Patent Application
20190048368
The invention relates to a method for homo-ethanol production from lactose using a genetically modified lactic acid bacterium, where cells of the bacterium are provided with a substrate comprising dairy waste supplemented with an amino nitrogen source, such as hydrolysed corn steep liquor as a source of soluble protein, peptides and free amino acids. Additionally the invention provides the genetically modified lactic acid bacterium that is adapted for homo-ethanol production from lactose in dairy waste, such as whey, deproteinized whey permeate or permeate mother liquor, which is the byproduct after lactose extraction from whey permeate, and for its use for homo-ethanol production. The genetically modified lactic acid bacterium comprises both genes (lacABCD, lacEF, lacG) encoding enzymes catalysing a lactose catabolism pathway; and transgenes (pdc and adhB) encoding enzymes catalysing the conversion of pyruvate to ethanol. The lactic acid bacterium is further genetically modified by inactivation or deletion of those genes in its genome that encode polypeptides having lactate dehydrogenase (EC 1.1.1.27/EC 1.1.1.28); phosphotransacetylase (EC 2.3.1.8), and bifunctional alcohol dehydrogenase (EC 1.1.1.1 and EC 1.2.1.10) activity.
Currently there is a growing demand for liquid fuels that can be produced in a sustainable manner from renewable raw materials. The potential that lies in using microorganisms for converting various feedstocks, e.g. plant biomass, into useful compounds including fuels, has already been recognized, and intense research is being carried out to establish robust and economically feasible processes for production of biofuels. Microbially produced ethanol presently dominates the biofuel market, and it is mainly produced from either refined sugar or starch derived sugar. Although much focus has been on developing bio-processes, which are reliant on non-food plant biomass as feedstock, there are many challenges, including the high cost of enzymes needed for degrading the biomass, the recalcitrance of lignocellulose, a lack of microbial catalysts with sufficient robustness to withstand the inhibitors generated in pre-treatment or that have a sufficiently broad spectrum of carbohydrate utilization. As an alternative, one cheap abundant feedstock is cheese whey and its various processed forms, such as whey permeate or whey powder. Whey represents about 85-95% of the milk volume and retains 55% of milk nutrients; and is a liquid byproduct of cheese production, obtained when draining the cheese curd. The worldwide production of cheese whey in 2012 was reported to be 4×107 tons and about 50% hereof was used for animal feed or otherwise disposed of as waste. The latter is a serious problem as whey is discarded as liquid waste and has a high BOD (biochemical oxygen demand) and COD (chemical oxygen demand). The composition of whey varies according to the source of the milk and the technology used for its production. Normally it contains approximately 90% water, 4% lactose, 1% protein, 0.7% minerals and small amounts of vitamins. Separation of whey proteins generates whey-permeate and further extraction of lactose leads to permeate mother liquor (residual whey permeate, RWP), which comprises about 60% lactose on a dry weight basis, as a leftover product (Ling, 2008). Fermentation of the main carbon source in whey (lactose) to ethanol has been studied for the last 30 years and most of the research has been focused on yeasts that naturally metabolize lactose. There are, however, problems associated with yeasts, and these include a generally low robustness, slow fermentation-rate, and substrate-inhibition effects, which is why there is a need for better performing microbial candidates.
Lactococcus lactis, which is well-known for its role in cheese production, has great potential as a cell factory, due to properties such as its high glycolytic flux, ability to metabolize a broad range of carbohydrates, well-characterized metabolic network and ease of genetic manipulation. Its long record of safe use is also an important asset, especially for production of food ingredients. Normally, most of the carbon flux in L. lactis is directed to lactate (homolactic fermentation). However, it can be successfully engineered to produce ethanol by knocking out alternative product pathways and introducing pyruvate decarboxylase and alcohol dehydrogenase heterologously (Solem et al 2013). A potential drawback of using L. lactis as a cell factory is its fastidious nature, i.e. its many nutritional growth requirements, which could perhaps make it less attractive for some industrial applications, e.g. for production of low-priced chemicals where, for competitive reasons, it is important to keep costs at a minimum. Accordingly, there exists a need to develop cheap fermentation media based on nutrient-rich waste substrates that can circumvent problems associated with using L. lactis as a production host organism for ethanol.
According to a first embodiment, the invention provides a method for the production of ethanol, comprising the steps of:
According to a second embodiment, the invention provides for a use of a genetically engineered lactic acid bacterium for the production of ethanol from an aqueous culture medium comprising a lactose rich substrate (such as whey, whey permeate or residual whey permeate), and an amino nitrogen source (such as acid hydrolysed corn steep liquor (CSLH) as defined herein); wherein the genetically engineered lactic acid bacterium comprises transgenes encoding:
According to a third embodiment, the invention provides the genetically modified lactic acid bacterium for production of ethanol for the production of ethanol from an aqueous culture medium comprising residual a lactose rich substrate (such as whey, whey permeate or whey permeate), and an amino nitrogen source (such as acid hydrolysed corn steep liquor (CSLH)); where the bacterium is as defined above.
Amino acid sequence identity: The term “sequence identity” as used herein, indicates a quantitative measure of the degree of homology between two amino acid sequences of substantially equal length. The two sequences to be compared must be aligned to give a best possible fit, by means of the insertion of gaps or alternatively, truncation at the ends of the protein sequences. The sequence identity can be calculated as ((Nref−Ndif)100)/(Nref), wherein Ndif is the total number of non-identical residues in the two sequences when aligned and wherein Nref is the number of residues in one of the sequences. This sequence identity obtained using the BLAST program e.g. the BLASTP program (Pearson W. R and D. J. Lipman (1988)) (www.ncbi.nlm.nih.gov/cgi-bin/BLAST). Sequence alignment is performed with the sequence alignment method ClustalW with default parameters as described by Thompson J., et al 1994, available at http://www2.ebi.ac.uk/clustalw/.
Preferably, the numbers of substitutions, insertions, additions or deletions of one or more amino acid residues in the polypeptide as compared to its comparator polypeptide is limited, i.e. no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additions, and no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 deletions. Preferably the substitutions are conservative amino acid substitutions: limited to exchanges within members of group 1: Glycine, Alanine, Valine, Leucine, Isoleucine; group 2: Serine, Cysteine, Selenocysteine, Threonine, Methionine; group 3: Proline; group 4: Phenylalanine, Tyrosine, Tryptophan; Group 5: Aspartate, Glutamate, Asparagine, Glutamine.
Corn steep liquor: has CAS Number: 66071-94-1 and is a by-product of corn wet-milling industry. It contains proteins, amino acids, vitamins and minerals and contains approx. 50% (w/w) solids.
Deleted gene: the deletion of a gene from the genome of a bacterial cell leads to a loss of function of the gene and hence where the gene encodes a polypeptide the deletion results in a loss of expression of the encoded polypeptide. Where the encoded polypeptide is an enzyme, the gene deletion leads to a loss of detectable enzymatic activity of the respect polypeptide in the bacterial cell.
Functional gene: gene that is capable of expressing an active enzyme encoded by the gene. Loss of a gene or loss of the function of a gene results in an inability to express the active enzyme encoded by the gene. A loss of function may be the result of a failure to transcribe the gene; or may be a failure to translate the transcribed gene into an active enzyme (e.g. due to mutations). When an enzyme loses more than 60% activity, preferably more than 90% activity; it is deemed to be inactive, in the sense that it no longer has a significant influence on product flux in the sugar metabolism pathway.
gi number: (genInfo identifier) is a unique integer which identifies a particular sequence, independent of the database source, which is assigned by NCBI to all sequences processed into Entrez, including nucleotide sequences from DDBJ/EMBL/GenBank, protein sequences from SWISS-PROT, PIR and many others.
Native gene: endogenous gene in a bacterial cell genome (where the genome includes plasmids), where the gene is homologous to the host bacterium.
Transgenes encoding polypeptides having pyruvate decarboxylase (PDC) activity (EC 4.1.1.1) and alcohol dehydrogenase (ADHB) activity (EC 1.1.1.1) confer on a cell the ability to convert pyruvate to ethanol via acetaldehyde.
Whey and whey permeate and residual whey permeate: whey is a byproduct of cheese manufacture; and comprises whey proteins having a high nutritional value and lactose. Removal of whey proteins, typically by means of ultrafiltration or diafiltration produces a whey protein concentrate and whey permeate that is lactose-rich. The lactose content of the whey permeate is dependent on the treatment conditions and typically it can reach as high as hundreds of grams per liter by reverse osmosis, such as 200 g/L. Removal of fat from whey, or from lactose-rich permeate, typically by centrifugation, yields a fat-free composition (whey or permeate). Residual whey permeate (also called permeate mother liquor) is obtained after the extraction of lactose from whey permeate (typically by lactose crystallisation); and has a lower lactose content of about 150 g/L.
Lactococcus lactis is a homo-lactic fermentative lactic acid bacterium, directing about 90% of metabolic flux to lactate when grown on fast fermentable sugars, such as glucose (
In order to limit metabolic flux towards lactate in the lactic acid bacterium of the invention, the activity of the enzymes of the lactate pathway are reduced by inactivating or deleting one or more genes, for example ldh, ldhX and ldhB, encoding enzymes of this pathway. In order to additionally prevent metabolic flux to acetate, the activity of the acetate pathway is reduced by inactivating or deleting the gene encoding phosphotransacetylase (pta), which converts acetylphosphate to acetate. Additionally, the native alcohol dehydrogenase gene (adhE) encoding a bifunctional alcohol dehydrogenase, ADHE (EC 1.1.1.1 and EC 1.2.1.10) may be inactivated or deleted in order to maximize the production of ethanol with a high yield. So long as the native alcohol dehydrogenase (ADHE) is active, another byproduct (acetoin) is formed in order to balance the cofactors (2 NADH is formed per glucose by glycolysis, while 4 NADH is required for the complete reduction to ethanol by ADHE, which means only half of the carbon flux can be diverted to ethanol). Therefore, the removal of ADHE activity is beneficial for high yield ethanol production.
The lactic acid bacterium of the invention, further comprises codon-optimized transgenes (pdc and adhB), sourced from Zymomonas moblis, encoding pyruvate decarboxylase (EC 4.1.1.1) and alcohol dehydrogenase (EC 1.1.1.1) enzymes, respectively. Expression of these encoded PDC and ADHB enzymes in cells of the lactic acid bacterium of the invention, where the lactate and acetate pathways are inactivated or deleted, confers the ability for homo-ethanol production and for growth under anaerobic growth conditions (see Strain CS4435 in Table 3 of Example 1). The above described genetic modifications in the lactic acid bacterium of the invention provide the cells with a metabolic pathway for the use of (extracellular) glucose as substrate for homo-ethanol production.
The lactic acid bacterium of the invention, however, further comprises genes encoding enzymes that facilitate the uptake of extracellular lactose and its entry into the glycolytic pathway; such that lactate can be used as substrate for homo-ethanol production. Preferably, the cells of the lactic acid bacterium comprise the following genes: a gene encoding a lactose specific phosphotransferase system (PTS) e.g., a lacEF gene, whereby phosphorylated lactose is assimilated by the cells; a gene encoding a phospho-β-galactosidase (e.g. a lacG gene) that hydrolyzes lactose phosphate to glucose and galactose-6-phosphate (gal-6-P), where the glucose moiety enters into glycolysis; genes encoding the tagatose-6-P pathway, e.g., the lacAB, lacC, lacD genes encoding galactose-6-phosphate isomerase, D-tagatose-6-phosphate kinase, and tagatose 1,6-diphosphate aldolase, respectively, whereby gal-6-P is degraded and enters the glycolytic pathway as glyceraldehyde-3-phosphate.
Cells of the lactic acid bacterium of the invention are shown to be efficient producers of ethanol from lactose via homo-ethanol fermentation with a growth rate that is close to that on a glucose-containing medium (Example 1).
The characteristics of the individual genes that may be deleted or introduced in order to produce a lactic acid bacterium of the invention are detailed below.
The lactic acid bacterium of the invention is characterised by knockouts of one or more endogenous native gene encoding a polypeptide having lactate dehydrogenase activity causing a block in the lactate synthesis pathway in the bacterium. Deletion of at least one gene (e.g. ldh) encoding a lactate dehydrogenase enzyme (E.C 1.1.1.27 or EC 1.1.1.28) provides a lactic acid bacterium of the invention that is depleted in lactate production. For example, where the lactic acid bacterium of the invention belongs to a given genus, the deleted endogenous gene is one encoding a polypeptide having lactate dehydrogenase activity in that genus. Preferably the polypeptide having lactate dehydrogenase activity (EC 1.1.1.27 or EC 1.1.1.28) has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% amino acid sequence identity to one of the following sequences: SEQ ID NO: 2 in a Lactococcus species (e.g. Lactococcus lactis); SEQ ID NO: 4, 6, or 8 in a Lactobacillus species (e.g. Lactobacillus acidophilus); SEQ ID NO: 10 in a Lactobacillus species (e.g. Lactobacillus delbrueckii); SEQ ID NO. 12, 14 or 16 in a Lactobacillus species (e.g. Lactobacillus casei), SEQ ID NO. 18 or 20 in a Lactobacillus species (e.g. Lactobacillus plantarum); SEQ ID NO: 22 in a Pediococcus species (e.g. Pediococcus pentosaceus), SEQ ID NO: 24 or 26 in a Leuconostoc species (e.g. Leuconostoc mesenteroides), SEQ ID NO: 28 in a Streptococcus species (e.g. Streptococcus thermophilus), SEQ ID NO: 30 or 32 in a Oenococcus species (e.g. Oenococcus oeni), and SEQ ID NO: 34 or 36 in a Bacillus species (e.g. Bacillus coagulans).
In one embodiment, an additional endogenous gene, encoding a polypeptide having lactate dehydrogenase enzymatic activity (E.C 1.1.1.27 or EC1.1.1.28), is deleted from the lactic acid bacterium of the invention. For example, where the lactic acid bacterium of the invention belongs to the genus Lactococcus, the deleted gene (ldhX) encodes a polypeptide having at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% amino acid sequence identity to SEQ ID NO: 38.
In one embodiment, an additional endogenous gene, encoding a polypeptide having lactate dehydrogenase enzymatic activity (EC 1.1.1.27 or EC 1.1.1.28), is deleted from the lactic acid bacterium of the invention. For example, where the lactic acid bacterium of the invention belongs to the genus Lactococcus, the deleted gene (ldhB) encodes a polypeptide having at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% amino acid sequence identity to SEQ ID NO: 40. Further, where the lactic acid bacterium of the invention belongs to the genus Lactococcus, the three genes (ldh, ldhB and ldhX) encoding a polypeptide having at least 70% amino acid sequence identity to SEQ ID NO: 2, 38 and 40 respectively may be deleted.
In one embodiment, the lactic acid bacterium of the invention is characterised by knockout of the endogenous native gene encoding a phosphotransacetylase (EC 2.3.1.8), causing a block in the acetate synthesis pathway in the bacterium. Deletion of a gene (e.g. pta) encoding a phosphotransacetylase enzyme provides a lactic acid bacterium of the invention that is blocked in acetate production. For example, where the lactic acid bacterium of the invention belongs to a given genus, the deleted endogenous gene is one encoding a polypeptide having phosphotransacetylase activity (EC 2.3.1.8) in that genus. Preferably the polypeptide having phosphotransacetylase activity has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% amino acid sequence identity to one of the following sequences: SEQ ID NO: 42 in a Lactococcus species (e.g. Lactococcus lactis); SEQ ID NO: 44, 46, 48, and 50 in a Lactobacillus species (e.g. Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus casei, Lactobacillus plantarum), SEQ ID NO: 52 in a Pediococcus species (e.g. Pediococcus pentosaceus), SEQ ID NO: 54 in a Leuconostoc species (e.g. Leuconostoc mesenteroides), SEQ ID NO: 56 in a Streptococcus species (e.g. Streptococcus thermophilus), SEQ ID NO: 58 Oenococcus species (e.g. Oenococcus oeni), and SEQ ID NO: 60 in a Bacillus species (e.g. Bacillus coagulans).
In one embodiment, the lactic acid bacterium of the invention is characterised by knockout of the endogenous native gene encoding bifunctional alcohol dehydrogenase activity (EC 1.1.1.1 and EC 1.2.1.10) causing a block in the ethanol synthesis pathway in the bacterium. Deletion of the gene encoding an alcohol dehydrogenase enzyme provides a lactic acid bacterium of the invention that is blocked in ethanol production.
For example, where the lactic acid bacterium of the invention belongs to a given genus, the deleted endogenous gene (e.g. adhE) is one encoding a polypeptide having bifunctional alcohol dehydrogenase activity (EC 1.1.1.1 and EC 1.2.1.10) in that genus. Preferably the polypeptide having alcohol dehydrogenase activity has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% amino acid sequence identity to one of the following sequences: SEQ ID NO: 62 in a Lactococcus species (e.g. Lactococcus lactis); SEQ ID NO: 64 in a Lactobacillus species (e.g. Lactobacillus acidophilus); SEQ ID NO: 66 or 68 in a Lactobacillus species (e.g. Lactobacillus casei); SEQ ID NO: 70 in a Lactobacillus species (e.g., Lactobacillus plantarum), SEQ ID NO: 72 in a Leuconostoc species (e.g. Leuconostoc mesenteroides), SEQ ID NO: 74 in a Streptococcus species (e.g. Streptococcus thermophilus), SEQ ID NO: 76 in a Oenococcus species (e.g. Oenococcus oeni), and SEQ ID NO: 78 in a Bacillus species (e.g. Bacillus coagulans).
The bacterium of the invention comprises a transgene encoding a polypeptide having pyruvate decarboxylase (PDC) activity (EC 4.1.1.1) that converts pyruvate to acetaldehyde. The amino acid sequence of the polypeptide has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% sequence identity to the amino acid sequence of the pyruvate decarboxylase (SEQ ID NO: 80) encoded by a codon optimized derivative of the Zymomonas mobilis pdc gene.
The bacterium of the invention comprises a transgene encoding a polypeptide having alcohol dehydrogenase (ADH) activity (EC 1.1.1.1) that converts acetaldehyde to ethanol, but is not able to use acetyl-CoA as substrate (the acetaldehyde being formed by pyruvate decarboxylase activity). The expression of these heterologous enzymes, PDC and ADHB, enables the complete cofactor balance thereby facilitating maximum ethanol production. The amino acid sequence of the polypeptide has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% sequence identity to the amino acid sequence of the pyruvate decarboxylase (SEQ ID NO: 82) encoded by a codon optimized derivative of the Zymomonas mobilis adhB gene.
The lactic acid bacterium of the invention comprises the following native genes or transgenes required for lactose assimilation and catabolism:
1) a first and second gene encoding a first and a second polypeptide component together conferring lactose-specific phosphotransferase system (PTS) activity (EC 2.7.1.69), whereby phosphorylated lactose is assimilated by the cells. The amino acid sequence of the first polypeptide component has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% sequence identity to the amino acid sequence of the phosphotransferase system EIICB component (SEQ ID NO: 84) encoded by the Lactococcus lactis lacE gene; and the amino acid sequence of the second polypeptide component has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% sequence identity to the amino acid sequence of the phosphotransferase system EIIA component (SEQ ID NO: 86) encoded by the Lactococcus lactis lacF gene; and
2) a gene encoding a polypeptide having phospho-β-D-galactosidase activity (EC 3.2.1.85) that hydrolyzes lactose-6-phosphate to glucose and galactose-6-phosphate (gal-6-P), whereby the glucose moiety can then enter the glycolytic pathway. The amino acid sequence of the polypeptide has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% sequence identity to the amino acid sequence of the phospho-β-D-galactosidase (SEQ ID NO: 88) encoded by the Lactococcus lactis lacG gene.
Additionally, the following genes encoding enzymes in the tagatose-6-P pathway, whereby gal-6-P is degraded and enters the glycolytic pathway as glyceraldehyde-3-phosphate, are required:
3) a first and second gene encoding a first and a second polypeptide subunit together conferring galactose-6-phosphate isomerase activity (EC 5.3.1.26). The amino acid sequence of the first polypeptide subunit has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% sequence identity to the amino acid sequence of the first subunit of the galactose-6-phosphate isomerase (SEQ ID NO: 90) encoded by the Lactococcus lactis lacA gene; and the amino acid sequence of the second polypeptide subunit has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% sequence identity to the amino acid sequence of the second subunit of the galactose-6-phosphate isomerase (SEQ ID NO: 92) encoded by the Lactococcus lactis lacB gene; and
4) a gene encoding a polypeptide having D-tagatose-6-phosphate kinase activity (EC 2.7.1.114). The amino acid sequence of the polypeptide has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% sequence identity to the amino acid sequence of the D-tagatose-6-phosphate kinase (SEQ ID NO: 94) encoded by the Lactococcus lactis lacC gene; and
5) a gene encoding a polypeptide having tagatose 1,6-diphosphate aldolase activity (EC 4.1.2.40). The amino acid sequence of the polypeptide has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% sequence identity to the amino acid sequence of the tagatose 1,6-diphosphate aldolase (SEQ ID NO: 96) encoded by the Lactococcus lactis lacD gene; and
6) optionally a gene encoding a polypeptide having lactose transport regulator activity. The amino acid sequence of the polypeptide has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% sequence identity to the amino acid sequence of the lactose transport regulator (SEQ ID NO: 98) encoded by the Lactococcus lactis lacR gene.
Lactococcus lactis is characterised by homo-lactic fermentation when grown on glucose (
The strain CS4435L, derived from strain CS4435, comprises and expresses the entire lactose catabolism pathway, encoded by genes on the Lactococcal plasmid, pLP712 (55.395 kbp) (
The lactic acid bacterium according to the invention, that comprises genes encoding a pathway for homo-ethanol production, as described in section I, is a member of a genus of lactic acid bacteria selected from the group consisting of Lactococcus, Lactobacillus, Pediococcus, Leuconostoc, Streptococcus, Oenococcus, and Bacillus, preferably Lactococcus. The lactic acid bacterium of the invention may for example be a species of lactic acid bacteria selected from the group consisting of Lactococcus lactis, Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus casei, Lactobacillus plantarum, Pediococcus pentosaceus, Leuconostoc mesenteroides, Streptococcus thermophilus, Oenococcus oeni and Bacillus coagulans.
Residual whey permeate (RWP) is the permeate mother liquor after extracting lactose from whey permeate, and is a waste product of the dairy industry. However, its use as a feedstock for ethanol production has the potential to create a sustainable and economical bioprocess for ethanol production. RWP comprises lactose, as a source of energy, as well as the amino acids essential for L. lactis growth, although in relatively low concentrations (Table 5).
A growth medium, based on RWP alone, was found insufficient to support either fermentative growth or ethanol production by the genetically modified lactic acid bacterium of the invention (Example 2). A supplement to the RWP medium to provide a source of complex amino nitrogen was found essential, since fermentative growth and ethanol production were obtained when a supplement of yeast extract (0.5% w/v) was provided, while ammonium salts were insufficient (Example 2).
The use of YE as a supplement to RWP, while effective, does not provide a cost-effective growth medium for producing ethanol using lactic acid bacteria, for a number of reasons, as explained herein. Firstly, due to its high price (currently 7000˜10000 $/ton), it would increase the total ethanol production costs using the genetically modified lactic acid bacterium of the invention on RWP supplemented media by an estimated 30%, and for this reason alone there exists a need to find cheaper alternative sources of complex amino nitrogen.
Corn steep liquor having CAS Number: 66071-94-1 (CSL), which is a byproduct of the corn milling industry, is a cheaper amino nitrogen source that currently costs around 500 $/ton. CSL has a pH of about 4.0 and consists predominantly of naturally occurring nutritive materials such as water-soluble proteins, amino acids (e.g., alanine, arginine, aspartic acid, cysteine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tyrosine, valine), vitamins (e.g., B-complex), carbohydrates, organic acids (e.g., lactic acid), minerals (e.g., Mg, P, K, Ca, S), enzymes and other nutrients. However, even though CSL is a source of complex amino nitrogen, when the genetically modified lactic acid bacterium of the invention was supplied with the RWP medium supplemented with CSL in an amount corresponding to 0.1% to 2.5% (w/v)), the final biomass concentration obtained was still very low and almost no ethanol was produced.
Surprisingly, the hydrolysis of CSL, for example acid hydrolysis, yielded a hydrolysed CSL composition (CSLH) that when used as a supplement to RWP provided an effective low cost medium for ethanol production by fermentation using the genetically modified lactic acid bacterium of the invention (Table 4, in Example 2). The need to use hydrolysed CSL as a supplement was unexpected, since many native strains of lactic acid bacteria, including the strain CS4435L used in the examples, comprise a cell-envelope bound protease enabling their growth on milk, which is also low in free amino acids and peptides. Only limited acid hydrolysis was required to increase the content of free peptides and amino acids in the CSL, sufficient to produce a suitable CSLH supplement. The growth and high yield of ethanol produced by the genetically modified lactic acid bacterium of the invention on RWP supplemented with the CSLH (obtained by limited acid hydrolysis) is consistent with the fact that cells of these lactic acid bacteria comprise both intracellular peptidases as well as various uptake systems for peptides as well as free amino acids that facilitate the assimilation of amino nitrogen in this form.
Alternative methods for improving growth and ethanol production were also tested using RWP supplemented with treated-forms of CSL (see example 4,
Down-stream processing of ethanol produced by fermentation, typically involves a distillation step. In order to minimize distillation costs it is important that the ethanol content of the fermentation broth is at least 4% (w/w) ethanol. Employing a fed-batch fermentation process, a fermentation broth comprising over 4.1% (w/w) ethanol was obtained when the genetically modified lactic acid bacterium of the invention was cultured on RWP supplemented with CSLH, and subsequent feeding with lactose (Example 3, and Table 4).
Accordingly, the invention provides a method for ethanol production employing a genetically engineered lactic acid bacterium comprising the steps of:
a. introducing a genetically modified lactic acid bacterium into an aqueous culture medium;
b. incubating the culture of (a);
c. recovering ethanol produced by said culture in step (b), and optionally
d. isolating the recovered ethanol;
wherein the aqueous culture medium comprises a lactose rich substrate (such as whey permeate or residual whey permeate), and a amino nitrogen source (such as acid hydrolysed corn steep liquor, as defined herein), and
wherein the genetically engineered lactic acid bacterium comprises transgenes encoding:
According to one embodiment of the method for ethanol production according to the invention, the aqueous culture medium comprises whey permeate or residual whey permeate, combined with acid hydrolysed corn steep liquor: wherein the final lactose content of the culture medium is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 g/L lactose; between any one of 2-200 g/L, 5-180 g/L, 5-150 g/L, 5-100 g/L, 10-100 g/L, 5-90 g/L, 10-90 g/L, 5-80 g/L, 10-80 g/L lactose; preferably equal to or less than any one of 150 g/L, 140 g/L, 130 g/L, 120 g/L, 110 g/L, 100 g/L, 90 g/L, and 80 g/L lactose. The desired lactose content of the aqueous culture medium is obtainable by diluting the whey permeate of residual whey permeate in the aqueous culture medium. For example, the aqueous culture medium may comprise any one of 1-80%, 5-80%, 10-80%, 20-80%, 20-60%, and 30-50% residual whey permeate.
The hydrolysed corn steep liquor (CSLH) component of the aqueous culture medium, is derived from corn steep liquor (CAS Number: 66071-94-1) by acid hydrolysis, for example hydrolysis with H2SO4, or HCl. The hydrolysed CSL is characterized by enhanced levels of soluble proteins, peptides and free amino acids (the concentrations of nearly all the 20 free amino acids is doubled compared with untreated corn steep liquor, especially arginine, glutamine, histidine, methionine, isoleucine, leucine, valine, and tyrosine. The peptides present in CSLH include oligopeptides of 2, 3, 4, 5 amino acid residues or even longer peptides; in particular the peptides Leu-Gly, Gly-Gly, Gly-Gly-Leu, Thr-Pro-Val-Gly-Lys.
A hydrolysed CSLH preparation, suitable for use as a supplement to a RWP medium, is one wherein the concentration of at least one free amino acid, selected from the group consisting on glutamine, histidine, methionine, leucine, isoleucine, and valine is at least 1.5 fold greater than the concentration of the corresponding amino acid in the original CSL from which the CSLH was derived by hydrolysis. Preferably, the concentration of the at least one free amino acid is at least 1.6, 1.7, 1.8, 1.9 or 2 fold greater than the concentration of the corresponding amino acid in the original CSL from which the CSLH was derived by hydrolysis. When the at least one free amino acid is histidine, the concentration of the histidine in a CSLH preparation having a 25% w/v solids content is at least 8 mM, preferably at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mM; while the mM concentration of the histidine in a 2.5% (w/v solids content) CSLH preparation is correspondingly 10 folded lower. Alternatively, when the at least one free amino acid is histidine, the concentration of the histidine in a CSLH preparation having a 25% w/v solids content is at least 2 mM, preferably at least 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 mM higher than the concentration of histidine in the original CSL from which the CSLH was derived by hydrolysis; while the mM concentration of the histidine in a 2.5% (w/v solids content) CSLH preparation is correspondingly 10 folded lower.
The RWP medium growth is supplemented with CSLH in an amount sufficient to support ethanol production using the genetically modified lactic acid bacterium of the invention. It has been observed that addition of acid hydrolysed treated CSL (H1) to give a final concentration of at least 0.5 w/v solids content was sufficient to enhance ethanol production and growth. Preferably, when the initial lactose concentration of the growth medium is between 5-80 g/L lactose, then the acid hydrolysed CSL (H1) is added to the medium in an amount to give a final w/v CSL solids concentration of at least 0.75, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25, 2.5, 2.75, 3.0%. Preferably, when the initial lactose concentration of the growth medium is between 60-200 g/L lactose, then acid hydrolysed CSL (H1) is added in an amount to give a final CSL w/v solids concentration of at least 2.00, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75 and 5.0%. The desired final concentration of CSLH in the aqueous culture medium is obtainable by diluting the CSLH into the aqueous culture medium. CSL is obtainable in a CSL w/v solids concentration of 40 to 60%; typically 50%. Accordingly, a dilution of 10 fold into the aqueous culture medium will give a CSL w/v solids concentration of 4 to 6%; typically 5%. Since the solids content of CSL remains unaltered by the acid hydrolysis, the final dilution of CSLH required to obtain the desired final concentration are the same as those for the original CSL from which the CSLH was derived.
According to one embodiment of the method for ethanol production according to the invention, the aqueous culture medium may be further characterized as a composition consisting of the components: whey permeate of residual whey permeate (as defined herein); hydrolysed corn steep liquor (as defined herein); water and optionally supplemented with yeast extract and/or an aqueous solution of lactose.
The ethanol produced by fermentation using the genetically modified lactic acid bacterium of the invention can be recovered from the fermentation medium by steps including distillation.
Methods for detecting and quantifying ethanol produced by a micro-organism of the invention include high performance liquid chromatography (HPLC) combined with Refractive Index detection to identify and quantify ethanol (as described by Solem et al., 2013) relative to a standard, as described and illustrated in the examples.
Integration and self-replicating vectors suitable for cloning and introducing one or more gene encoding one or more a polypeptide having an enzymatic activity required for homo-ethanol production in a lactic acid bacterium of the invention are commercially available and known to those skilled in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989). Cells of a lactic acid bacterium are genetically engineered by the introduction into the cells of heterologous DNA (RNA). Heterologous expression of genes encoding one or more polypeptide having an enzymatic activity required for homo-ethanol production in a lactic acid bacterium of the invention is demonstrated in Example 1.
A nucleic acid molecule, that encodes one or more polypeptide having an enzymatic activity required for homo-ethanol production according to the invention, can be introduced into a cell or cells and optionally integrated into the host cell genome using methods and techniques that are standard in the art. For example, nucleic acid molecules can be introduced by standard protocols such as transformation including chemical transformation and electroporation, transduction, particle bombardment, etc. Expressing the nucleic acid molecule encoding the enzymes of the claimed invention may also be accomplished by integrating the nucleic acid molecule into the genome.
Deletion of endogenous genes in a host lactic acid bacterium to obtain a genetically modified lactic acid bacterium according to the invention can be achieved by a variety of methods; for example by transformation of the host cell with linear DNA fragments containing a locus for resistance to an antibiotic, or any other gene allowing for rapid phenotypic selection, flanked by sequences homologous to closely spaced regions on the cell chromosome on either side of the gene to be deleted, in combination with the immediate subsequent deletion or inactivation of the recA gene. By selecting for a double-crossover event between the homologous sequences, shown by the antibiotic resistance or other detectable phenotype, a chromosome disruption can be selected for which has effectively deleted an entire gene. Inactivation or deletion of the recA gene prevents recombination or incorporation of extrachromosomal elements from occurring, thereby resulting in a bacterial strain which is useful for screening for functional activity or production of genetically engineered proteins in the absence of specific contaminants. The deletion of the genes ldh, ldhB, ldhX, pta, and adhE from L. lactis given in Example 1, illustrates one method for deleting these genes.
The deletion of endogenous genes in a host lactic acid bacterium to obtain a genetically modified lactic acid bacterium according to the invention can also be achieved by the more traditional approach involving mutagenesis and screening/selection. For instance, LDH (lactate dehydrogenase) mutants can be screened out using solid medium containing 2,3,5-triphenyl tetrazolium following mutagenesis using for instance N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or UV radiation. Alternatively, after mutagenesis, low-acid producing strains could be selected using a combination of bromide and bromate as described by Han et al., 2013. ALDB (α-acetolactate decarboxylase) mutants can be obtained easily after mutagenesis, for instance using NTG, or grown in the medium containing an unbalanced concentration of leucine versus valine and isoleucine in the medium (Goupil et al., 1996). ADHE (ethanol dehydrogenase) mutants can be screened in the presence of various concentrations of acetaldehyde.
The genetic modifications required to produce a Lactococcus lactis strain that is capable of homo-ethanol production from lactose in dairy waste and to efficiently direct the flux towards this end product are described below.
The plasmid-free strain Lactococcus lactis subsp. cremoris MG1363 or its derivatives were used for the studies described herein [18]. The Escherichia coli strain ABLE-C (E. coli C lac (LacZ−)[Kanr McrA− McrCB− McrF− Mrr− HsdR (rk− mk−)][F′ proAB lacIqZΔM15 Tn10(Tetr)]) (Stratagene) was used only for facilitating DNA cloning steps. The lactose-metabolism plasmid pLP712 (55,395 bp) was extracted from the dairy isolate NCDO712 based on the method of Andersen (1983).
E. coli strains were grown aerobically at 30° C. in Luria-Bertani broth (Sambrook et al. 2001). For growth experiments L. lactis was grown in 100 ml flasks without shaking in defined SA medium (Jensen et al., 1993), where glucose was replaced by lactose, or residual whey permeate medium (RWP). RWP, which was provided from Arla Foods Ingredients Group P/S (http://www.arlafoodsingredients.com/), is the mother liquor from lactose production and its composition can be seen in Table 3. When required, yeast extract (Sigma-Aldrich, USA) was used as an amino nitrogen source. Antibiotics were added in the following concentrations: erythromycin: 200 μg/ml for E. coli and 5 μg/ml for L. lactis, tetracycline: 8 μg/ml for E. coli and 5 μg/ml for L. lactis, chloramphenicol: 20 μg/ml for E. coli and 5 μg/ml for L. lactis.
For nitrogen source optimization tests, 50 g/L lactose (diluted RWP) was mixed with different concentrations of nitrogen sources (NH4Cl, yeast extract, CSL or CSLH) in 25 ml tube with a volume of 10 ml.
For ethanol production tests, RWP was diluted and used as the main substrate for fermentation without the addition of any vitamins or salts, except 2.5% (w/v) CSLH. L. lactis strain CS4435L was grown in a 125 ml flask with 100 ml of medium with slow magnetic stirring and no aeration. The cultivation was carried out at 30° C.
All manipulations were performed according to Sambrook et al (1989). PCR primers used can be seen in Table 2. PfuX7 polymerase was used for PCR applications (Nørholm, 2010). Chromosomal DNA from L. lactis was isolated using the method described for E. coli with the modification that cells were treated with 20 μg of lysozyme per ml for 2 hours. Cells of E. coli were transformed using electroporation. L. lactis was made electro competent as described previously by Holo and Nes (1989), with the following modifications: the cells were grown with 1% glycine, and at an optical density of 0.5 (600 nm) ampicillin was added to a final concentration of 20 μg ml−1 and incubation was continued at 30° C. for 30 minutes.
The plasmid vector pCS1966 (Solem et al., 2008) was used for deleting genes in L. lactis. Plasmids employed for deleting chromosomal genes were prepared by PCR amplifying approximately 800 base pairs (bp) regions upstream and downstream of the L. lactis chromosomal region to be deleted using the PCR primers and chromosomal DNA isolated from L. lactis. The primers used for amplifying the upstream and downstream regions are indicated in Table 2 as “geneX ups.” and geneX dwn”. The amplified fragments and the plasmid, pCS1966, were then digested with the respective restriction enzymes indicated in the primer table, prior to inserting the fragment into the plasmid. The resulting plasmids were transformed into the parent strain individually and gene deletion was performed as described by Solem et al., (2008). Specifically, the plasmids were transformed into the strains via electroporation, and the strains comprising the plasmids integrated into the chromosome were selected for on M17 plates supplemented with glucose and erythromycin. Afterwards, the transformants were purified and plated on SA glucose medium (Jensen et al., 1993) plates supplemented with 5-fluoroorotate, thereby selecting for strains in which the plasmid had been lost by homologous recombination. The successful deletions were verified by PCR (Solem et al., 2008).
1.4 Deleting Genes from the Lactococcus lactis Subsp. Cremoris
The following genes were deleted from the Lactococcus lactis subsp. cremoris parent strain ldhX, ldhB, ldh, pta, and adhE. The genes were deleted using gene deletion plasmids derived from pCS1966 designated as: pCS4026 (ldhX), pCS4020 (ldhB), pCS4104 (ldh), pCS4230 (pta), pCS4273 (adhE), constructed as described above (Example 1.2).
Deletion of the genes from the Lactococcus lactis subsp. cremoris parent strain was verified by PCR amplification of the respective gene using primers 774/777 (ldhX), 769/771 (ldhB), 788/789 (ldh), 880/881 (pta) and 929/930 (adhE).
The strain containing the three lactate dehydrogenase deletions (ldh, ldhB, ldhX) was named CS4099 or MG1363Δ3ldh. CS4234 (MG1363 Δ3ldhΔpta) was derived from CS4099, by additionally the deleting a phosphotransacetylase gene, pta using pCS4230. CS4363 (MG1363 Δ3ldhΔptaΔadhE) was derived from CS4234, by additionally the deleting the native adhE gene using pCS4273, using the gene deletion methods described in section 1.3.
L. lactis strains
E. coli/L. lactis shuttle vector, TetR,
E. coli/L. lactis shuttle vector, CamR
1.5 Introducing Codon-Optimized Genes Encoding Pyruvate Decarboxylase and Alcohol Dehydrogenase B into L. lactis Strain MG1363 Δ3ldhΔptaΔadhE
The pyruvate decarboxylase gene (pdc) was amplified using primers 690/829 and the alcohol dehydrogenase gene (adhB) was amplified using 830/791. In both cases the templates were synthetic codon-optimized genes based on Zymomonas mobilis pdc and adhB genes (GenScript). An SP (synthetic promoter)-pdc-adhB fragment was amplified from CS4116 chromosomal DNA using primers 947/894, phosphorylated using T4 polynucleotide kinase and ligated to pTD6 amplified using 891/892. The ligation was transformed directly into CS4363 (MG1363 Δ3ldhΔptaΔadhE). CS4435 is a strain comprising the plasmid pCS4268 comprising and expressing the pdc-adhB genes. The plasmid pTD6 is a derivative of pAK80 (Solem et al., 2013) containing a gusA reporter gene. A PCR fragment was amplified from pAK80 using primers ptd45/ptd46. The gusA gene was amplified from E. coli MG1655 using primers ptd1/ptd2. After digesting both fragments with BglII and AvrII the fragments were ligated and transformed into E. coli strain MC1000. The resulting plasmid was named pTD5. The erythromycin marker of pTD5 was replaced by a tetracycline marker by PCR amplifying pTD5 using primers ptd49/ptd50, digesting the product with StuI/ApaI and ligating it with a StuI/ApaI fragment containing the tetracycline resistance genes amplified from pG+host8 (29) using primers ptd51/ptd52. The ligation was introduced in E. coli MC1000 and the result was plasmid pTD6.
1.6 Introducing Genes Encoding the Lactose Catabolism Pathway into Lactococcus lactis Subsp. Cremoris Strain MG1363 Δ3LdhΔPtaΔadhE
The wild type strain L. lactis MG1363, and its derivatives described herein (e.g. CS4435), are plasmid-free strains that cannot utilize lactose as a carbon source. The Lactococcus plasmid, pLP712 (55.395 kbp), comprises genes encoding the entire lactose catabolism pathway (Wegmann et al., 2012). The lactose-metabolism plasmid pLP712 (55,395 bp) was extracted from the dairy isolate NCDO712 based on the method of Andersen (1983); and then transformed into L. lactis strain CS4435 to give strain CS4435L.
The lactose catabolism pathway genes located on the pLP712 plasmid (
1. the lacAB genes encoding two subunit polypeptides that together have galactose-6-phosphate isomerase activity (EC 5.3.1.26); wherein the first subunit polypeptide has an amino acid sequence of SEQ ID NO: 90 encoded by the L. lactis lacA gene; and the second subunit polypeptide has an amino acid sequence of SEQ ID NO: 92 encoded by the L. lactis lacB gene;
2. the lacC gene encoding a polypeptide having D-tagatose-6-phosphate kinase activity (EC 2.7.1.114); wherein the polypeptide has an amino acid sequence of SEQ ID NO: 94);
3. the lacD gene encoding a polypeptide having tagatose 1,6-diphosphate aldolase activity (EC 4.1.2.40); wherein the polypeptide has an amino acid of SEQ ID NO: 96;
4. the lacEF genes encoding a two polypeptide components together having lactose-specific phosphotransferase system (PTS) activity (EC 2.7.1.69); wherein the first polypeptide component is a phosphotransferase system EIICB component having an amino acid sequence of SEQ ID NO: 84, encoded by the L. lactis lacE gene; and the second polypeptide component is a phosphotransferase system EIIA component having an amino acid sequence of SEQ ID NO: 86, encoded by the L. lactis lacF gene; and
5. the lacG gene encoding a polypeptide having phospho-β-D-galactosidase activity (EC 3.2.1.85); wherein the polypeptide has the amino acid sequence of the phospho-β-D-galactosidase of SEQ ID NO: 88; and
6. the lacR gene encoding a lactose transport regulator of SEQ ID NO: 98
The wild type strain MG1363 and its genetically modified derivative strains, as listed in Table 3, were cultivated in defined SA medium (Jensen et al., 1993) with glucose and samples of each culture were collected after 24 hours. Cell growth was regularly measured by OD600 and quantification of lactose, glucose, lactate, formate, acetate, ethanol, acetoin and 2,3-butanediol was carried out using an Ultimate 3000 high-pressure liquid chromatography system (Dionex, Sunnyvale, USA) equipped with a Aminex HPX-87H column (Bio-Rad, Hercules, USA) and a Shodex RI-101 detector (Showa Denko K.K., Tokyo, Japan). The column oven temperature was set at 60° C. and the mobile phase of consisted of 5 mM H2SO4, at a flow rate of 0.5 ml/min. CO2 is included for calculating the carbon balance. Values are averages of three independent experiments and standard deviations are indicated.
Lactococcus lactis is characterised by homolactic fermentation when grown on glucose, as illustrated for the wild-type strain MG1363 in Table 3. Following deletion of the three ldh genes, in strain CS4099, the dominant products become acetate and ethanol, while the production of acetate was eliminated by deleting the pta gene, in strain CS4234, however this strain still produced significant amounts of formate and acetoin, and small amounts of 2,3-butandiol. The additional deletion of the native adhE gene yielded strain CS4363 that was unable to grow under anaerobic conditions because of the defect in cofactor regeneration ability. However, its growth could be restored in the presence of O2, where NAD+ is recycled by NADH oxidase (NoxE) which results in acetoin as the main fermentation product. The introduction of pdc and adhB genes, however, creating strain CS4435, restored complete cofactor-recycling and bacterial growth under anaerobic conditions, and the outcome was a genetically modified L. lactis strain capable of homo-ethanol production from glucose as carbon source (Table 3). The native adhE gene encodes a bifunctional ADHE that can use both acetyl-CoA and acetaldehyde as substrate, while the transgene adhB, encoding an ADHB derived from from Z. mobilis only uses acetaldehyde. As a consequence, the cofactors are completely balanced in the heterologous ethanol forming pathway and nearly all the carbon balance is converted to ethanol.
The strain CS4435L, derived from strain CS4435, comprises the Lactococcal plasmid, pLP712 (55.395 kbp) that encodes the entire lactose catabolism pathway (
a= Conversion efficiency was calculated based on the theoretical maximal yield (0.538 ethanol/g lactose).
b= The composition of SA medium (Jensen et al. 1993) comprises 19 amino acids, vitamins and salts. Glucose was replaced by lactose.
c= Diluted RWP (residual whey permeate) and 0.5% (w/v) YE (yeast extract).
d-f= Diluted RWP (residual whey permeate) and 2.5% (w/v) CSLH (corn steep liquor hydrolysate), prepared by condition H1.
m= Fed-batch was performed with initial 80 g/L lactose and the details can be found in FIG. 7.
Waste stream residual whey permeate (RWP) is the permeate mother liquor after extracting lactose from whey permeate. The composition of the RWP, which was supplied by Arla Foods Ingredients Group P/S (http://www.arlafoodsingredients.com) was determined and shown in Table 5. The sugar components of a filtered sample of RWP were determined as described in example 1.6, and the amino acid composition was determined by the steps of hydrolysis of the filtered sample with 6 M HCl; separation by ion exchange chromatography and detection after oxidation and derivatization with o-phthaldialdehyde, as described by Barkholt et al., (1989).
aResidual whey permeate is a concentrate of the residue remaining after lactose extraction from whey permeate.
Although strain CS4435L both grew and produced ethanol when cultured on defined SA medium (Jensen et al., 1993) comprising 7.2 g/L lactose; the strain was unable to grow or produce ethanol formation when cultured on RWP alone, even though an initial the lactose content of 50 g/L lactose was provided (
Since the price of YE is too high to provide the basis for a low cost medium; corn steep liquor (CSL) was tested as a cheap source of complex nitrogen. CSL was purchased from Sigma-Aldrich (St. Louis, Mo.) with 40-60% solid content. RWP medium supplemented with CSL, at concentrations ranging from 0.1% to 2.5% (w/v), however, only supported low levels of cell growth of strain CS4435L, and levels of ethanol produced were also very low. A combination of 0.1% (w/v) YE with CSL, led to only a small stimulation of growth (
In order to enhance the available amino nitrogen content of CSL, samples of CSL were subjected to various degrees of acid hydrolysis. The acid hydrolysis was performed with very small amounts of sulfuric acid (0.05-0.5% concentrated H2SO4 added to CSL having a 20-30% w/v solid content). The following hydrolysis conditions were applied to produce corn steep liquor hydrolysates (CSLH). H1 condition: original CSL was diluted 2 times with water and then 50 μl concentrated sulfuric acid was mixed with 100 ml diluted CSL. The mixture was kept at 121° C. for 15 mins and subsequently the pH was adjusted to 6.8-7.1 with the addition of 10 M NaOH solution. H2 condition: original CSL was diluted 2 times with water and then 250 μl concentrated sulfuric acid was mixed with 100 ml diluted CSL. The mixture was kept at 121° C. for 15 mins and subsequently the pH was adjusted to 6.8-7.1 with the addition of 10 M NaOH solution. H3 condition: original CSL was diluted 2 times with water and then 500 μl concentrated sulfuric acid was mixed with 100 ml diluted CSL. The mixture was kept at 121° C. for 15 mins and subsequently pH was adjusted to 6.8-7.1 with the addition of 10 M NaOH solution.
When comparing the growth and ethanol production of CS4435L cultured on RWP supplemented with the CSL hydrolysates (CSLH), the use of H1 hydrolysate (CSL treated with 0.05% H2SO4) gave the greatest increase in production levels (
Analysis of the free amino acid composition of CSL revealed that hydrolysis of corn steep liquor increases the free amino acid content of CSL by circa 2 fold in comparison with untreated corn steep liquor.
A direct correlation was observed between the amount of CSLH added to the RWP medium and the final cell biomass and ethanol produced (
The composition of two tested low cost growth media composed of RWP supplemented with hydrolysed CSL is shown in Table 7.
The low cost growth medium composed of RWP, used in an amount conferring a lactose concentration of either 50 g/L or 80 g/L when supplemented with 2.5% or 5% CSLH (H1) respectively, provides lactose as substrate for ethanol production and amino acids in sufficient amounts to support ethanol production and growth. The 2.5% or 5% CSLH, hydrolysed under the conditions of H1 is sufficient to enhance the levels of available complex amino nitrogen (soluble polypeptides, peptides and free amino acids), and in particular the levels of the essential amino acids glutamine, histidine, methionine, leucine, isoleucine, and valine.
Ethanol production by L. lactis strain CS4435L and cell growth during fermentation on the low cost medium, RWP supplemented with 2.5% (w/v) CSLH (H1), was monitored in order to determine the yields obtainable by this process. Cell growth reached a final cell density (OD600nm) of 4.0 after 14.5 hours in medium with 2.5% (w/v) CSLH (H1); and the initial lactose content of 40 g/L lactose was completely consumed within 31 hours (
Enhanced ethanol titers obtainable with L. lactis strain CS4435L, during fermentation on the low cost medium, RWP supplemented with 2.5% (w/v) CSLH (H1), were achieved by raising the initial lactose content of the medium. When the initial lactose content of the medium was raised to 80 g/L lactose, the lactose was totally consumed after 55 hours fermentation (
In order to enhance lactose supply during fermentation, without further elevating the lactose concentration in the low cost medium, fed-batch fermentation method was implemented, whereby lactose was added during fermentation. Specifically, fed-batch was performed with diluted RWP comprising a lactose content of 80 g/L lactose supplemented with 2.5% (w/v) CSLH; and 500 g/L lactose stock solution was used for feeding. The feeding was performed when the lactose concentration was lower than 10 g/L and after rapid injection it returned to around 20 g/L.
Using this fermentation strategy using L. lactis strain CS4435L on the low cost medium (
In order for L. lactis strain CS4435L to grow and produce ethanol when cultured on diluted residual whey permeate medium and 80 g/L lactose; it requires a source of amino nitrogen (Example 2). Example 2 further shows that CSL can only serve as a cheap source of amino nitrogen when it is hydrolysed. The present example compares three different methods of hydrolysing CSL included in the growth medium for L. lactis strain CS4435L, and their respective impacts on ethanol production. The CSI was either hydrolysed with acid or it was proteolytically hydrolysed.
Surprisingly, growth media comprising acid hydrolysed CSLH supported significantly higher levels of ethanol production than proteinase-treated CSL. Thus, L. lactis strain CS4435L cultures grown in media supplemented with CSLH produced 24 g/L ethanol in 39 h; while cultures grown in media supplemented with 70 IU/L or 700 IU/L proteinase-treated CSL, only produced 8.9 g/L and 10.7 g/L ethanol in 48 h, respectively.
| Number | Date | Country | Kind |
|---|---|---|---|
| 16157325.8 | Feb 2016 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/054347 | 2/24/2017 | WO | 00 |
