Field

The invention relates to a process for producing a valuable compound through anaerobic fermentation, to a set-up for running an anaerobic fermentation process suitable for producing a valuable compound, and to the use of a lactic acid sensitive biosensor in a set up for running an anaerobic fermentation process suitable for producing a valuable compound to reduce the dosage of antimicrobial compound.

Background

By functionally replacing fossil-fuel derived compounds, microbial production of chemicals and transport fuels can contribute to a transition to a sustainable, low-carbon global economy. Ethanol and biogas are important biofuels made from biomass. The total industrial production of fuel ethanol, which reached ca. 100 billion liters in 2015, is predicted to increase further. The yeast Saccharomyces cerevisiae is the established microbial cell factory for conversion of starch and sucrose derived hexose units to ethanol, as it combines a high ethanol yield and productivity with robustness under process conditions. Efforts in yeast strain improvement and process optimization of corn-starch and cane sugar based bioethanol production have further improved product yields and productivity. Furthermore, intensive metabolic and evolutionary engineering studies have yielded yeast strains capable of efficiently fermenting the pentose sugars xylose and arabinose, thus paving the way for yeast based ‘second-generation’ bioethanol production from lignocellulosic hydrolysates.

The production of biogas via the anaerobic digestion of organic material is a rapidly growing source of renewable energy. The process is complex; a combined action of several biotechnological processes determines the stability, efficiency and yield of the biogas produced. An optimal process design is still under active research done at laboratory and pilot plants. Substrates like grass, manure or sludge can be used as feed for the biogas production due to their high yield potential

Bacterial contamination represents a persistent problem in the production of both biogas and ethanol. It reduces the efficiency of the fermentation, increased fermenation time and increased cost biofuel. Gram-positive bacteria, particularly lactic acid bacteria (l_AB) are the most common bacterial contaminants found in ethanol production. See“The Alcohol Textbook” - a reference for the beverage, fuel and industrial alcohol industries”, 4 ^th edition, edited by KA Jaques, TP Lyons and DR Kelsall, 2003, Nottingham University Press, more particularly“Bacterial contamination and control in ethanol production” by N.V. Narendranath, pages 287-298.

A common solution to combat bacterial contamination is the use of virginiamycin, which is a streptogramin antibiotic similar to pristinamycin and quinupristin/dalfopristin. The ethanol industry is moving away from use of antibiotics as it increases cost. Furthermore, it may end up in the DDGS. The extent of microbial contamination depends on the hygiene of the ethanol plant and the organic material. When plants are run in a hygienic fashion, antibiotics may not be necessary at all, or to a lesser extent, but because antibiotics are typically added at the start of the fermentation, it is likely that - on average - too much antibiotics are added.

Ideally, one would dose antibiotic only when needed, i.e. when it is apparent that there is bacterial growth. However, demonstrating such bacterial growth by e.g. using plate count would take a considerable amount of time (for colonies to grow), during which time bacterial growth may already have reached a stage at which it is too late to add antibiotics. Because of this, antibiotics are typically added prophylactically, i.e. at the start of the fermentation. Therefore, there is a need to reduce the use of antibiotic in biofuel production.

Summary of the invention

The invention provides a process for producing a valuable compound through anaerobic fermentation of a composition comprising an organic material, said process comprising:

(a) providing a fermentation reactor having an inlet and outlet, and at least one feed inlet for feeding an antimicrobial compound into the reactor,

(b) providing a lactic acid sensitive sensor which is capable of making contact with the composition;

(c) fermenting, in said fermentation reactor, said composition in the presence of a yeast which is capable to convert a C6 sugar and/or a C5 sugar; and optionally

(d) recovery of the valuable compound,

whereby an antimicrobial compound is fed to the fermentation reactor when the concentration of lactic acid in the composition medium exceeds a pre-determined value.

The use of a lactic acid sensitive sensor to control the feeding of antimicrobial compound enables real-time measurement of lactic acid, and on demand feeding of antimicrobial compound. This results in an overall reduction of antibiotics because lower amounts can be dosed. Furthermore, they are added only when necessary (i.e. in case of bacterial contamination) and prevents feeding antibiotics in cases where it would not be necessary. The feeding of the antimicrobial compound can be done manually, or it can be controlled by a means for controlling the feed of the antimicrobial compound, which means can be a programmable means which is operatively connected to the lactic acid sensitive sensor. An example of a suitable sensor is a biosensor, for example based on an enzyme such as lactate oxidase, which enzyme is preferably immobilized. A particularly suitable sensor includes a voltammetric sensor.

Detailed description

In one aspect the invention provides a process for producing a valuable compound through anaerobic fermentation of a composition comprising an organic material, said process comprising:

(a) providing a fermentation reactor having an inlet and outlet, and at least one feed inlet for feeding an antimicrobial compound into the reactor,

(b) providing a lactic acid sensitive sensor which is capable of making contact with the composition;

(c) fermenting, in said fermentation reactor, said composition in the presence of a yeast which is capable to convert a C6 sugar and/or a C5 sugar; and optionally (d) recovery of the valuable compound,

whereby an antimicrobial compound is fed to the fermentation reactor when the concentration of lactic acid in the composition medium exceeds a pre-determined value.

The inventor has realized that measuring the lactic acid concentration in real time, by using a lactic acid sensitive sensor which is in direct contact with the fermentation medium, the dosing of antibiotics can be reduced to only those instances where there is bacterial growth. This “on demand” addition of antimicrobial compound prevents feeding antibiotics in cases where it would not be necessary. In the art, even when an antimicrobial compound is added prophylactically, i.e. at the start of the fermentation, lactic acid is always formed, and typically increases over time. A possible cause for this is that the antimicrobial compound is not stable at the fermentation temperature and degrades, thereby allowing bacteria to grow. In contrast, according to the process of the invention the antimicrobial compound can be dosed at a lower concentration because it is dosed when contamination levels are still low. In the early stages of contamination, when the bacteria have not yet grown out fully and when the lactic acid concentration is therefore low, a low dosage antimicrobial compound is sufficient to control the outgrow of the bacteria.

Throughout the present specification and the accompanying claims, the words "comprise" and "include" and variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

The articles“a” and“an” are used herein to refer to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By way of example,“a fermentation reactor” may mean one fermentation reactor or more than one fermentation reactors.

The fermentation reactor may be an industrial type and scale fermentor. It may also be a shakeflask, bottle, or any other container, as long as it has an inlet and an outlet and at least one feed inlet for feeding an antimicrobial compound into the reactor, and can be used for anaerobic fermentation. The inlet and outlet may be the same. For example, if the fermentation is carried out in a bottle, the opening of the bottle serves both as inlet and outlet.

The lactic acid sensitive sensor (henceforward referred to as“sensor”) is able to detect at least L-lactic acid. When the sensor is in contact with the composition comprising an organic material (also referred to as“fermentation medium”) it sensor may give a reading e.g. in the form of a value which is displayed on a screen. The sensitivity of the sensor is such that it is preferably able to detect concentrations of at least 0.01 g/L lactic acid.

In one embodiment the sensor is able to distinguish between the presence or absence of lactic acid, whereby the sensor preferably has a threshold sensitivity which is below the pre-determined value. Such sensor may give a“yes/no” reading (“no” meaning no lactic acid;“yes” meaning the presence of lactic acid).

In another embodiment the sensor is able to provide the concentration of lactic acid. This concentration may be displayed on a screen or monitor.

In the context of the invention“anaerobic fermentation” is defined as a fermentation which is run in the absence of oxygen or in which substantially no oxygen is consumed, preferably less than about 5, about 2.5 or about 1 mmol/L/h, more preferably 0 mmol/L/h is consumed (i.e. oxygen consumption is not detectable), and includes micro-anaerobic (or micro-aerobic) fermentation.

In an embodiment the feeding of the antimicrobial compound is done manually. For example, a person may monitor the reading of the sensor and, when the reading exceeds a predetermined value, add an antimicrobial compound via the feed inlet for feeding an antimicrobial compound into the reactor, for instance by using a scoop or by emptying a container.

Alternatively, the feeding of the antimicrobial compound can be controlled by a means for controlling the feed of the antimicrobial compound. Such means can include a pump or screw which is able to feed antimicrobial compound via the feed inlet for feeding an antimicrobial compound into the reactor.

In an embodiment, said means for controlling the feed of the antimicrobial compound is programmable and operatively connected to the sensor. In this embodiment, feeding of the antimicrobial compound is based on a reading of the sensor to which it is operatively connected. The means is operated such that when the concentration of lactic acid exceeds a pre-determined value, it triggers a response which results in feeding of antimicrobial compound. This enables the use of in-line, on demand feeding of antimicrobial compound.

In the context of the invention“operatively connected” means the sensor and the means for controlling the feed of the antimicrobial compound are connected in a way to perform the designated function, in casu feeding of antimicrobial compound when the concentration of lactic acid in the fermentation medium exceeds a pre-determined value.

In an embodiment the sensor is a biosensor which is preferably based on an enzyme such as lactate oxidase, which enzyme is preferably immobilized.

In another embodiment the sensor is a voltammetric sensor. Voltammetric (or amperomeric) sensors are known in the art, see e.g. Kavita Rathee et at, Biosensors based on electrochemical lactate detection: A comprehensive review (2016), Biochemistry and Biophysics Reports, 5, 35-54. A preferred electrochemical biosensor is based on the enzyme lactic acid oxidase (LOX or LOD) or lactic acid dehydrogenase (LDH or LD), most preferably lactic acid oxidase. Rhathee et al extensively describe the principles of amperometric detection of lactic acid and how to immobilise the enzyme. Voltammetric sensors have the advantage that they are able to provide the concentration of lactic acid. This give the operator of a fermentation plant more freedom to select the value at which antimicrobial compound is to be fed to the reactor. Voltammetric sensors also have the advantage that the speed of formation of lactic acid can be monitored, i.e. the kinetics of lactic acid formation. Therefore, the invention is understood to include an embodiment wherein lactic acid is fed to the fermentation reactor when lactic acid is formed at a rate (g/L/h) which exceeds a pre-determined value.

The pre-determined value (or set-point or threshold value) can be between 0.01 and 20 g/L lactic acid (relative to the volume of fermentation media). Ideally this value is as low as possible as this will give the most sensitive and immediate response to bacterial contamination. The pre-determined value may be between 0.02 and 5 g/L, or between 0.03 and 1 g/L, or between 0.05 and 0.5 g/L, or at or around 0.1 g/L. The skilled person may select a higher value in the event that the composition comprising an organic material already comprises some lactic acid before the fermentation has started.

Suitable antimicrobial compounds include erythromycin, tylosin, virginiamycin, formaldehyde, nisin, penicillin, chlorine dioxide, peracetic acid, hops acids, and mixtures thereof. Lactrol is typically used at concentrations around 1 ppm, and a typical range is 0.5-2.0 ppm. However, sometimes much higher concentrations are used, up to 20 ppm when ethanol plants are struggling with infections.

The skilled person knows how much antimicrobial compound is to be fed when the concentration of lactic acid in the fermentation medium exceeds a pre-determined value. Factors which determine the amount of feeding include the scale of the fermentor, the stage of the fermentation, the type of antimicrobial compound, and the recommended dose of the antimicrobial compound which is used.

In an embodiment the organic material comprises lignocellulosic biomass or hydrolysate thereof, such as a corn stover hydrolysate or a corn fiber hydrolysate.

By a "hydrolysate" is meant a polysaccharide-comprising material (such as corn stover, corn starch, corn fiber, or lignocellulosic material, which polysaccharides have been depolymerized through the addition of water to form mono and oligosaccharide sugars. Hydrolysates may be produced by enzymatic or acid hydrolysis of the polysaccharide-containing material.

Lig nocellulose herein includes hemicellulose and hemicellulose parts of biomass. Lig nocellulose includes lignocellulosic fractions of biomass. Suitable lignocellulosic materials may be found in the following list: orchard primings, chaparral, mill waste, urban wood waste, municipal waste, logging waste, forest thinnings, short-rotation woody crops, industrial waste, wheat straw, oat straw, rice straw, barley straw, rye straw, flax straw, soy hulls, rice hulls, rice straw, corn gluten feed, oat hulls, sugar cane, corn stover, corn stalks, corn cobs, corn husks, switch grass, miscanthus, sweet sorghum, canola stems, soybean stems, prairie grass, gamagrass, foxtail; sugar beet pulp, citrus fruit pulp, seed hulls, cellulosic animal wastes, lawn clippings, cotton, seaweed, trees, softwood, hardwood, poplar, pine, shrubs, grasses, wheat, wheat straw, sugar cane bagasse, corn, corn husks, corn hobs, corn kernel, fiber from kernels, products and by-products from wet or dry milling of grains, municipal solid waste, waste paper, yard waste, herbaceous material, agricultural residues, forestry residues, municipal solid waste, waste paper, pulp, paper mill residues, branches, bushes, canes, corn, corn husks, an energy crop, forest, a fruit, a flower, a grain, a grass, a herbaceous crop, a leaf, bark, a needle, a log, a root, a sapling, a shrub, switch grass, a tree, a vegetable, fruit peel, a vine, sugar beet pulp, wheat midlings, oat hulls, hard or soft wood, organic waste material generated from an agricultural process, forestry wood waste, or a combination of any two or more thereof. Lignocellulose, which may be considered as a potential renewable feedstock, generally comprises the polysaccharides cellulose (glucans) and hemicelluloses (xylans, heteroxylans and xyloglucans). In addition, some hemicellulose may be present as glucomannans, for example in wood-derived feedstocks. The enzymatic hydrolysis of these polysaccharides to soluble sugars, including both monomers and multimers, for example glucose, cellobiose, xylose, arabinose, galactose, fructose, mannose, rhamnose, ribose, galacturonic acid, glucuronic acid and other hexoses and pentoses occurs under the action of different enzymes acting in concert. In addition, pectins and other pectic substances such as arabinans may make up considerably proportion of the dry mass of typically cell walls from non-woody plant tissues (about a quarter to half of dry mass may be pectins). Lignocellulosic material may be pre-treated.

Pretreatment may comprise exposing the lignocellulosic material to an acid, a base, a solvent, heat, a peroxide, ozone, mechanical shredding, grinding, milling or rapid depressurization, or a combination of any two or more thereof. This chemical pretreatment is often combined with heat- pretreatment, e.g. between 150-220°C for 1 to 30 minutes.

In another embodiment the organic material comprises starch or hydrolysate thereof, such as a corn starch hydrolysate.

Such pre-treated material is commonly subjected to enzymatic hydrolysis to release sugars that may be fermented according to the invention. This may be executed with conventional methods, e.g. contacting with cellulases, for instance cellobiohydrolase(s), endoglucanase(s), beta-glucosidase(s) and optionally other enzymes. The conversion with the cellulases may be executed at ambient temperatures or at higher temperatures, at a reaction time to release sufficient amounts of sugar(s). The result of the enzymatic hydrolysis is hydrolysis product comprising C5/C6 sugars, herein designated as the sugar composition.

The composition comprising organic material may also comprise sludge or biomass from purification, fermentation or digestion processes, or manure.

The fermentation in step (c) is preferably run at a temperature that is optimal for the yeast. Thus, the fermentation process is performed at a temperature which is less than about 50°C, less than about 42°C, or less than about 38°C, preferably at a temperature which is lower than about 35°C, about 33°C, about 30 or about 28°C and at a temperature which is higher than about 20°C, about 22°C, or about 25°C.

The valuable compound may be ethanol or biogas, preferably ethanol.

The process optionally comprises recovering the valuable product. For the recovery existing technologies can be are used. Existing methods of recovering ethanol from aqueous mixtures commonly use fractionation and adsorption techniques. For example, a beer still can be used to process a fermented product, which contains ethanol in an aqueous mixture, to produce an enriched ethanol- containing mixture that is then subjected to fractionation (e.g., fractional distillation or other like techniques). Next, the fractions containing the highest concentrations of ethanol can be passed through an adsorber to remove most, if not all, of the remaining water from the ethanol. In an embodiment in addition to the recovery of fermentation product, the yeast may be recycled.

Recovery of biogas is known in that art. It may simply be collected from the headspace of the reactor.

The yeast is capable to convert a C6 sugar and/or a C5 sugar. Examples of C5 sugars (also referred to as“pentose” or“pentose sugar”) are xylose, ribose, and arabinose, or derivatives thereof. Examples of C6 sugars (also referred to as“hexose” or“hexose sugar” are glucose, galactose, and mannose, preferably glucose and galactose, more preferably glucose.

In an embodiment the yeast is selected from the list consisting of Saccharomyces, Kluyveromyces, Candida, Scheffersomyces, Pichia, Schizosaccharomyces, Hansenula, Ogataea, Kloeckera, Schwanniomyces, Issatchenkia (such as I. orientalis) and Yarrowia, preferably the yeast is Saccharomyces cerevisiae. A preferred yeast is a Saccharomyces, preferably S. cerevisiae. The invention further provides a set up for running an anaerobic fermentation process suitable for producing a valuable compound comprising:

a fermentation reactor having an inlet and outlet and at least one feed inlet for feeding an antimicrobial compound into the reactor;

a programmable means for controlling the feed of said antimicrobial compound into the reactor; and

a lactic acid sensitive sensor which is capable of making contact with a fermentation medium when in progress;

whereby the lactic acid sensitive sensor is operatively connected to the programmable means for feeding the antimicrobial compound into the reactor.

This set up is useful in the process of the invention. All embodiments relating to the elements of the process of the invention such as the sensor, the fermentation reactor, the antimicrobial compound, the means for controlling the feed of said antimicrobial compound into the reactor, how the means and the sensor may be linked, and the valuable compound as described above in the process of the invention equally apply to the set-up of the invention. A“set up” may be an ethanol or biogas fermentation plant, or part thereof. It may consist of a single fermentation reactor, or two or more reactors, which may be operated in series or parallel.

The invention further provides the use of a lactic acid sensitive sensor to control the dosage of an antimicrobial compound in a set up for running an anaerobic fermentation process suitable for producing a valuable compound. All embodiments relating to the elements of the process of the invention such as the sensor, the fermentation reactor, the antimicrobial compound, the means for controlling the feed of said antimicrobial compound into the reactor, how the means and the sensor may be linked, and the valuable compound as described above in the process of the invention equally apply to the use of the invention.

EXAMPLES

Example 1

A series of shake flasks is prepared with corn starch hydrolysate as fermentation medium. To shake flasks B to G lactrol is added according to Table 1 .

To shake flask A, 1 and 2 no initial lactrol is added. Shake flasks 1 and 2 are connected to a three-electrode amperometric lactate biosensor as used in, and described by Imani et at,“A wearable chemical-electrophysiological hybrid biosensing system for real-time health and fitness monitoring”, Nature Communications (2016) DOI: 10.1038/ncomms1 1650. The sensor is placed such that it is in contact with the fermentation medium and can be read by visual inspection. The sensor is operatively connected to a programmable pump system which is able to dose lactrol to the shake flask. The pump connected to shake flask 1 is programmed such that lactrol is dosed to the shake flask when the lactic acid concentration exceeds 0.1 g/L lactic acid. The pump connected to shake flask 2 is programmed such that lactrol will be dosed to the shake flask when the lactic acid concentration exceeds 0.01 g/L lactic acid. If the lactic acid concentration exceeds 0.1 g/L (for shake flask 1 ) or 0.01 g/L (for shake flask 2) the pump starts feeding lactrol in an amount corresponding to 0.1 ppm. When this amount of lactrol has been fed, the pump shuts off until the lactic acid concentration again exceeds 0.1 g/L or 0.01 g/L.

The shake flasks are inoculated with Ethanol Red, a commercially available ethanol yeast and incubated at 32°C for 48 hours. After 48 hours of fermentation, the amount of lactic acid and ethanol, and the bacterial plate count are determined by methods known in the art. Results are in Table 1.

It can be seen that using the lactic acid biosensor, the total amount of added lactrol is lower, whereas the plate count and amount of lactic acid are lower.

Example 2

An effective amount of alpha-amylase is added to 800 gram water and mixed well. Next, 200 gram corn flour is added to the aqueous mixture. The pH is adjusted the pH 5.5 with 2N H2SO4 or 4M KOH. The obtained mixture is transferred into a 2-litre bottle and capped. Thereafter, the bottle is incubated at 80°C in an oven or water bath for 4 hours under constant mixing. Then, the bottle is cooled to room temperature.

Next, 300 gram of the obtained corn starch hydrolysate is added to a series of shake flasks A to E. 0.4 gram urea is added to each shake flask and mixed well. The pH is adjusted to 4.5 with 2N H2SO4.

Then, an effective amount of glucoamylase and 150 mg Ethanol Red (a commercially available ethanol yeast) is added and the shake flasks are incubated at 32°C for 6-8 hours.

At the start of the fermentation, to shake flasks A to E, 1 ml of a lactobacillus culture containing 1 ⁸ - 1 ⁹ colonies is added for controlled formation of lactic acid. To shake flask A no antibiotics are added. To shake flask B, 100 mg neomycin and 100 mg penicillium G is initially added. To shake flask C, 50 mg neomycin and 50 mg penicillium G is initially added and an additional 50 mg neomycin and 50 mg Penicillium G is added after increase in lactic acid (when lactic acid increase exceeds 0.05 g/l) measured with a three-electrode amperometric lactate biosensor. To shake flask D, 25 mg neomycin and 25 mg penicillium G is initially added and an additional 25 mg neomycin and 25 mg penicillium G is added after increase in lactic acid (when lactic acid increase exceeds 0.05 g/l) measured with a three-electrode amperometric lactate biosensor. To shake flask E, 10 mg neomycin and 10 mg penicillium G is initially added and an additional 10 mg neomycin and 10 mg penicillium G is added after increase in lactic acid (when lactic acid increase exceeds 0.05 g/l) measured with a three-electrode amperometric lactate biosensor.

After fermentation, the amount of lactic acid and the bacterial plate count are determined by methods known in the art. Results are shown in Table 2.

It can be seen that using the lactic acid biosensor, the total amount of added antibiotics is lower, whereas the plate count and amount of lactic acid are lower compared to when no antibiotics are added or when all antibiotics are added only initially. Table 1 :

* added initially; ** fed via the programmable pump system.

Table 2: