creates a stable ecological niche for e.g. the psychrophilic and mesophilic member of the community.
• the at least one thermophilic microorganism of the community is located in the fermentation slurry.
• This setup is in particular preferred when using a psychrophilic algae for producing omega-3 fatty acids or a when using thermophilic acetic acid producers of the class Clostridia, e.g. Moorella thermoacetica.
• most microalgae grow autotrophically, which means that light provides the required energy to fix carbon from carbon dioxide.
• microalgae can also grow on organic carbon sources such as acetic acid or other carboxylic acids or carbohydrates in the absence of light when oxygen is present (heterotrophic growth). When microalgae grow on light and organic carbon sources the mode of growth is called mixotrophic.
• Heterotrophic and, in particular, mixotrophic metabolism allows for higher growth rates, higher biomass density and higher lipid content compared to autotrophic growth. Furthermore, the fitness of microalgae that grow heterotrophically in darkness is lower than that of microalgae that grow mixotrophically because light, in particular wavelengths between 420 to 470 nm (blue) and 660 to 680 nm (red), induces cell division and gene expression among others.
• light is provided to enable autotrophic or mixotrophic metabolism of at least one lipid forming microorganism.
• the bioreactor is equipped with a light source to provide light for microalgal metabolism.
• Light can be injected to a biorecator in various ways. The most common way is to provide an external light source and to illuminate the bioreactor from outside i.e. the microorganisms are illuminated through the translucent reactor walls. Another option is to provide a light source within the bioreactor, e.g. point sources or light fibers.
• Typical light intesities for autotrophic and mixotrophic algal metabolism range between 1 to 2000 pmolnr 2 s 1 preferentialy between 50 to 200 pmolnr 2 s 1 . .
• 3000 K high power LEDs were chosen as light source with an emission spectrum suitable for photosynthesis. The spectrum peaks at 442.5 nm (blue) and 615 nm (red) whereby the power density is higher in the red region.
• Flexible fibers with an outer diameter of for example 1 .5 mm are used to direct the light of the LED into the reactor’s interior. The physical properties of the fiber allow radial light emission by internal scattering.
• the advantage is that the partial oxygen pressure increases and thus the oxygen transfer rate increases as well.
• the volumetric oxygen transfer coefficients is determined using the following method.
• a dissolved oxygen probe is used to measure dissolved oxygen probe in the reactor.
• nitrogen is sparged or purged until the dissolved oxygen concentration (DO) falls below the detection limit without air flow through the membrane.
• DO dissolved oxygen concentration
• a second step nitrogen flow is stopped and air flow is increased through the membrane to set- point; the increase of the DO is measured and the kla value is calculated from the initial increase of the DO concentration.
• the pressure applied to the membrane may be increased by e.g. using an additional pressure drop (e.g.
• a pressure retention valve or a tubing with a very small inner diameter at the outlet of the reactor.
• the flow rate was the same as before and the kla-value increased.
• a kla value of 0.459 1/h may be determined without artificial backpressure (i.e. 0.05 mbar pressure drop over the length of the membrane at set air flow rate)
• a kla value 0.998 1/h may be obtained.
• the kla value by merging an intended elevated pressure in the compartment of the oxygen transfer fluid (i.e. inside the membrane) thereby increasing the partial pressure of oxygen with the approach to circulate the fluid.
• an oxygen containing fluid is pumped through the membrane continuously in a cycle.
• the oxygen content in the fluid is measured and is connected to a control loop, in which air or pure oxygen is metered into the fluid such that the consumed oxygen is replenished in the fluid.
• the cycling system needs to be pressurized only once.
• the pumps solely need to counterbalance the pressure drop in the piping.
• the oxygen transfer rate and the oxygen concentration in the fluid can be adapted and controlled online. This combination reduces operational costs and allows the online impact of the oxygen transfer rate by controlling the pressure and the oxygen concentration.
• oleaginous microorganisms e.g. algae or oleaginous yeast
• This induction is done by nitrogen limitation of the microorganisms
• microbial biomass i.e. where microorganisms proliferate
• nitrogen containing source such as ammonia, ammonium salts, nitrate, amino acids or urea
• lipid production phase which starts after exposure to nitrogen-starvation conditions (i.e. where the nitrogen source is depleted).
• these oleaginous microorganisms grow in the same reactor and/or in community with other microorganisms such as the producer of cellulolytic enzymes which continuously require a supply of nitrogen for growth and production of cellulolytic, hemicellulolytic and/or lignocellulolytic enzymes (or cellulosomes) different nitrogen requirement prevail.
• the nitrogen source is supplied locally in the reactor to the microbial community.
• a nitrogen source is provided in the rector via a nitrogen containing fluid through a membrane. The nitrogen diffuses through the membrane into the aqueous fermentation broth thereby creating continuous nitrogen input or a local niche containing nitrogen.
• the continuous enzyme production in the nitrogen rich compartment is enabled whereas a compartment with nitrogen-depletion conditions is present in the reactor where lipid formation is taking place at the same time.
• the same oleaginous microbial strain can form microbial biomass i.e. grow in the nitrogen rich zone of the reactor and simultaneously but spatially separated in the same reactor accumulate lipids in the nitrogen depleted region of the reactor.
• Possible fluids for the supply of nitrogen through the membrane are aqueous ammonia solutions or aqueous solutions of ammonium salts with controlled pH values i.e. high pH values to shift the ammonium/ammonia equilibrium to the ammonia side.
• Further fluids can also be gaseous e.g. air containing ammonia vapors.
• the local nitrogen supply may be merged with temperature and oxygen gradients with one or more membranes creating compartments (as exemplified in Fig. 12).
• a solution is to create local pH gradients in the reactor. These pH gradients can be created by diffusing acids or bases through the membrane into the reactor. Thereby the pH gradient is locally changed on the surface of the membrane If for example a tubular membrane is used different aqueous or gaseous fluids can be circulated through the membrane. Possible fluids to locally increase the pH value are aqueous ammonia solution, or caustic solutions such as NaOH or gaseous ammonia vapors Possible fluids to decrease the pH value locally are carbon dioxide, nitrogen dioxide, chlorine or alternatively, the acids such as hydrochloric acid (see also Fig 13).
• a sterile UV sampling device is provided.
• the sampling device is suitable for obtaining samples from fermentation broths which contain solids such as plant biomass, plant biomass particles, lignocellulosic solids. Such solid components or solid particles may by larger than 1 pm, preferably larger than 10 pm, more preferably larger than 50 pm, even more preferably larger than 100 pm.
• the specific UV sampling device uses a UV barrier for preventing microbial contamination between the samplings from the fermentation broth.
• One conventional system which allows the sterile sampling from bioreactors designed for processes based on soluble carbon sources is based on a non-return valve serving as contamination barrier after sampling.
• this system is not suited for a contamination free sampling in fermentations where plant biomass particles are present, as these particles may prevent a hermetic closing of the backpressure valve and cause blockage.
• Biomass particles are characterized by their much larger size (in the range of millimeters) than microbial cells.
• the non-return valve was exchanged through a UV barrier.
• This device is a box in which a UV source is mounted emitting light at a wave length between 200 to 300 nm, preferentially at 255 nm. Through the box a tube, made of quartz glass is directed, which is connected to the sampling tubing.
• Figure 1 a schematic illustration of the‘Lactate Platform’
• Figure 2 a schematic representation of the production of butyric acid using a synthetic microbial consortium
• Figure 3 a process for producing butyric acid from lignocellulosic biomass using a cross kingdom microbial consortium
• Figure 4 a process for the direct production of propionic and acetic acid from lignocellulosic biomass
• Figure 5 a further process for the direct production of short chain fatty acids from lignocellulosic biomass
• Figure 6 a schematic representation of a further process flow for the fermentation of cellulose to lactate and/or acetate and subsequently to short chain fatty acids
• Figure 7 a schematic representation of a process flow for the fermentation of cellulose to lactate and/or acetate and subsequently to lipids by lipid producing microorganisms
• Figure 8 a schematic representation of a reactor system used for conducting the process according to the invention
• Figure 9 a schematic representation of a sampling device based on a UV source
• Figure 10 a schematic representation of a reactor system used for conducting the process according to the invention
• Figure 1 1 a schematic representation providing a temperature gradient in the reactor system
• Figure 12 a schematic representation of the system providing a (bio-available) nitrogen gradient or nitrogen and temperature gradients in the bioreactor
• Figure 13 a schematic representation of the system providing a pH gradient in the bioreactor
• Figure 14 a schematic representation of the adjustment of the pH of an aqueous fluid in the reactor via a membrane
• Figure 15 a summary of pathways used by microorganisms used in the process according to the invention.
• Figure 16 a schematic representation of the compound flux in a two-stage steam pretreatment process of beech wood
• Figure 17 a schematic representation of pathways in a co-culture comprising an algae
• Figure 19 images of a fungal-microalgae biofilm grown on a membrane surface
• Figure 20 time-resolved formation of a spatial pH-gradient in a solid water / agarose/sodium chloride mixture
• Figure 21 time-resolved formation of a pH gradient in a biofilm-mimicking agarose coating on the cathode.
• FIG. 1 shows a schematic illustration of the‘Lactate Platform.
• T. reesei was introduced as in-situ producer of cellulolytic enzymes.
• the heterogeneous mixture of carbohydrates is metabolically tunneled to mainly lactic acid by lactic acid bacteria.
• Lactic acid as central intermediate in the metabolism was selected to reduce the required metabolic flexibility of the target product forming microorganisms.
• a designed artificial pathway distributed among different microorganisms was developed to integrate acetic acid as additional carbon source, to increase the metabolic flux to the target product, reduce the formation of side products and enable the cultivation of microorganisms which are unable to utilize carbohydrates but metabolize lactic acid and/or acetic acid.
• FIG. 2 is a schematic representation of a bioprocess for the production of butyric acid.
• a two-stage steam pretreatment was applied to beech wood to alter the lignin structure, improve the accessibility of the cellulose and dissolve the hemicellulose.
• a mainly cellulose containing solid fraction and a prehydrolyzate containing soluble xylo-oligomers, acetic acid and various inhibitory compounds (formic acid, phenolics) was obtained.
• Acetyl side chains were released from the xylopuranose backbone by enzymatic hydrolysis.
• a helical coiled, tubular polydimethylsiloxane membrane was installed in the stirred tank reactor, permeable for oxygen and continuously flushed with air. T.
• reesei formed an aerobic biofilm on top of the membrane and secreted cellulases and hemicellulases (EGI: endoglucanase I, CBHI: cellobiohydrolase I, CHBII: cellobiohydrolase II, BXL: beta-xylosidase, XLN: betaendoxylanase).
• EGI endoglucanase I
• CBHI cellobiohydrolase I
• CHBII cellobiohydrolase II
• BXL beta-xylosidase
• XLN betaendoxylanase
• Figure 3 illustrates fed-batch experiments for synthesis of butyric acid using a cross-kingdom microbial consortium.
• L. pentosus was inoculated at the beginning of phase I after T. reesei was inoculated 48 hours in advance.
• C. tyrobutyricum was introduced.
• A, B, C and E L. pentosus as LAB.
• D L. pentosus and L. brevis as LAB.
• Figure 4 illustrates the process for the direct production of propionic and acetic acid from lignocellulosic biomass.
• L. pentous was inoculated at the beginning of phase I after T. reesei was inoculated 48 hours in advance. At the beginning of phase II V. criceti was introduced.
• Figure 5A shows a process scheme for the direct production of propionic acid, butyric acid, valeric acid, hexanoic acid by integrating M. elsdenii as a strict anaerobic strain into the consortium.
• M. elsdenii uses lactic acid and acetic acid as well as sugar molecules as carbon source.
• Figure 5B depicts a two-stage steam-pretreated beech wood solids (3% w/w) that is mixed with a feed of the corresponding prehydrolyzate over a period of 200 h.
• the dashed blue line shows the xylose fed with the prehydrolyzate recalculated from xylooligomers.
• a 2.7 liter reactor with an OTR of 0.34 h- 1 was used.
• the consortium used in B1 comprises T. reesei, L. pentosus, M. elsdenii, in B2) T. reesei, L. pentosus, C. tyrobutyricum, M. elsdenii, and in B3) T. reesei, L. pentosus, V. criceti, M. elsdenii.
• the goal was to produce a targeted mixture of VFAs by inserting additional microorganisms to the community.
• the competition and different metabolic end products should affect the VFA mixture.
• the consortium of B1 produced acetic, butyric and hexanoic acid.
• V. criceti produces acetic and propionic acid. Both are alternative substrates for M. elsdenii.
• M. elsdenii was able to reutilize both acids and increased concentrations of valeric acid were published.
• Our intention of this experiment was to shift the produced VFA mixture towards valeric acid in comparison to B1 .
• the conditions were the same as described for the production of butyric or acetic/propionic acid using C. tyrobutyricum or V. criceti, respectively (please refer to section: Membrane Bioreactor).
• Figure 6 shows a schematic representation of the process flow for the subsequent secondary fermentation of lactate to volatile fatty acids, such as acetate, propionate and butyrate.
• lactate volatile fatty acids
• volatile fatty acids such as acetate, propionate and butyrate.
• the heterogeneous carbohydrate mixture is ‘metabolically tunneled’ to lactic acid using a fungal-bacterial consortium. Without ‘tunneling’ a high level of metabolic flexibility of the product forming microorganism is required to selectively convert the heterogeneous mixture to the target product.
• Microcrystalline cellulose was converted to a model lactate broth by T. reesei and L.
• Figure 7 shows a schematic representation of a second process flow for the fermentation of cellulose to lactate and/or acetate and subsequently to short chain fatty acids by lipid producing microorganisms such as algae.
• a) aerobic microorganism for producing cellulolytic enzymes for converting biomass to sugar molecules e.g. T. reesei, A. niger, etc.
• a) aerobic microorganism for producing cellulolytic enzymes for converting biomass to sugar molecules e.g. T. reesei, A. niger, etc.
• acetic acid e.g. Lactobacillus pentosus, Acetobacterium woodii
• lipid producing microorganism such as algae for conversion of lactic acid and/or acetic acid to lipids
• aerobic microorganism for producing cellulolytic enzymes for converting biomass to sugar molecules (e.g. T. reesei, A.
• short chain fatty acids e.g. butyric acid, propionic acid, acetic acid etc.
• lipid producing microorganism such as algae for anaerobic conversion of short chain fatty acids to lipids.
• FIG. 8 shows a reactor system that can be used for conducting the process according to the invention.
• the reactor system comprises a bioreactor with a membrane, an expansion vessel optionally with a sparger, a pump, an oxygen probe and suitable valves and piping.
• the reactor system is constructed such that the oxygen transfer over the membrane is effected such that an oxygen containing fluid is circulated and the oxygen diffuses from the fluid through the membrane into the culturing medium.
• the oxygen containing fluid is applied with a high flow rate, what in turn requires rather large amounts of fluid.
• the oxygen concentration of the fluid flowing through the membrane may have an influence on the metabolic activity of the aerobic microorganisms.
• an oxygen containing fluid is pumped through the membrane continuously in a cycle.
• the oxygen content in the fluid is measured and is connected to a control loop, in which air or pure oxygen is metered into the fluid such that the consumed oxygen is replenished in the fluid.
• the cycling system is pressurized in order to impact the partial pressure of oxygen by control of the system pressure. The system needs to be pressurized only once.
• the pumps solely need to counterbalance the pressure drop in the piping.
• the oxygen transfer rate and the oxygen concentration in the fluid can be adapted and controlled online.
• the circulation of the oxygen containing fluid has the advantages that only the consumed oxygen has to be replenished.
• the metering of the oxygen into the fluid can be done in several ways. As shown in Fig. 8 A (left side) the fluid is a gas and oxygen is metered directly into the circulating fluid stream. According to Fig. 8B (right side) the oxygen is dissolved in the fluid and the oxygen containing fluid is circulated. The metering of the oxygen can be realized by using a sparger.
• the reactor system comprises a bioreactor which is connected to an expansion vessel.
• the expansion vessel is required since the volume of the circulating fluid is not always constant.
• Figure 9 shows a schematic representation of a sampling device for the sterile sampling of fermentation broths containing plant biomass particles.
• the system is based on a UV source ensuring a sterility barrier.
• the sample from the fermentation broth is harvested through a quartz glass tube. This zone of the quartz glass tube is illuminated by the UV source between the sampling operations to ensure the destruction of microorganisms.
• the sampling procedure is as follows: 1 .
• the UV source is turned off, 2.
• the 3-way valve is turned to the‘sampling’ position, i.e. such that a sample from the fermentation broth can be pulled into the sampling loop, 3.
• the sample loop is filled with fermentation broth, 4.
• the 3- way valve is turned back into the position‘push back’, 5.
• the 3-way valve is turned to the‘sampling’ position, 7.
• the sample in the sampling loop is harvested, 8.
• the 3-way valve is turned back into the‘push back’ position, 8.
• the fermentation broth that was again sucked into the tubing is pushed back into the reactor sing sterile air or nitrogen, 9.
• the UV source is turned on again (this can also be controlled by a timer and does not necessarily have to burn all the time).
• Figure 10 shows a reactor system for providing an oxygen gradient as well as a temperature gradient over the biofilm and membrane.
• the temperature of the circulating oxygen containing fluid is adjusted in a chiller before circulated through the membrane forming a temperature and oxygen gradient in the reactor.
• Figure 1 1 A-D illustrate how a temperature gradient affects the growth of the consortium of microorganism.
• a temperature gradient is required in cases when the temperature optimum of enzymes secreted by mesophilic microorganism is above the temperature tolerated by the producing microorganisms B (Fig. 1 1 A). This causes the enzymatic hydrolysis to be slower under suboptimal conditions and the process has a lower productivity.
• the selection of microorganisms for product formation is limited since thermophilic microorganisms cannot be co-cultivated if they do not have overlapping tolerance ranges.
• the fluid transporting the oxygen into the reactor is cooled for example in a suitable cooling unit (see Fig. 10).
• the membrane When the cooled oxygen containing fluid passes the membrane not only an oxygen gradient is formed but also a temperature gradient is formed (see Fig. 1 1 B).
• the temperature in the biofilm on the membrane may be lower than in the fermentation slurry or broth. This allows for a higher temperature in the fermentation broth and the enzymatic hydrolysis may be carried out at higher temperatures.
• microorganisms with different temperature optima may be co-cultivated.
• microorganisms A and B are co-cultivated by growing in compartments with different temperatures. Although the microorganisms are characterized by missing intersection of the tolerated temperature range (see Fig. 1 1 A) their co-cultivation is enabled in one reactor. At last one aerobic (ligno-) cellulolytic enzyme producing microorganism B is cultivated in the compartment located close to the membrane. At least one microaerophilic, facultative anaerobic or anaerobic microorganism C is cultivated in the compartment with increased temperature.
• thermophilic microorganism A can be co-cultivated with further meso- and/or thermophilic microorganism B, C.
• a two-layered biofilm is generated on the membrane surface, wherein each of the microorganism A, B and C can find an ecological niche in the biofilm reactor.
• the at least one thermophilic microorganism of the community is located in the fermentation slurry.
• a psychrophilic algae for producing omega-3 fatty acids and thermophilic acetic acid producer of the class of Clostridia, e.g. Moorella thermoacetica may be employed.
• Figure 12 illustrates how a nitrogen gradient (here in form of ammonia), an oxygen gradient and optionally a temperature gradient affect the growth and the product formation of the different microorganisms in the microbial consortium used for lipid production.
• a nitrogen gradient is required to allow the simultaneous enzyme production by the cellulolytic fungus, such as T. reesei, and the lipid production by an oleaginous microorganism (oleas MO), such as algae (Fig 12A).
• oleas MO oleaginous microorganism
• Nitrogen and oxygen is necessary for the production of cellulolytic enzymes.
• the nitrogen limitation which the oleas MO encounters in the nitrogen depleted zone, induces the production of lipids in the aerobic zone.
• the nitrogen gradient across the biofilm also allows the simultaneous growth and product formation by the oleas MOs.
• These nitrogen and oxygen gradients can also be combined with temperature gradients across the biofilm and membrane (Fig 12B, C1 C2, C3, D).
• the fermentation broth is kept at a higher temperature e.g. for a faster enzymatic hydrolysis (see Fig. 1 1 ) than the oxygen and nitrogen transporting fluid, i.e. a heat flux from the bulk liquid to the oxygen/nitrogen transporting fluid across the biofilm and the membrane is created (Fig 12B).
• the enzymatic hydrolysis runs at the highest temperature.
• the oleas MO grows at the intermediate temperature and the cellulolytic enzyme producer at the lowest temperature.
• the oleas MOs need lower temperatures for growth and product formation than the cellulolytic enzyme producer set-ups as illustrated in Fig C1 , C2 and C3 as well as D apply.
• D a heat flux from the oxygen/nitrogen transporting fluid to the bulk liquid across the biofilm and the membrane is created i.e. the oxygen/nitrogen transporting fluid is at a higher temperature (Fig 12D).
• a second membrane is necessary. Through both membranes oxygen is fed but only through one oxygen and a nitrogen source while through the second oxygen and optionally nitrogen.
• the cellulolytic enzyme producer grows and produces enzymes.
• the oleas MO is immobilized and produces lipids where the nitrogen is depleted (and proliferates where nitrogen is present closer to the membrane in case nitrogen is fed through the membrane).
• the oleas MO grows/forms lipids at a lower temperature than the cellulolytic microorganism, i.e. oxygen transporting fluid in the membrane on which the cellulolytic microorganism grows is colder than the fluid in the membrane on which the lipid producer grows.
• the bulk liquid can be at the same low temperature level as for the oleas MO (Fig C1 ) or at a higher temperature (Fig C3), i.e. at the growth temperature of the cellulolytic enzyme producer or even at a higher temperature (Fig C2).
• Figure 13 illustrates the creation of pH gradients in the biofilm to create niches for the respective microorganisms in the consortium.
• a base e.g. ammonia
• the pH value is increased in the boundary layer on the membrane and a pH gradient to the lower pH value of the bulk liquid is formed (Fig 13A).
• an acid is fed through the membrane the pH value is decreased in the boundary layer on the membrane and an increasing gradient toward the bulk liquid is formed.
• the microbial strains can thus form a biofilm in the zone of their optimum pH value.
• Figure 14 illustrates a pH change induced by the feeding of a base (here NH 3 ) or an acid (here C0 2 ) through the membrane into the reactor filled with an aqueous solution. Volume 200 ml_, stirred at 300 rpm with a magnetic stir bar, tubular membrane made of PDMS, outer diameter 3.18mm, wall thickness 0.80mm, length 3 m. Carbon dioxide fed to the membrane closed at the end with an overpressure of 1 .2 bar; 10 % NH 3 solution continuously circulated in the membrane.
• Figure 15 illustrates a summary of the pathways of the microorganisms producing acetic and lactic acid as well as butyric and propionic acid.
• Clostridium tyrobutyricum can assimilate glucose to butyric acid and carbon dioxide. If C. tyrobutyricum co-produce butyric and acetic acid from glucose using electron bifurcation the energy gain is with 3.25 mol ATP per mol glucose higher in comparison to sole butyric acid formation (3.0 mol ATP per mol glucose). C. tyrobutyricum assimilates 3 mol lactate and 1 mol of acetate to 2 mol butyric acid with carbon dioxide and hydrogen formation. Acetobacterium woodii converts lactic acid to acetic acid. A. woodii can utilize 4 mol lactate and 4 mol carbon dioxide to produce 6 mol acetate and 4 mol carbon dioxide.
• Figure 16 illustrates the mass flux of glucan, xylan, acetic acid and lignin for a two-stage steam- pretreatment procedure of beech wood.
• a two-stage pretreatment procedure was applied to (i) partially recover dissolved acetic acid and xylan (as xylooligomers) in a prehydrolyzate and (ii) alter the structure of the solids to allow maximum glucose yields in enzymatic hydrolysis.
• beech wood was steam-pretreated at 180 °C for 24.8 minutes.
• the solids and the prehydrolyzate were utilized in CBP processes with (A) T. reesei, L. pentosus and C. tyrobutyricum, yielding 196.5 kg butyric acid per ton of raw beech wood (217.2 kg total VFA/t), (B) T. reesei, L. pentosus and V. criceti, yielding 1 13.6 kg propionic acid per ton and 133.3 kg acetic acid per ton.
• Figure 17 provides an Overview of pathways in a co-culture of T. reesei and C. reinhardtii using cellulose as carbon source and light as energy source.
• the thickness of the arrows indicates increased production of cellulolytic enzymes by T. reesei compared to C. reinhardtii and increased cellobiose consumption of C. reinhardtii compared to T. reesei, respectively.
• Figure 18 shows Confocal Laser Scanning Microscopy Images of Fungal-Microalgal Biofilm Grown on the Surface of the Aerated-Membrane with and without Illumination.
• A,B CLSM z- stack images about five days after inoculation of T.
• Chlorophyll a Chi a
• chlorophyll b Chi b
• Total Chlophyll Total Chi
• Inskeep and Bloom Extinction Coefficients of Chlorophyll a and b in N,N-Dimethylformamide and 80% Acetone. Plant Physiol. 77, 483 ⁇ 185,1985).
• the total amount or carotenoids (Chi x) is calculated using the equation from Wellburn (The Spectral Determination of Chlorophylls a and b, as well as Total Carotenoids, Using Various Solvents with Spectrophotometers of Different Resolution. J. Plant Physiol. 144, 307-313, 1994).
• Figure 19 shows a Photograph and Confocal Laser Scanning Microscopy Image of Fungal- Microalgal Biofilm Grown on the Surface of the Aerated-Membrane.
• A Photograph of microalgal-fungal biofilm attached to the membrane which is helically coiled around the membrane-holding structure next to the light fiber. A slightly green second layer in the upper part of the biofilm is on top of a basic layer.
• B Confocal laser scanning microscopy (CLSM) z-stack of the biofilm shown in (A). Higher z values indicate an increased distance from the surface of the membrane (source of oxygen). The image was taken about one week after inoculation of both microorganisms.
• the light fiber provides light for microalgal metabolism. Approximately 80 % of the light coupled into the fiber is emitted through radial emission at a fiber length of about one meter. In order to increase the amount of light emitted in the reactor even more both ends of the fiber instead of only one are connected to one LED.
• the number of photons in the 400 to 700 nm range emitted from the fiber (called photosynthetically active radiation (PAR)) was quantified using a Sky light meter with a SKL 215 PAR 'Quantum' sensor (Skye Instruments, Llandrindod Wells, United Kingdom). While about 5 pmolm-2s 1 was measured in the upper part closer to the lid, at the bottom about 1 pmolm-2s _1 was measured.
• Figure 20 shows the time-resolved formation of a spatial pH gradient in a solid water/agarose/sodium chloride mixture in a bioreactor exploiting electrolysis of water at two electrodes.
• Electrodes made of V4A stainless steel tubes (outer diameter: 8 mm, inner diameter: 6 mm) are installed in the bioreactor 8 cm apart from each other. A constant voltage of 3.0 V is applied across the electrodes and the current is limited to 0.1 A. After (B) 65 minutes and (D) 399 minutes of applied voltage the pH was measured at various spots.
• the test setup as illustrated in Fig. 20 was built to demonstrate the feasibility to use electricity to create a spatial pH gradient in the bioreactor.
• a bioreactor was equipped with two electrodes.
• the aqueous 15 gL-1 agarose solution was used to mimick the biofilm as both have a high content of water and are highly viscous.
• 1 gL 1 sodium chloride was added to deionized water. Such a salt concentration is typical for standard cultivation media.
• the pH indicator bromothymol blue was added to visualize pH changes (Fig. 20).
• the pH was set to either pH 4.0 (Fig. 20A) or pH 9.0 (Fig. 20C).
• the next test experiment targeted the formation of a pH gradient locally in a biofilm-mimicking agarose gel (15 gL 1 agarose) which covered the cathode (see Fig. 21 ).
• the test vessel was filled with bromothymol blue stained water with 1 gL 1 sodium chloride (without agarose) and the pH was set to 4.1 .
• Two electrodes were located in the fermenter, but only the cathode was covered with the biofilm- mimicking mixture. The uncovered anode was located 8 cm apart from the cathode in the bioreactor.
• a magnetic stirrer was used to continuously stir at 300 rpm.
• the next step is to enable online control of the pH in the biofilm by embedding microsensors for pH measurements in the biofilm and to process the signal to regulate the voltage across the electrodes.
• both electrodes can be covered with a biofilm to provide two niches with extreme pH values at the electrodes and an intermediate pH value in the stirred liquid between the electrodes.
• Figure 21 shows the time-resolved formation of a pH gradient in a biofilm-mimicking agarose coating on the cathode (in a bioreactor equipped with two electrodes). The cathode was covered with an agar layer made of bromothymol blue stained water/agarose/sodium chloride mixture, the anode was uncovered.
• Both electrodes are located in a water/sodium choride solution stained with bromothymol blue with an initial pH of 4.1 .
• the electrodes are made of V4A stainless steel tubes (outer diameter: 8 mm, inner diameter: 6 mm) which are installed 8 cm apart from each other. A constant voltage of 3.0 V is applied across the electrodes and the current is limited to 0.1 A.
• the reactor was stirred at 300 rpm using a magnetic stirrer.
• the pH was set to either pH 4.0 or pH 9.0.
• MRS medium contained (gL-1 ) peptone from casein: 10, meat extract: 10, yeast extract: 5, glucose: 20, Tween 80: 1 , K2HP04: 2, sodium acetate: 5, ammonium citrate: 2 and MgS04 7 H20: 0.2 whereby the pH value was adjusted to 6.2-Q.5 with hydrochloric acid.
• C. tyrobutyricum precultures were cultivated in reinforced clostridial medium (RCM).
• C. tyrobutyricum precultures were cultivated in reinforced clostridial medium (RCM).
• the composition of the RCM medium in gL-1 ): Yeast extract: 13, peptone: 10, glucose: 5, soluble starch: 1 , sodium chloride: 5, sodium acetate: 3, L-Cystein-HCI: 0.5, agar: 0.5. V.
• criceti precultures were cultivated in: Trypticase (BBL) 5 gL-1 , yeast extract 3.0 gL-1 , N a- (DL)- lactate 7.5 gL-1 , Na-thioglycolate 0.75 gL-1 , Tween 80 1 .0 gL-1 , Glucose 1 .0 gL-1 , Na-resazurin solution (0.1 % (w/v)) 0.5 mL.
• the medium for C. tyrobutyricum and V. criceti was boiled under continuous flushing with nitrogen, the pH value was adjusted to 6.8, aliquoted to 100 ml_ serum bottles (50 ml_ in each) and was autoclaved at 121 °C for 20 minutes.
• A. woodii precultures were cultured in DSMZ-medium 135.
• the medium was made using individual components.
• NH 4 C1 1 .0 g, KH 2 P0 0.33 g, K 2 HP0 0.45 g, MgS0 x 7 H 2 0 0.1 g, Aceto bacterium 363 trace element solution (DSMZ-medium 141 ) 20 ml_, yeast extract 2 g, Na- resazurin solution (0.1 % (w/v)) 0.5 ml_ and water 1 liter were boiled and cooled to room temperature under 80 % N 2 and 20 % C0 2 gas mixture.
• Solid sodium bicarbonate (10 g) was added and the pH value was equilibrated by aeration with the gas mixture until a pH of 7.4.
• the medium was aliquoted into serum bottles (60 ml_ in each) under anaerobic conditions and sterilized at 121 ' ⁇ for 20 minutes in the autoclave.
• the composition of the Acetobacterium trace element solution nitrilotriacetic acid 1 .5 gl_ 1 , MgS0 x 7 H 2 0 3.0 gl_ 1 , MnS0 x H 2 0 0.5 gl_ 1 , NaCI 1 .0 gl_ 1 , FeS0 4 x 7 H 2 0 0.1 gl_ 1 , CoS0 4 x 7 H 2 0 0.18 gl_ 1 , CaCI 2 x 2 H 2 0 0.1 gl_ 1 , ZnS0 4 x 7 H 2 0 0.18 gl_ 1 , CuS0 4 x 5 H 2 0 0.01 gl_ 1 , KAI(S0 4 ) 2 x 12 H 2 0 0.02 gl_ 1 , H 3 B0 3 0.01 gl_ 1 , Na 2 Mo0 x 2 H 2 0 0.01 gl_ 1 , NiCI 2 x 6 H 2 0 0.
• Nitrilotriacetic acid was dissolved in distilled water, the pH value adjusted to 6.5 with KOH, the minerals added and the final pH adjusted to 7.0 with KOH.
• the composition of the vitamin solution was as follows: Biotin 2.0 mgL ⁇ 1 , Folic acid 2.0 mgL ⁇ 1 , Pyridoxine-HCI 10.0 mgL ⁇ 1 , Thiamine-HCI x 2 H 2 0 5.0 mgL ⁇ 1 , Riboflavin 5.0 mgL ⁇ 1 , Nicotinic acid 5.0 mgL ⁇ 1 , D-Ca-pantothenate 5.0 mgL ⁇ 1 , Vitamin B I2 0.1 mgL ⁇ 1 , p-383 Aminobenzoic acid 5.0 mgL ⁇ 1 , Lipoic acid 5.0 mgL ⁇ 1 .
• T. reesei Rut-C30 (ATCC 56765) was purchased from the VTT, Finnland. Spores from a seven-day old potato dextrose agar slant culture were incubated at 28 °C and stored at 4 q C.
• Mandels medium containing 7.5 gL-1 microcrystalline cellulose (Avicel PH- 101 , Sigma Aldrich, Buchs, Switzerland) was inoculated with spores from the agar slant described above in an Erlenmeyer flask for four days at 28 °C and 150 rpm.
• Mandels medium contained the following ingredients (gL-1 ) KH 2 P0 : 2, (NH 4 ) 2 S0 : 1 .4, MgS0 7 H 2 0: 0.3, CaCI 2 6H 2 0: 0.4, urea: 0.3, peptone: 0.75, yeast extract: 0.25, and 1 mLL-1 trace element stock.
• the trace element stock contained (gL-1 ) FeS0 4 7 H 2 0: 5, MnS0 4 H 2 0: 1 .6, ZnS0 7 H 2 0: 1 .4, CoCI 2 6H 2 0: 3.7, and 10 mLL-1 concentrated hydrochloric acid and was sterile filtered. To avoid precipitation, 100x CaCI 2 and MgS0 solutions were autoclaved separately before merging with the remaining ingredients.
• TAP medium contained per liter: TRIS, 2.42 g, TAP salt stock, 25 mL; phosphate solution, 0.375 mL, six Kropat’s trace element stock solutions, 1 mL each and glacial acetic acid, 1 mL.
• the medium was autoclaved for 20 minutes at 121 C.
• TAP salt stock solution contained (in gL _1 ): NH 4 CI, 15.0; MgS0 7 H 2 0, 4.0; CaCI 2 2 H 2 0, 2.0.
• the phosphate solution contained 28.8 g K 2 HP0 and 14.4 g KH 2 P0 and water was added to a volume of 100 mL.
• Kropat’s trace element stock solutions contained: 1 ) 25 mM EDTA-Na 2 ; 2) 28.5 mM (NH 4 ) 6 M0 7 0 24 ; 3) 2 mM CuCI 2 2 H 2 0 and 2 mM EDTA; 4) 2.5 mM ZnS0 4 7 H 2 0 and 2.75 mM EDTA; 5) 6 mM MnCI 2 4 H 2 0 and 6 mM EDTA; 6) 20 mM FeCL 6 H 2 0, 22 mM EDTA and 22 mM Na 2 CC>3.
• the membrane bioreactors are based on Multifors 2 and Labfors 5 bioreactors (Infors HT, Bottmingen/Basel, Switzerland) with 0.5 and 2.7 liter working volume, respectively. Both reactors were modified with a tubular polydimethylsiloxane (PDMS) membrane.
• PDMS polydimethylsiloxane
• the membrane area to volume ratio was 0.3 cm2ml_-1 at 0.5 liter scale.
• Mono-Lumen Tubing, 0.64x1 .19x0.28 (Dow Corning, USA) enabled a kLa value of 0.34 h-1 in the Multifors reactors.
• the membrane area to volume ratio was 0.3 cm2mL-1 in the Labfors.
• the membrane was continuously flushed with air with 140 mLmin- 1 per liter liquid phase.
• the temperature was set to 30 °C and the pH value to 6.0 using 4 N phosphoric acid and 4 M sodium hydroxide.
• Liquid samples were collected using a UV lamp as contamination barrier. Neither was an external cellulolytic enzyme mixture added nor was nitrogen applied at any time.
• the reactor was autoclaved for 20 minutes at 121 °C with microcrystalline cellulose or pretreated beech wood. The remaining medium components were added when the reactor was inoculated with 5 % (v/v) T. reesei.
• the lactic acid bacteria were inoculated to an optical density (OD 60 o) of 0.5 whereby the cells were centrifuged at 3000 rpm for 10 minutes and suspended in fresh Mandels medium. This time was set as time zero. Additional 48 hours after inoculation of the LAB the C. tyrobutyricum or V. criceti was inoculated to 5 % (v/v) from a two-day old pre-culture. For fed-batch experiments the feed solution was sterile filtrated and fed with a constant feeding rate.
• Beech wood chips Fagua sylvatica from a local forest were air-dried (dry matter 94 %) and milled to ⁇ 1 .5 mm.
• the raw biomass composed of (% (w/w)) glucan 39.95 ⁇ 1 .25, xylan 19.0 ⁇ 0.5, acetic acid 7.3 ⁇ 0.2, acid-insoluble lignin 25.6 ⁇ 0.7, acid-soluble lignin 5.3 ⁇ 0.1 , ash 0.5 and extractives 0.9.
• a two-stage steam pretreatment was applied with a custom-built steam gun (Industrieanlagen Treatsgesellschaft m.b.H., Austria) to target the partial recovery of soluble xylooligosaccharides from hemicellulose in the prehydrolyzate and simultaneously maximize the glucose yield in enzymatic hydrolysis of the solids.
• Beech wood was heated to 180‘O by the injection of saturated steam and pretreated for a residence time of 24.8 minutes (severity of 3.75).
• the liquid phase was removed through a circular nozzle located slightly above the lower ball valve. Subsequently the pressure was slowly released to 2.0 bar which is below the known pressure to see an effect of the explosion36.
• the lower ball valve opened and the solids were removed from the reactor.
• the solids composed of (% (w/w)) 49.0 ⁇ 0.62 glucan, 7.55 ⁇ 0.1 xylan, 4.2 ⁇ 0.1 acetic acid, 29.9 ⁇ 0.73 acid-insoluble lignin and 3.68 ⁇ 0.37 acid-soluble lignin.
• the recovered solids were treated at 230 ' ⁇ for 14.1 minutes and the pressure was abruptly released.
• the solids contained (in % (w/w)) 55.0 ⁇ 1 .4 glucan, 2.1 ⁇ 0.1 xylan, 1 2 ⁇ 0.2 acetic acid, 35.4 ⁇ 0.8 acid436 insoluble lignin and 2.5 ⁇ 0.1 acid-insoluble.
• the 180 °C liquid fraction contained (in gL-1 ) acetic acid: 2.13, formic acid: 0.4, xylose: 1 .1 , xylooligosaccharides calculated as xylan: 22.35.
• acetic acid 2.13, formic acid: 0.4, xylose: 1 .1 , xylooligosaccharides calculated as xylan: 22.35.
• additional 5.57 gL-1 acetic acid was released from deacetylation of xylooligomers.
• the Severity was calculated according to Overend et al.
• Beech wood was first pretreated at 180 ' ⁇ for 25 minutes. Acetic acid bound as acetyl side chain in hardwood to the xylopuranose backbone was released during this treatment when steam condensation formed a pre-hydrolysate. The formation of free acetic acid lowered the pH value and auto catalyzed the acid-catalyzed release of acetic acid. The pre-hydrolysate was extracted under pressure which contained, based on the raw material, 46 % xylan recovered as xylooligomers, 5 % xylan degraded to formic and furfural, 41 % acetic acid and solubilized phenolic compounds from lignin.
• the remaining wet solids were pretreated again under harsher conditions (230 °C, 14.1 min) which allowed the maximum release of glucose by subsequent enzymatic hydrolysis.
• the inhibitory filtrate was separated from the wet solids which contained 88 % glucan, 9 % xylan and 15 % acetic acid and 78 % lignin with respect to the raw material.
• 3 % (w/w) of pretreated solids was added all in once and the corresponding prehydrolyzate was fed with a constant feeding rate to enable the in situ fungal detoxification.
• L. brevis was added, 8.0 gL-1 butyric acid accumulated (0.32 gg-1 , 91 .9 % selectivity) which corresponds for butyric acid to 165.4 kg/t and total VFAs to 181 .0 kg/t.
• a consortium composed of T. reesei, L pentosus and V. criceti accumulated 6.7 gL-1 acetic acid and 5.6 gL-1 propionic acid (0.49 gg-1 ). This corresponds to 139.6 kg acetic acid per ton raw biomass and 1 18.9 kg propionic acid per ton raw biomass.
• Acetic acid, lactic acid, propionic acid, butyric acid, formic acid, glucose, cellobiose, xylose, hydroxymethylfurfural and furfural were quantified by high performance liquid chromatography (Waters 2695 Separation Module, Waters Corporation, Milford, MA, USA) using an Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA) at 65 °C with 5 mM H2SO4 as the mobile phase a flow at 0.6 mLmin-1 , a refractive index detector (Waters 410) at 40 °C and a photo diode array detector (Waters 2998) at 210 nm. The detection limit was 0.05 gL-1 . Structural carbohydrates and lignin were measured according to standardized methods.
• Chlorophyll a, chlorophyll b, total chlorophyll and total carotenoids in biofilms of T. reesei and C. reinhardtii were quantified by measuring the optical density at 480, 647 and 664.5 nm with a spectrophotometer (Thermo Spectronic UV-1 (Thermo Fisher Scientific, Waltham, USA)). Prior to the measurement the pigments were extracted using dimethylformamide (DMF). The preparation of the samples is as follows. Samples with a specific volume or mass were centrifuged in a 1 .5 ml_ Eppendorf vial, 1 ml_ DMF was added to the pellet and the vials were agitated using a vortex at room temperature for 15 minutes.
• DMF dimethylformamide
• the vials were centrifuged at 10.000 rpm on a benchtop centrifuge. Ideally, the pellet should be white or colorless and the DMF light green. When the pellet was still green another vortex step for 5 to 10 minutes with recentrifugation was performed. The supernatant was transferred to another vial to measure the optical density. If the measured optical density was not in the linear absorbance range of the spectrophotometer a dilution with DMF was performed. The measurements were performed in DMF resistant cuvettes. The chlorophyll a, chlorophyll b and total chlorophyll contents were calculated using the equations from Inskeep and Bloom and the amount of carotenoids using the equation from Wellburn.
• a confocal laser scanning microscope from Leica SP8 (Leica Microsystems, Germany) was used to analyze stained biofilm samples.
• the setup was equipped with a white light laser set to 30 % laser power to excite the sample.
• the scan speed was 400 Hz and the objective HC PL APO CS2 40x/1 .30 oil with a magnification of 40 was installed.
• Autofluorescence from photosynthetic pigments was detected in the red channel (excitation at 650 nm and emission at 680 to 700 nm).
• Fluorescein diacetate was used as viability stain. Fluorescein was detected in the green channel (excitation 495 nm and emission at 514 to 521 nm).
• the fluorescence projections of the biofilm were generated using the Leica LAS software.

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