Immobilization ofLactobacillus bulgaricus in a hollowfiber bioreactor for production of lactic acid from acid whey permeate

Lactobacillus bulgaricus was immobilized in the shell side of an industrial hollow-fiber ultrafiltration module. Acid whey permeate, containing 46 g/L lactose supplemented with 10 g/L yeast extract, was pumped through the tube side at dilution rates of 0.2–2.5/h. At a cell concentration of 100 g/L, productivity was 1.5–5 g lactic acid/L/h.     

Conversion of aqueous ammonia-treated corn stover to lactic acid by simultaneous saccharification and cofermentation

Treatment of corn stover with aqueous ammonia removes most of the structural lignin, whereas retaining the majority of the carbohydrates in the solids. After treatment, both the cellulose and hemicellulose in corn stover become highly susceptible to enzymatic digestion. In this study, corn stover treated by aqueous ammonia was investigated as the substrate for lactic acid production by simultaneous saccharification and cofermentation (SSCF). A commercial cellulase (Spezyme-CP) and Lactobacillus pentosus American Type Culture Collection (ATCC) 8041 (Spanish Type Culture Collection [CECT]-4023) were used for hydrolysis and fermentation, respectively. In batch SSCF operation, the carbohydrates in the treated corn stover were converted to lactic acid with high yields, the maximum lactic acid yield reaching 92% of the stoichiometric maximum based on total fermentable carbohydrates (glucose, xylose, and arabinose). A small amount of acetic acid was also produced from pentoses through the phosphoketolase pathway. Among the major process variables for batch SSCF, enzyme loading and the amount of yeast extract were found to be the key factors affecting lactic acid production. Further tests on nutrients indicated that corn steep liquor could be substituted for yeast extract as a nitrogen source to achieve the same lactic acid yield. Fed-batch operation of the SSCF was beneficial in raising the concentration of lactic acid to a maximum value of 75.0 g/L.     

Continuous production of succinic acid by a fumarate-reducing bacterium immobilized in a hollow-fiber bioreactor

Enterococcus faecalis RKY1, a fumarate-reducing bacterium, was immobilized in an asymmetric hollow-fiber bioreactor (HFBR) for the continuous production of succinic acid. The cells were inoculated into the shell side of the HFBR, which was operated in transverse mode. Since the pH values in the HFBR declined during continuous operation to about 5.7, it was necessary to change the feed pH from 7.0 to 8.0 after 24 h of operation in order to enhance production of succinic acid. During continuous operation with a medium containing fumarate and glycerol, the productivity of succinate was 3.0–10.9 g/(L·h) with an initial concentration of 30 g/L of fumarate, 4.9–14.9 g/(L·h) with 50 g/L of fumarate, and 7.2–17.1 g/(L·h) with 80 g/L of fumarate for dilution rates between 0.1 and 0.4 h⁻¹. The maximum productivity of succinate obtained by the HFBR (17.1 g of succinate /[L·h]) was 1.7 times higher than that of the batch bioconversions (9.9 g of succinate /[L·h]) with 80 g/L of fumarate. Furthermore, the long-term stability of the HFBR was demonstrated with a continuously efficient production of succinate for more than 15 d (360 h).     

Succinic Acid Production by Actinobacillus succinogenes Using Spent Brewer's Yeast Hydrolysate as a Nitrogen Source

To develop a cost-effective fermentation medium, spent brewer's yeast hydrolysate was evaluated as a nitrogen source for succinic acid production by Actinobacillus succinogenes NJ113 in glucose-containing media. Autolysis and enzymatic hydrolysis were used to hydrolyze the spent brewer's yeast cells to release the nutrients. The results showed that enzymatic hydrolysis was a more effective method due to the higher succinic acid yield and cell growth. However, the incomplete glucose consumption indicated existence of nutrient limitation. Vitamins were subsequently identified as the main limiting factors for succinic acid production using enzymatically hydrolyzed spent brewer's yeast as a nitrogen source. After the addition of vitamins, cell growth and succinic acid concentration both improved. As a result, 15 g/L yeast extract could be successfully replaced with the enzymatic hydrolysate of spent brewer's yeast with vitamins supplementation, resulting in a production of 46.8 g/L succinic acid from 68 g/L glucose.     

Conditions that promote production of lactic acid byZymomonas mobilis in batch and continuous culture

This study documents the similar pH-dependent shift in pyruvate metabolism exhibited byZymomonas mobilis ATCC 29191 and ATCC 39676 in response to controlled changes in their steady-state growth environment. The usual high degree of ethanol selectivity associated with glucose fermentation by Z.mobilis is associated with conditions that promote rapid and robust growth, with about 95% of the substrate (5% w/v glucose) being converted to ethanol and CO₂, and the remaining 5% being used for the synthesis of cell mass. Conditions that promote energetic uncoupling cause the conversion efficiency to increase to 98% as a result of the reduction in growth yield (cell mass production). Under conditions of glucose-limited growth in a chemostat, with the pH controlled at 6.0, the conversion efficiency was observed to decrease from 95% at a specific growth rate of 0.2/h to only 80% at 0.042/h. The decrease in ethanol yield was solely attributable to the pH-dependent shift in pyruvate metabolism, resulting in the production of lactic acid as a fermentation byproduct. At a dilution rate (D) of 0.042/h, decreasing from pH 6.0 to 5.5 resulted in a decrease in lactic acid from 10.8 to 7.5 g/L. Lactic acid synthesis depended on the presence of yeast extract (YE) or tryptone in the 5% (w/v) glucose-mineral salts medium. At D = 0.15/h, reduction in the level of YE from 3 to 1 g/L caused a threefold decrease in the steady-state concentration of lactic acid at pH 6. No lactic acid was produced with the same mineral salts medium, with ammonium chloride as the sole source of assimilable nitrogen. With the defined salts medium, the conversion efficiency was 98% of theoretical maximum. When chemostat cultures were used as seed for pH-stat batch fermentations, the amount of lactic acid produced correlated well with the activity of the chemostat culture; however, the mechanism of this prolonged induction     

Fermentation metabolism and kinetics in the production of organic acids byPropionibacterium acidipropionici

The genusPropionibacterium acidipropionici was grown under pH-controlled batch fermentation conditions for the production of acetic and propionic acids using 19.1 g/L glucose as a carbon source. The optimum pH range was found to be between 5.5 and 6.5. Bacterial metabolism and fermentation pathways were altered at pH values outside this range. Lactic acid was produced as a key intermediate, with the final acetic and propionic acid production entirely dependent on the cell's ability to metabolize the lactic acid. Most of the glucose in the medium was consumed in less than 20 h of fermentation and converted to lactic acid. Batch fermentation at pH 6 showed that lactic acid was completely utilized to produce 8.5 g/L propionic acid and 5.7 g/L acetic acid. However, the bacteria were unable to metabolize lactic acid at pH 7, resulting in 0.7 g/L propionic acid and 7.0 g/L acetic acid in the fermenter. A kinetic study of batch fermentation at pH 6 showed two distinct growth phases during the fermentation. Most of the cell growth was achieved in the exponential growth stage when glucose was consumed as a main substrate. A nonexponential growth stage was observed when lactic acid was utilized as a carbon source, producing propionic and acetic acids as secondary metabolites.     

Continuous production of lactic acid from glucose and lactose in a cell-recycle reactor

Growth and lactic acid production ofLactobacillus delbreuckii were compared using glucose and lactose as carbon sources. A continuous-flow stirred-tank fermenter was coupled with a cross-flow filtration unit to permit operation at high-cell concentrations. At steady state, yeast extract requirements for lactic-acid production were lower when glucose was used as a substrate than with lactose fermentation. Once steady state was obtained, with glucose feed, it was possible to lower the yeast extract concentration without affecting biomass concentration and lactic acid production. The lacticacid concentration that inhibited cell growth and lactic acid production was found to depend on the choice of a carbon substrate.     

Lactic acid fermentation in cell-recycle membrane bioreactor

Traditional lactic acid fermentation suffers from low productivity and low product purity. Cell-recycle fermentation has become one of the methods to obtain high cell density, which results in higher productivity. Lactic acid fermentation was investigated in a cell-recycle membrane bioreactor at higher substrate concentrations of 100 and 120 g/dm³. A maximum cell density of 145 g/dm³ and a maximum productivity of 34 g/(dm³…h) were achieved in cell-recycle fermentation. In spite of complete consumption of substrate, there was a continuous increase in cell density in cell-recycle fermentation. Control of cell density in cell-recycle fermentation was attempted by cell bleeding and reduction in yeast extract concentration.     

Effect of starting xylose concentration on the microaerobic metabolism of Debaryomyces hansenii: the use of carbon material balances

Xylitol production by Debaryomyces hansenii NRRL Y-7426 was performed on synthetic medium varying the initial xylose concentration between 50 and 300 g/L. The experimental results of these tests were used to investigate the effect of substrate level on xylose consumption by this yeast. Satisfactory values of product yield on substrate (0.74–0.83 g/g) as well as volumetric productivity (0.481–0.694 g/L·h) were obtained over a wide range of xylose levels (90–200 g/L), while a worsening of kinetic parameters took place at higher concentration, likely due to a substrate inhibition phenomenon. The metabolic behavior of D. hansenii was studied, under these conditions, through a carbon material balance to estimate the fractions of xylose consumed by the cell for different activities (xylitol production, biomass growth, and respiration) during the lag, exponential, and stationary phases.     

Nutritional requirements of a strain of bacillus thuringiensis subsp. kurstaki and use of gruel hydrolysate for the formulation of a new medium for δ-endotoxin production

Nutritional requirements of a strain ofBacillus thuringiensis (Bt) subsp.kurstaki were elucidated for δ-endotoxin production. The effect of some principal nutrients was deeply investigated, showing several nutritional and metabolite limitations in Bt growth and δ-endotoxin synthesis. This led us to formulate a new medium based on the hydrolysate of gruel, a cheap and abundant byproduct of semolina factories, supporting growth and δ-endotoxin synthesis. After hydrolysis of gruel by α-amylase, followed by proteolysis using alcalase, the resultant soluble material substituted glucose very well for Bt δ-endotoxin production. Indeed, 15 g/L total sugars coming from that hydrolysate, supplemented by 5.4 g/L ammonium sulfate as nitrogen source and either 5 g/L yeast extract or 3 g/L peptone from casein or 3 g/L casaminoacids or 0.25 g/L cysteine or aspartic acid, were the principal components of this new medium in which almost 1 g/L of δ-endotoxin in 4.5 g/L total dry biomass was produced.     

<Emphasis Type="SmallCaps">d</Emphasis>(−)-Lactic Acid Production by <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> B512 Using Different Carbon and Nitrogen Sources

Sugar concentration from sugarcane juice and yeast autolysate increased lactic acid production more than the other agro-industrial substrates tested. The concentrations of these two components were further optimized using the Plackett–Burman design and response surface method. A second-order polynomial regression model estimated that a maximal lactic acid production of 66.11 g/L would be obtained when the optimal values of sugar and yeast autolysate were 116.9 and 44.25 g/L, respectively. To validate the optimization of the medium composition, studies were carried out using the optimized conditions to confirm the result of the response surface analysis. After 48 h, lactic acid production using the shake-flask method was at 60.2 g/L.     

Lactic acid production from cellulosic material by synergetic hydrolysis and fermentation

The hydrolysis process on corncob residue was catalyzed synergetically by the cellulase from Trichoderma reesei and the immobilized cellobiase. The feedback inhibition to cellulase reaction caused by the accumulation of cellobiose was eliminated efficiently. The hydrolysis yield of corncob residue was 82.5%, and the percentage of glucose in the reducing sugar reached 88.2%. The glucose in the cellulosic hydrolysate could be converted into lactic acid effectively by the immobilized cells of Lactobacillus delbrueckii. When the enzymatic hydrolysis of cellulose and the fermentation of lactic acid were coupled together, no glucose was accumulated in the reaction system, and the feedback inhibition caused by glucose was also eliminated. Under the batch process of synergetic hydrolysis and lactic acid fermentation with 100 g/L of cellulosic substrate, the conversion efficiency of lactic acid from cellulose and the productivity of lactic acid reached 92.4% and 0.938 g/(L·h), respectively. By using a fed-batch technique, the total concentration of cellulosic substrate and lactic acid in the synergetic process increased to 200 and 107.5 g/L, respectively, whereas the dosage of cellulase reduced from 20 to 15 IU/g of substrate in the batch process. The results of the bioconversion of renewable cellulosic resources were significant.     

Production of lactic acid from cheese whey by batch and repeated batch cultures of Lactobacillus sp. RKY2

The fermentative production of lactic acid from cheese whey and corn steep liquor (CSL) as cheap raw materials was investigated by using Lactobacillus sp. RKY2 in order to develop a cost-effective fermentation medium. Lactic acid yields based on consumed lactose were obtained at more than 0.98 g/g from the medium containing whey lactose. Lactic acid productivities and yields obtained from whey lactose medium were slightly higher than those obtained from pure lactose medium. The lactic acid productivity gradually decreased with increase in substrate concentration owing to substrate and product inhibitions. The fermentation efficiencies were improved by the addition of more CSL to the medium. Moreover, through the cell-recycle repeated batch fermentation, lactic acid productivity was maximized to 6.34 g/L/h, which was 6.2 times higher than that of the batch fermentation.     

A new approach to improving the performance ofZymomonas in continuous ethanol fermentations

Although most fermentation ethanol is currently produced in traditional batch processes with yeast, the ethanologenic bacteriumZymomonas mobilis is recognized as an alternative process organism for fuel alcohol production. Different strategies for improving the productivity of ethanol fermentations are reviewed. In batch and open-type continuous fermentations the advantage of replacing yeast byZymomonas relates principally to the 10% higher fermentation efficiency (product yield), whereas in high cell density, closed-type continuous systems (operating with cell recycle or retention) the superior kinetic properties ofZymomonas can be exploited to affect about a five-fold improvement in volumetric productivity. Unlike yeast, the rate of energy supply (conversion of glucose to ethanol) inZymomonas is not strictly regulated by the energy demand and a nongrowing culture exhibits a maintenance energy coefficient that is at least 25 times higher than yeast. As an alternative to process improvement through genetic engineering of the process organism this investigation has taken a biochemical and physiological approach to increasing the kinetic performance ofZ. mobilis through manipulation and control of the chemical environment. Energetically “uncoupled” phenotypes with markedly increased specific rates of ethanol production were generated under conditions of nutritional limitation (nitrogen, phosphate, or potassium) in steady-state continuous culture. The pH was shown to influence energy coupling inZymomonas affecting the maintenance coefficient (m _e ) rather than the max growth yield coefficient (Y _x ^sάx ). Whereas the pH for optimal growth ofZ. mobilis (ATCC 29191) in a complex medium was 6.0–6.5, the specific rate of ethanol production in continuous fermentations was maximal in the range 4.0–4.5. Fermentation conditions are specified for maximizing the specific productivity of aZymomonas-based continuous ethanol fermentation where the potential exists for improving the volumetric productivity in dense culture fermentations with an associated 35–40% reduction in capital costs of fermentation equipment and an estimated savings of 10–15% on cost of product recovery (distillation), and 3–7% on overall production costs based on the projected use of inexpensive feedstocks.     

Efficient Non-sterilized Fermentation of Biomass-Derived Xylose to Lactic Acid by a Thermotolerant Bacillus coagulans NL01

Xylose is the major pentose and the second most abundant sugar in lignocellulosic feedstock. Its efficient utilization is regarded as a technical barrier to the commercial production of bulk chemicals from lignocellulosic biomass. This work aimed at evaluating the lactic acid production from the biomass-derived xylose using non-sterilized fermentation by Bacillus coagulans NL01. A maximum lactic acid concentration of about 75 g/L was achieved from xylose of 100 g/L after 72 h batch fermentation. Acetic acid and levulinic acid were identified as important inhibitors in xylose fermentation, which markedly reduced lactic acid productivity at 15 and 1.0 g/L, respectively. But low concentrations of formic acid (<2 g/L) exerted a stimulating effect on the lactic acid production. When prehydrolysate containing total 25.45 g/L monosaccharide was fermented with B. coagulans NL01, the same preference for glucose, xylose, and arabinose was observed and18.2 g/L lactic acid was obtained after 48 h fermentation. These results proved that B. coagulans NL01 was potentially well-suited for producing lactic acid from underutilized xylose-rich prehydrolysates.     

Production of polyhydroxyalkanoate from sesame seed wastewater by sequencing batch reactor cultivation process of Haloferax mediterranei

Due to the high cost of bioplastic production, sesame wastewater, generated from the sesame seed hulling process, was investigated to be used as inexpensive and renewable carbon source for the production of biodegradable polyhydroxyalkanoate (PHA) by extreme Haloferax mediterranei. The sesame wastewater (SWW) was hydrolyzed using different concentrations of hydrochloric acid (0.4. 1.00 and 2.00 M) at different period of times (15, 60 and 90 min). The concentration of salt (NaCl) and nitrogen source (NH₄Cl and yeast) required for H. mediterranei cells growth and the accumulation of PHA biopolymer was optimized. A maximum 0.53 g/L concentration of PHA was achieved when the SWW extract media was supplemented with 100 g/L NaCl and 6.0 g/L yeast extract. The cultivation was scaled-up using sequencing batch reactor (SBR) fermentation under non-sterile conditions. The SBR results showed that SWW needs an auxiliary carbon source to obtain high PHA production. Consequently, the system fed with SWW and glucose produced higher PHA (20.9 g/L) than the system fed with SWW.     

Continuous production of lactic acid in a cell recycle reactor

The production of lactic acid from glucose has been demonstrated using a CSTR (continuous stirred-tank reactor) with cell recycle. Studies were conducted withLactobacillus delbrueckii at a fermentation temperature of 42°C and a pH of 6.25. A cell density of 140 g dry weight/L and a volumetric productivity of 150 g/L.h, with complete glucose consumption, were obtained. It was not possible to obtain a lactic acid concentration above 60 g/L because of product inhibition. A cell purge was not necessary to maintain high viability bacteria culture or to obtain a steady state. At steady state the net cell growth appeared to be negligible. The specific glucose consumption for cell maintenance was 0.33 g glucose/g cells-h.     

Lactic acid bacteria production from whey

The main purpose of this work was to isolate and characterize lactic acid bacteria (LAB) strains to be used for biomass production using a whey-based medium supplemented with an ammonium salt and with very low levels of yeast extract (0.25 g/L). Five strains of LAB were isolated from naturally soured milk after enrichment in whey-based medium. One bacterial isolate, designated MNM2, exhibited a remarkable capability to utilize whey lactose and give a high biomass yield on lactose. This strain was identified as Lactobacillus casei by its 16S rDNA sequence. A kinetic study of cell growth, lactose consumption, and titratable acidity production of this bacterial strain was performed in a bioreactor. The biomass yield on lactose, the percentage of lactose consumption, and the maximum increase in cell mass obtained in the bioreactor were 0.165 g of biomass/g of lactose, 100%, and 2.0 g/L, respectively, which were 1.44, 1.11, and 2.35 times higher than those found in flask cultures. The results suggest that it is possible to produce LAB biomass from a whey-based medium supplemented with minimal amounts of yeast extract.     

Fermentation of xylose into acetic acid by Clostridium thermoaceticum

For optimum fermentation, fermenting xylose into acetic acid by Clostridium thermoaceticum (ATCC 49707) requires adaptation of the strain to xylose medium. Exposed to a mixture of glucose and xylose, it preferentially consumesxylose over glucose. The initial concentration of xylose in the medium affects the final concentration and the yield of acetic acid. Batch fermentation of 20 g/L of xylose with 5g/L of yeast extract as the nitrogen source results in a maximum acetate concentration of 15.2 g/L and yield of 0.76 g of acid/g of xylose. Corn steep liquor (CLS) is a good substitute for yeast extract and results in similar fermentation profiles. The organism consumes fructose, xylose, and glucose from a mixture of sugars in batch fermentation. Arabinose, mannose, and galactose are consumed only slightly. This organism loses viability on fed-batch operation, even with supplementation of all the required nutrients. In fed-batch fermentation with CSL supplementation, d-xylulose (an intermediate in the xylose metabolic pathway) accumulates in large quantities.     

Ethanol fermentation in a tower fermenter using self-aggregatingSaccharomyces uvarum

A self-aggregating strain ofSaccharomyces uvarum (U4) was used as a biocatalyst to carry out continuous ethanol fermentation in a tower fermentor equipped with a cell separator. Cell aggregates (2–3 mm) formed a stable packed bed in the fermentor, and the cell separator retained yeast cells effectively. Corn steep liquor was used as a nitrogen source for the fermentation of corn syrup and black strap molasses. An ethanol productivity of 54 g/L/h was reached using corn syrup at a dilution rate of 0.7/h, and sugar concentration in the feed was 15% (w/v). For molasses fermentation, an ethanol productivity of 22 g/L/h was obtained at a dilution rate of 0.7/h, and sugar concentration in the feed was 12.5% (w/v). Ethanol yields obtained from tower fermentation are higher than those obtained from flask fermentation (96% for corn syrup fermentation and 92% for molasses fermentation). No significant loss in fermentation activity was observed after 3 mo of operation.