pH-controlled Batch Fermentations Research Articles

Engineering Escherichia coli for cost-effective production of medium-chain fatty acids from soy whey using an optimized galactose-based autoinduction system

Medium-chain fatty acids (MCFAs) are essential chemical feedstocks. Microbial production of MCFAs offers an attractive alternative to conventional methods, but the costly media and external inducers limit its practical application. To address this issue and make MCFA production more cost-effective, an E.coli platform was developed using soy whey as a medium and galactose as an autoinducer. We first designed an efficient, stringent, homogeneous, and robust galactose-based autoinduction system for the expression of pathway enzymes by rationally engineering the promoter of the galactose-proton symporter (GalP). Subsequently, the intracellular acetyl-CoA availability and NADH regeneration were enhanced to improve the reversal of the β-oxidation cycle. The resulting strain yielded 8.20 g/L and 16.42 g/L MCFA in pH-controlled batch fermentation and fed-batch fermentation with glucose added using soy whey as medium, respectively. This study provided a cost-effective and promising platform for MCFA production, as well as future strain development for other value-added chemicals production.

Prebiotic Potential of a New Sweetener Based on Galactooligosaccharides and Modified Mogrosides.

This study was conducted to investigate the sweetness intensity and the potential fecal microbiome modulation of galactooligosaccharides in combination with enzymatically modified mogrosides (mMV-GOS), both generated through a patented single-pot synthesis. Sweetness intensity was performed in vivo by trained sensory panelists. The impact on the human fecal microbiome was evaluated by in vitro pH-controlled batch fermentation, and bacterial populations and organic acid concentrations were measured by qPCR and GC-FID, respectively. Significant growth (p ≤ 0.05) during the fermentation at 10 h of bacterial populations includes Bifidobacterium (8.49 ± 0.44 CFU/mL), Bacteroides (9.73 ± 0.32 CFU/mL), Enterococcus (8.17 ± 0.42 CFU/mL), and Clostridium coccoides (6.15 ± 0.11 CFU/mL) as compared to the negative control counts for each bacterial group (7.94 ± 0.27, 7.84 ± 1.11, 7.52 ± 0.37, and 5.81 ± 0.08 CFU/mL, respectively) at the same time of fermentation. Likewise, the corresponding significant increase in production of SCFA in mMV-GOS at 10 h of fermentation, mainly seen in acetate (20.32 ± 2.56 mM) and propionate (9.49 ± 1.44 mM) production compared to a negative control at the same time (8.15 ± 1.97 and 1.86 ± 0.24 mM), is in line with a positive control (short-chain fructooligosaccharides; 46.74 ± 12.13 and 6.51 ± 1.91 mM, respectively) revealing a selective fermentation. In conclusion, these substrates could be considered as novel candidate prebiotic sweeteners, foreseeing a feasible and innovative approach targeting the sucrose content reduction in food. This new ingredient could provide health benefits when evaluated in human studies by combining sweetness and prebiotic fiber functionality.

Profiles of Small Regulatory RNAs at Different Growth Phases of Streptococcus thermophilus During pH-Controlled Batch Fermentation.

Small regulatory RNA (sRNA) has been shown to play an important role under various stress conditions in bacteria, and it plays a vital role in regulating growth, adaptation and survival through posttranscriptional control of gene expression in bacterial cells. Streptococcus thermophilus is widely used as a starter culture in the manufacture of fermented dairy products. However, the lack of reliable information on the expression profiles and potential physiological functions of sRNAs in this species hinders our understanding of the importance of sRNAs in S. thermophilus. The present study was conducted to assess the expression profiles of sRNAs in S. thermophilus and to identify sRNAs that exhibited significant changes. A total of 530 potential sRNAs were identified, including 198 asRNAs, 135 sRNAs from intergenic regions, and 197 sRNAs from untranslated regions (UTRs). Significant changes occurred in the expression of 238, 83, 194, and 139 sRNA genes during the lag, early exponential growth, late exponential growth, and stationary phases, respectively. The expression of 14 of the identified sRNAs was verified by qRT-PCR. Predictions of the target genes of these candidate sRNAs showed that the primary metabolic pathways targeted were involved in carbon metabolism, biosynthesis of amino acids, ABC transporters, the metabolism of amino and nucleotide sugars, purine metabolism, and the phosphotransferase system. The expression of the predicted target genes was further analyzed to better understand the roles of sRNAs during different growth stages. The results suggested that these sRNAs play crucial roles by regulating biological pathways during different growth phases of S. thermophilus. According to the results, sRNAs sts141, sts392, sts318, and sts014 are involved in the regulation of osmotic stress. sRNAs sts508, sts087, sts372, sts141, sts375, and sts119 are involved in the regulation of starvation stress. sRNAs sts129, sts226, sts166, sts231, sts204, sts145, and sts236 are involved in arginine synthesis. sRNAs sts033, sts341, sts492, sts140, sts230, sts172, and sts377 are involved in the ADI pathway. The present study provided valuable information for the functional study of sRNAs in S. thermophilus and indicated a future research direction for sRNA in S. thermophilus. Overall, our results provided new insights for understanding the complex regulatory network of sRNAs in S. thermophilus.

Enzymatically synthesised fructooligosaccharides from sugarcane syrup modulate the composition and short-chain fatty acid production of the human intestinal microbiota

Fructooligosaccharides can be produced by direct enzymatic conversion from sucrose-rich sugarcane syrup (SS) consisting of 58.93% sucrose yielding 21.28 g FOS/100 g sucrose. This study evaluated the prebiotic effect of unpurified/purified SS containing FOS for the modulation of the human intestinal microbial composition and short-chain fatty acid production. The unpurified and purified FOS substrates, which were a mixture of 1-kestose, nystose and 1F-fructosylnystose, were supplemented into human faecal culture using a pH-controlled batch fermentation system and significantly increased the Bifidobacterium counts after 5 h fermentation, while Bacteroides/Prevotella counts were highest throughout 24 h fermentation. Meanwhile, Lactobacillus/Enterococcus exhibited a slight increase after 5 h fermentation before reaching a plateau afterwards. The steady Bacteroides/Prevotella growth and increased Bifidobacterium population promoted an increase in the production of short-chain fatty acids acetate (58 ± 2.70 mM), propionate (9.19 ± 5.94 mM) and butyrate (7.15 ± 2.28 mM). These results provide evidence that representative gut microbiota could utilise the enzymatically synthesised FOS to generate short-chain fatty acids as metabolites in pH-controlled conditions, thus FOS from SS are a potential prebiotic ingredient for foods and health drinks.

Metabolic Profiles of Carbohydrates in Streptococcus thermophilus During pH-Controlled Batch Fermentation.

Revealing the metabolic profiles of carbohydrates with their regulatory genes and metabolites is conducive to understanding their mechanism of utilization in Streptococcus thermophilus MN-ZLW-002 during pH-controlled batch fermentation. Transcriptomics and metabolomics were used to study carbohydrate metabolism. More than 200 unigenes were involved in carbohydrate transport. Of these unigenes, 55 were involved in the phosphotransferase system (PTS), which had higher expression levels than those involved in ABC protein-dependent systems, permeases, and symporters. The expression levels of the genes involved in the carbohydrate transport systems and phosphate transport system were high at the end-lag and end-exponential growth phases, respectively. In addition, 166 differentially expressed genes (DEGs) associated with carbohydrate metabolism were identified. Most genes had their highest expression levels at the end-lag phase. The pfk, ldh, zwf, and E3.2.1.21 genes involved in the glycolytic pathway had higher expression levels at the end-exponential growth phase than the mid-exponential growth phase. The results showed high expression levels of lacZ and galKTM genes and reabsorption of extracellular galactose. S. thermophilus MN-ZLW-002 can metabolize and utilize galactose. Overall, this comprehensive network of carbohydrate metabolism is useful for further studies of the control of glycolytic pathway during the high-density culture of S. thermophilus.

Metabolic Pathway Profiling in Intracellular and Extracellular Environments of Streptococcus thermophilus During pH-Controlled Batch Fermentations.

Elucidating the metabolite profiles during the growth of Streptococcus thermophilus is beneficial for understanding its growth characteristics. The changes in the intracellular and extracellular concentrations of carbohydrates, nucleotides, amino sugars, nucleoside sugars, fatty acids, and amino acids, as well as their metabolites over time, were investigated by metabolomics technology. Most metabolites of nucleotides were highly accumulated in the intracellular environment after the mid-exponential phase. Increases in the intracellular unsaturated fatty acids and N-acetyl-glucosamine and N-acetyl-muramoate recycling provided potential evidence that cell envelope remodeling occurred after the mid-exponential phase. At the later fermentation stages, potentially functional metabolite produced by glycine was highly accumulated in the intracellular environment. Additionally, potential toxic metabolites produced by phenylalanine and tyrosine could not be excreted into the extracellular environment in a timely basis. The accumulation of large amounts of these metabolites might be the primary cause of the overconsumption of amino acids and influence the growth of S. thermophilus.

Transcriptomic and proteomic profiling revealed global changes in Streptococcus thermophilus during pH-controlled batch fermentations.

Understanding global changes of physiological processes at the molecular level during the growth of Streptococcus thermophilus is essential for the rational design of cultivation media and the optimization of bioprocesses. Transcriptomics and proteomics were combined to investigate the global changes at the transcript and protein level during the growth of S. thermophilus. The expression of 1396 genes (FDR ≤ 0.001) and 876 proteins (P < 0.05) changed significantly over time. The most remarkable growth phase dependent changes occurred in the late-lag phase and were related to heterofermentation, glycolysis, peptidoglycan biosynthesis, conversion between amino acids and stress response. The present results could provide theoretical guidance for high-cell-density culture, help design cultivation media, and help attain a high biomass of S. thermophilus.

Metabolic profiles of cysteine, methionine, glutamate, glutamine, arginine, aspartate, asparagine, alanine and glutathione in Streptococcus thermophilus during pH-controlled batch fermentations

Elucidating the amino acid (AA) metabolism patterns of Streptococcus thermophilus has important effects on the precise design of nitrogen sources for high-cell-density culture. Transcriptomics and metabolomics were combined to reveal the cysteine, methionine, glutamate, glutamine, arginine, aspartate, asparagine and alanine metabolic pathways in S. thermophilus MN-ZLW-002, including glutathione. The changes in the synthesis, consumption and concentration of AAs and their metabolites, as well as regulatory genes with time were revealed. The metabolism of L-cysteine, L-glutamate, L-aspartate and L-alanine generated some potential functional metabolites. The metabolism of methionine and glutamate generated potential harmful metabolites. S. thermophilus MN-ZLW-002 can synthesize glutathione. Some potential functional metabolites have similar biological functions, indicating that S. thermophilus can resist environmental stresses through multiple mechanisms. The expression of some key genes in synthesis pathway of AA indicated that cysteine, methionine, asparagine, aspartate, arginine and lysine were insufficient or imbalance between nutrient components. The accumulation of large amounts of AA metabolites might be the primary cause of the overconsumption of AAs and influence the growth of S. thermophilus. The present study revealed the metabolic profiles of abovementioned AAs as well as those of regulatory genes and metabolites. These results were beneficial to the precise design of nitrogen sources and regulation of functional metabolites for the high-cell-density culture of S. thermophilus.

Assessment of the adequacy of different Mediterranean waste biomass types for fermentative hydrogen production and the particular advantage of carob (Ceratonia siliqua L.) pulp

The conversion of agro-industrial byproducts, residues and microalgae, which are representative or adapted to the Mediterranean climate, to hydrogen (H2) by C. butyricum was compared. Five biomass types were selected: brewery’s spent grain (BSG), corn cobs (CC), carob pulp (CP), Spirogyra sp. (SP) and wheat straw (WS). The biomasses were delignified and/or saccharified, except for CP which was simply submitted to aqueous extraction, to obtain fermentable solutions with 56.2–168.4 g total sugars L−1. In small-scale comparative assays, the H2 production from SP, WS, CC, BSG and CP reached 37.3, 82.6, 126.5, 175.7 and 215.8 mL (g biomass)−1, respectively. The best fermentable substrate (CP) was tested in a pH-controlled batch fermentation. The H2 production rate was 204 mL (L h)−1 and a cumulative value of 3.9 L H2 L−1 was achieved, corresponding to a H2 production yield of 70.0 mL (g biomass)−1 or 1.6 mol (mol of glucose equivalents)−1. The experimental data were used to foresight a potential energy generation of 2.4 GWh per year in Portugal, from the use of CP as substrate for H2 production.

Influence of extracellular pH on growth, viability, cell size, acidification activity, and intracellular pH of Lactococcus lactis in batch fermentations.

In this study, we investigated the influence of three extracellular pH (pHex) values (i.e., 5.5, 6.5, and 7.5) on the growth, viability, cell size, acidification activity in milk, and intracellular pH (pHi) of Lactococcus lactis subsp. lactis DGCC1212 during pH-controlled batch fermentations. A universal parameter (e.g., linked to pHi) for the description or prediction of viability, specific acidification activity, or growth behavior at a given pHex was not identified. We found viability as determined by flow cytometry to remain high during all growth phases and irrespectively of the pH set point. Furthermore, regardless of the pHex, the acidification activity per cell decreased over time which seemed to be linked to cell shrinkage. Flow cytometric pHi determination demonstrated an increase of the averaged pHi level for higher pH set points, while the pH gradient (pHi-pHex) and the extent of pHi heterogeneity decreased. Cells maintained positive pH gradients at a low pHex of 5.5 and even during substrate limitation at the more widely used pHex 6.5. Moreover, the strain proved able to grow despite small negative or even absent pH gradients at a high pHex of 7.5. The larger pHi heterogeneity at pHex 5.5 and 6.5 was associated with more stressful conditions resulting, e.g., from higher concentrations of non-dissociated lactic acid, while the low pHi heterogeneity at pHex 7.5 most probably corresponded to lower concentrations of non-dissociated lactic acid which facilitated the cells to reach the highest maximum active cell counts of the three pH set points.

Kinetic modeling of fermentative hydrogen production by Thermotoga neapolitana

A kinetic model of fermentative hydrogen (H2) production was developed for the hyperthermophilic marine bacterium Thermotoga neapolitana DSMZ 4359T using glucose as the substrate. The model was built based on anaerobic digestion model no.1 (ADM1) by IWA using Monod-like kinetic equations. Several series of pH controlled batch fermentation tests were performed under pure CO2 or N2 atmosphere in serum bottles to evaluate the growth kinetic parameters. The kinetic parameters were estimated by applying the non-linear least square method to Monod and Michaelis–Menten kinetic equations. The experimentally estimated value of k, kS, Y and kd were 0.839 h−1, 1.42 g/L, 0.1204 and 0.0043 h−1, respectively. The kinetic parameters were analyzed for sensitivities and subsequently calibrated and validated by a separate set of experimental results. The model was particularly effective in estimating the biomass growth, substrate consumption, and H2 production except for acetate and lactate production, due to capnophilic condition (under CO2 atmosphere).

Developing a kinetic model for co-culture of yogurt starter bacteria growth in pH controlled batch fermentation

Streptococcus thermophilus and Lactobacillus bulgaricus are yogurt starter cultures widely used in the dairy industry. Co-culture of these bacteria leads to higher biomass yield than their separate single strain culture because of the proto-cooperation between the two species. In the present study, a kinetic model was developed for the growth and lactic acid production by these two bacteria in pH-controlled co-cultures. This model quantifies the effects of pH, temperature, lactic acid, carbon and nitrogen substrate concentrations, and the influence of each bacterium on the growth of the other. The latter effect was described by considering the calculated concentration of metabolites produced by each of the bacterium which stimulate the growth of the other. The model was validated by the experimental data obtained from a set of 12 experiments which were designed using the response surface methodology.

Biohydrogen production from dark fermentation of cheese whey: Influence of pH

Hydrogen production from cheese whey through dark fermentation was investigated in this study in order to systematically analyse the effects of the operating pH. The effluents from pecorino cheese and mozzarella cheese production were the substrates used for the fermentation tests. Either CW only or a mixture of CW and heat-shocked activated sludge were used in mesophilic pH-controlled batch fermentation experiments. The results indicated that hydrogen production was strongly affected by multiple factors including the substrate characteristics, the addition of an inoculum as well as the pH. The process variables were found to affect to a varying extent numerous interrelated aspects of the fermentation process, including the hydrogen production potential, the type of fermentation pathways, as well as the process kinetics. The fermentation products varied largely with the operating conditions and mirrored the H2 yield. Significant fermentative biohydrogen production was attained at pHs of 6.5–7.5, with the best performance in terms of H2 generation potential (171.3 NL H2/kg TOC) being observed for CW from mozzarella cheese production, at a pH value of 6.0 with the heat-shocked inoculum.

Diversity Analysis, Batch Fermentation and Characterization of Nisin in Identified Strains of Lactococcus lactis spp. lactis

Thirty-one strains of Lactococcus lactis spp. lactis were identified out of 89 isolates of lactic acid bacteria (LAB) from dairy and nondairy sources. Of the 31 strains, 24 (46.1%) were obtained from dairy and seven (18.9%) from non-dairy sources. The cluster analysis of rep-PCR showed that (GTG)5-PCR followed by ERIC-PCR exhibited more discriminating potential than BOX-PCR. The obtained banding patterns characterized the polymorphism among strains. The strain level polymorphism was also obtained by the combined cluster analysis of (GTG)5, ERIC and BOX-PCR which exhibited a level of heterogeneity among strains but not with the sources of isolation. Among 31 strains, 17 strains were able to produce zones of inhibition against Lactobacillus acidophilus NCDC 015 and therefore considered as nisin producers. Nisin production by strains was further confirmed by PCR amplification of nisA/Z of 174 bp size. The nisin activity and cell growth observed to be higher in pH controlled batch fermentation than in uncontrolled fermentation. The nisin activity, cell concentration, and acidity were high in immobilized cell system than free cell batch fermentation. The hot acid and chloroform extraction method was found to be the efficient way for the partial purification of nisin from fermented broth.

Bioconversion of glycerol to 1,3-propanediol in thin stillage-based media by engineered Lactobacillus panis PM1

Thin stillage (TS) is a waste residue that remains after bioethanol production, and its disposal reflects the high costs of bioethanol production. Thus, the development of cost-effective ways to process TS is a pending issue in bioethanol plants. The aim of this study was to evaluate the utilization of TS for the production of the valuable chemical, 1,3-propanediol (1,3-PDO), by Lactobacillus panis PM1. Different fermentation parameters, including temperature, pH and strains [wild-type and a recombinant strain expressing a NADPH-dependent aldehyde reductase (YqhD)gene] were tested in batch and fed-batch cultivations. The highest 1,3-PDO concentration (12.85g/L) and yield (0.84g/g) were achieved by batch fermentation at pH-4.5/30°C by the YqhDrecombinant strain. Furthermore, pH-controlled batch fermentation reduced the total fermentation period, resulting in the maximal 1,3-PDO concentration of 16.23g/L and yield of 0.72g/g in TS without an expensive nutrient or nitrogen (e.g., yeast extract, beef extract, and peptone) supplementation. The addition of two trace elements, Mg(2+) and Mn(2+), in TS increased 1,3-PDO yield (0.74g/g) without 3-hydroxypropionaldehyde production, the only intermediate of 1,3-PDO biosynthetic pathway in L. panis PM1. Our results suggest that L. panis PM1 can offer a cost-effective process that utilizes the TS to produce a value-added chemical, 1,3-PDO.

Characterization of antifungal organic acids produced by Lactobacillus harbinensis K.V9.3.1Np immobilized in gellan–xanthan beads during batch fermentation

Antifungal activity of lactic acid bacteria (LAB) has been observed in many food fermentation processes. Organic acids produced during the growth of these microorganisms play a major role in antagonism towards molds. The present study characterizes antifungal compounds produced during growth of Lactobacillus harbinensis K.V9.3.1Np that was previously isolated from raw cow milk and found to possess strong antifungal activities. A pH-controlled batch fermentation in enriched milk protein medium with immobilized cells of this bacterium was carried out in the presence of disrupted cells of Debaryomyces hansenii UBOCC-A-211003 and Kluyveromyces lactis UBOCC-A-212021. After seven days of fermentation, cell-free culture supernatants with robust antifungal activities against D. hansenii, Penicillium expansum and Penicillium roqueforti were obtained. These cell-free supernatants contained high amounts of organic acids such as lactic and acetic acids as well as hexanoic acid (9 mM), which the latter revealed to be the most efficient antifungal compound. The antifungal activity of hexanoic acid was influenced by the environmental conditions including the nature of the matrix and the pH. Under the conditions tested, even if organic acids were key antifungal agents, they were only part of a complex mixture of various molecules that acted in a synergistic manner.

Metabolic engineering of Klebsiella oxytoca M5a1 to produce optically pure d-lactate in mineral salts medium

Klebsiella oxytoca strains were constructed to produce optical pure d-lactate by pH-controlled batch fermentation in mineral salts medium. The alcohol dehydrogenase gene, adhE, and the phospho-transacetylase/acetate kinase A genes, pta-ackA, were deleted from the wild type. KMS002 (ΔadhE) and KMS004 (ΔadhE Δpta-ackA) exhibited d-lactate production as a primary pathway for the regeneration of NAD+. Both strains produced 11–13g/L of d-lactate in medium containing 2% (w/v) glucose with yields of 0.64–0.71g/g glucose used. In sugarcane molasses, KMS002 and KMS004 produced 22–24g/L of d-lactate with yields of 0.80–0.87g/g total sugars utilized. Both strains also utilized maltodextrin derived from cassava starch and produced d-lactate at a concentration of 33–34g/L with yields of 0.91–0.92g/g maltodextrin utilized. These d-lactate yields are higher than those reported for engineered E. coli strains.

Enhanced production of nukacin D13E in Lactococcus lactis NZ9000 by the additional expression of immunity genes

Nukacin D13E (D13E) is a variant of type-A(II) lantibiotic nukacin ISK-1 produced by Staphylococcus warneri ISK-1. D13E exhibited a twofold higher specific antimicrobial activity than nukacin ISK-1 against a number of Gram-positive bacteria. We previously reported the heterologous production of D13E in Lactococcus lactis NZ9000 under the control of nisin-controlled gene expression system. In this study, we demonstrated enhanced production of D13E by the additional expression of immunity genes, nukFEG. The nukacin ISK-1 immunity, conferred by the ABC transporter complex, NukFEG, and the lantibiotic-binding protein, NukH, was not overwhelmed by D13E. The additional NukFEG resulted in a fourfold increase in the immunity level of the strain and a 5.2-fold increase in D13E production. The additional NukFEGH-expressing strain with the highest D13E immunity showed reduced level of production. Further improvement in D13E production was achieved by using pH-controlled batch fermentation.

Oxalate-degrading activity in Bifidobacterium animalis subsp. lactis: impact of acidic conditions on the transcriptional levels of the oxalyl coenzyme A (CoA) decarboxylase and formyl-CoA transferase genes.

Oxalic acid occurs extensively in nature and plays diverse roles, especially in pathological processes. Due to its highly oxidizing effects, hyperabsorption or abnormal synthesis of oxalate can cause serious acute disorders in mammals and can be lethal in extreme cases. Intestinal oxalate-degrading bacteria could therefore be pivotal in maintaining oxalate homeostasis and reducing the risk of kidney stone development. In this study, the oxalate-degrading activities of 14 bifidobacterial strains were measured by a capillary electrophoresis technique. The oxc gene, encoding oxalyl-coenzyme A (CoA) decarboxylase, a key enzyme in oxalate catabolism, was isolated by probing a genomic library of Bifidobacterium animalis subsp. lactis BI07, which was one of the most active strains in the preliminary screening. The genetic and transcriptional organization of oxc flanking regions was determined, unraveling the presence of two other independently transcribed open reading frames, potentially responsible for the ability of B. animalis subsp. lactis to degrade oxalate. pH-controlled batch fermentations revealed that acidic conditions were a prerequisite for a significant oxalate degradation rate, which dramatically increased in cells first adapted to subinhibitory concentrations of oxalate and then exposed to pH 4.5. Oxalate-preadapted cells also showed a strong induction of the genes potentially involved in oxalate catabolism, as demonstrated by a transcriptional analysis using quantitative real-time reverse transcription-PCR. These findings provide new insights into the characterization of oxalate-degrading probiotic bacteria and may support the use of B. animalis subsp. lactis as a promising adjunct for the prophylaxis and management of oxalate-related kidney disease.

Residual yoghurt whey for lactic acid production

Abstract The operation in global yoghurt market provokes frequently the reject and withdrawn of yoghurt derivates out of shelf life. Yoghurts that have past their ‘best before’ date constitute a waste that has to be environmentally treated. Its use as a source for lactic acid production by Lactobacillus casei is studied in this work, being also a proposal that can reduce the end-of-cycle impact of the residue. Production of yoghurt generates a residue highly supplemented with sugar and fruits syrups that can be metabolized to lactic acid, but no data are available in the literature about this potential transformation into a commercial valuable lactic acid. Bioconversion of total sugars around 44% was achieved in pH controlled batch fermentation after 34 h. Control of pH and yeast extract supplementation in the preculture stage have shown a remarkable enhancement of fermentation performance due to the reduction in the toxicity and the increase in nitrogen content. Successive bioconversions of lactose, glucose and sucrose have been shown, with a sugar consumption profile giving a hierarchical mode consumption in the order glucose > sucrose > lactose.