Production of Lactic Acid from Sucrose: Strain Selection, Fermentation, and Kinetic Modeling

Lactic acid is an important product arising from the anaerobic fermentation of sugars. It is used in the pharmaceutical, cosmetic, chemical, and food industries as well as for biodegradable polymer and green solvent production. In this work, several bacterial strains were isolated from industrial ethanol fermentation, and the most efficient strain for lactic acid production was selected. The fermentation was conducted in a batch system under anaerobic conditions for 50 h at a temperature of 34 °C, a pH value of 5.0, and an initial sucrose concentration of 12 g/L using diluted sugarcane molasses. Throughout the process, pulses of molasses were added in order to avoid the cell growth inhibition due to high sugar concentration as well as increased lactic acid concentrations. At the end of the fermentation, about 90% of sucrose was consumed to produce lactic acid and cells. A kinetic model has been developed to simulate the batch lactic acid fermentation results. The data obtained from the fermentation were used for determining the kinetic parameters of the model. The developed model for lactic acid production, growth cell, and sugar consumption simulates the experimental data well.     

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.     

Keratinase Production by Endophytic <Emphasis Type="Italic">Penicillium</Emphasis> spp. Morsy1 Under Solid-State Fermentation Using Rice Straw

Among all endophytic keratinolytic fungal isolates recovered from marine soft coral Dendronephthya hemprichii, Penicillium spp. Morsy1 was selected as the hyperactive keratinolytic strain under solid substrate fermentation of different agriculture and poultry wastes. The optimization of extraction process, physicochemical parameters affecting the keratinase production in solid-state fermentation, and the purified keratinase parameters were studied. Maximum keratinase activity (1,600 U g⁻¹, initial dry substrate) was recovered from moldy bran with 0.1% Tween 80. The optimized production conditions were rice straw as carbon source, pH of medium 6, growth temperature 26 °C, initial moisture content of 80% (v/w), inoculum size of 10⁵ spores ml⁻¹, and an average particle size of the substrate 0.6 mm (3,560 U g⁻¹, initial dry substrate after 5 days of fermentation). Two types of keratinase (Ahm1 and Ahm2) were purified from the culture supernatant through ammonium sulfate precipitation, DEAE-Sepharose, and gel filtration chromatography. Enzyme molecular weights were 19 kDa (Ahm1) and 40 kDa (Ahm2). The kinetic parameters of purified keratinases were optimized for the hydrolysis of azokeratin by Ahm1 (pH 7.0–8.0, stable in pH range of 6.0 to 8.0 at 50 °C) and Ahm2 enzymes (pH 10.0–11.0, stable in pH range of 6.0 to 11.0 at 60–65 °C). Whereas inhibitors of serine (phenylmethylsulfonyl fluoride) and cysteine (iodoacetamide) proteases had minor effects on both Ahm1 and Ahm2 activity, both keratinases were strongly inhibited by chelating agents EDTA and EGTA. These findings suggest that serine and cysteine residues are not involved in the catalytic mechanisms, and they are metalloproteases.     

Application of a pH Feedback-Controlled Substrate Feeding Method in Lactic Acid Production

Substrate concentration in lactic acid fermentation broth could not be controlled well by traditional feeding methods, including constant, intermittent, and exponential feeding methods, in fed-batch experiments. A simple feedback feeding method based on pH was proposed to control pH and substrate concentration synchronously to enhance lactic acid production in fed-batch culture. As the linear relationship between the consumption amounts of alkali and that of substrate was concluded during lactic acid fermentation, the alkali and substrate in the feeding broth were mixed together proportionally. Thus, the concentration of substrate could be controlled through the adjustment of pH automatically. In the fed-batch lactic acid fermentation with Lactobacillus lactis-11 by this method, the residual glucose concentration in fermentation broth was controlled between 4.1 and 4.9 g L⁻¹, and the highest concentration of lactic acid, maximum cell dry weight, volumetric productivity of lactic acid, and yield were 96.3 g L⁻¹, 4.7 g L⁻¹, 1.9 g L⁻¹ h⁻¹, and 0.99 g lactic acid per gram of glucose, respectively, compared to 82.7 g L⁻¹, 3.31 g L⁻¹, 1.7 g L⁻¹ h⁻¹, and 0.92 g lactic acid per gram of glucose in batch culture. This feeding method was simple and easily operated and could be feasible for industrial lactic acid production in the future.     

Kinetic Analysis and pH-Shift Control Strategy for Propionic Acid Production with <Emphasis Type="Italic">Propionibacterium Freudenreichii</Emphasis> CCTCC M207015

The production of propionic acid by Propionibacterium freudenreichii CCTCC M207015 was investigated in a 7.5-l stirred-tank fermentor. Batch fermentations by P. freudenreichii CCTCC M207015 at various pH values ranging from 5.5 to 7.0 were studied. Based on the analysis of the time course of specific cell growth rate (μ _x) and specific propionic acid formation rate (μ _p), a two-stage pH-shift control strategy was proposed. At first 48 h, pH was controlled at 6.5 to obtain the maximal μ _x, subsequently pH 6.0 was used to maintain high μ _p to enhance the production of propionic acid. By applying this pH-shift control strategy in propionic acid fermentation, the maximal propionic acid and glucose conversion efficiency had a significant improvement and reached 19.21 g/l and 48.03%, respectively, compared with those of constant pH operation (14.58 g/l and 36.45%). Fed-batch fermentation with pH-shift control strategy was also applied to produce propionic acid; the maximal propionic acid yield and glucose conversion efficiency reached 25.23 g/l and 47.76%, respectively.     

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.     

Effects of pH and Temperature on Recombinant Manganese Peroxidase Production and Stability

The enzyme manganese peroxidase (MnP) is produced by numerous white-rot fungi to overcome biomass recalcitrance caused by lignin. MnP acts directly on lignin and increases access of the woody structure to synergistic wood-degrading enzymes such as cellulases and xylanases. Recombinant MnP (rMnP) can be produced in the yeast Pichia pastoris αMnP1-1 in fed-batch fermentations. The effects of pH and temperature on recombinant manganese peroxidase (rMnP) production by P. pastoris αMnP1-1 were investigated in shake flask and fed-batch fermentations. The optimum pH and temperature for a standardized fed-batch fermentation process for rMnP production in P. pastoris αMnP1-1 were determined to be pH 6 and 30 °C, respectively. P. pastoris αMnP1-1 constitutively expresses the manganese peroxidase (mnp1) complementary DNA from Phanerochaete chrysosporium, and the rMnP has similar kinetic characteristics and pH activity and stability ranges as the wild-type MnP (wtMnP). Cultivation of P. chrysosporium mycelia in stationary flasks for production of heme peroxidases is commonly conducted at low pH (pH 4.2). However, shake flask and fed-batch fermentation experiments with P. pastoris αMnP1-1 demonstrated that rMnP production is highest at pH 6, with rMnP concentrations in the medium declining rapidly at pH less than 5.5, although cell growth rates were similar from pH 4–7. Investigations of the cause of low rMnP production at low pH were consistent with the hypothesis that intracellular proteases are released from dead and lysed yeast cells during the fermentation that are active against rMnP at pH less than 5.5.     

Utilization of Horticultural Waste for Laccase Production by <Emphasis Type="Italic">Trametes versicolor</Emphasis> Under Solid-State Fermentation

Horticultural waste collected from a landscape company in Singapore was utilized as the substrate for the production of laccase under solid-state fermentation by Trametes versicolor. The effects of substrate particle size, types of inducers, incubation temperature and time, initial medium pH value, and moisture content on laccase production were investigated. The optimum productivity of laccase (8.6 U/g substrate) was achieved by employing horticultural waste of particle size greater than 500 μm and using veratryl alcohol as the inducer. The culture was at 30 °C for 7 days at moisture content of solid substrate of 85% and initial pH 7.0. The decolorization was also investigated in order to assess the degrading capability of the ligninolytic laccase obtained in the above-mentioned cultures. The decolorization degree of a model dye, phenol red, was around 41.79% in 72 h of incubation. By far, this is the first report on the optimization of laccase production by T. versicolor under solid-state fermentation using horticultural waste as the substrate.     

Kinetic and Stoichiometric Parameters in the Production of Carotenoids by <Emphasis Type="Italic">Sporidiobolus salmonicolor</Emphasis> (CBS 2636) in Synthetic and Agroindustrial Media

With the objective of determining the kinetic behavior (growth, substrate, pH, and carotenoid production) and obtain the stoichiometric parameters of the fermentative process by Sporidiobolus salmonicolor in synthetic and agroindustrial media, fermentations were carried out in shaken flasks at 25°C, 180 rpm, and initial pH of 4.0 for 120 h in the dark, sampling every 6 h. The maximum concentrations of total carotenoids in synthetic (913 μg/L) and agroindustrial (502 μg/L) media were attained approximately 100 h after the start of the fermentative process. Carotenoid bioproduction is associated with cell growth and the ratio between carotenoid production and cell growth (Y _P/X) is 176 and 163 μg/g in the synthetic and agroindustrial media, respectively. The pH of the agroindustrial fermentation medium varied from 4.2 to 8.5 during the fermentation. The specific growth rate (μ _X) for S. salmonicolor in synthetic and agroindustrial media was 0.07 and 0.04 h⁻¹, respectively. The synthetic medium allowed for greater productivity, obtaining maximum cell productivity (P _x) of 0.08 g L⁻¹ h⁻¹ and maximum total carotenoid productivity (P _car) of 14.2 μg L⁻¹ h⁻¹. Knowledge of the kinetics of a fermentative process is of extreme importance when transposing a laboratory experiment to an industrial scale, as well as making a quantitative comparison between different culture conditions.     

Cellulase Production by <Emphasis Type="Italic">Streptomyces viridobrunneus</Emphasis> SCPE-09 Using Lignocellulosic Biomass as Inducer Substrate

An actinomycete strain, isolated from a soil sample under a sugar cane plantation in Brazil and identified as Streptomyces viridobrunneus SCPE-09, was selected as a promising cellulolytic strain, and tested for its ability to produce cellulases from agro-industrial residues. Sugar cane bagasse or wheat bran was tested as carbon source, and corn steep liquor tested as nitrogen source. Different concentrations of carbon and nitrogen were tested using factorial design to identify optimal cellulose production. The results showed that media containing wheat bran 2.0% (w/v) and corn steep liquid 0.19% (w/v) lead to the highest production, 2.0 U mL⁻¹ of CMCase, obtained on the fifth day of fermentation. The pH and temperature profile showed optimal activity at pH 4.9 and 50°C. As for thermostability, endoglucanases were most tolerant at 50°C, retaining more than 80% of maximal activity even after 2 h of incubation. Zymogram analyses using supernatant from growth under optimized conditions revealed the presence of two CMCase bands with apparent molecular masses of 37 and 119 kDa. The combination of pH tolerance and CMCase production from agro-industrial residues by S. viridobrunneus SCPE-09 offers promise for future bioethanol biotechnologies.     

Thermal Drying of <Emphasis Type="BoldItalic">Lactobacillus delbrueckii</Emphasis> subsp. <Emphasis Type="BoldItalic">bulgaricus</Emphasis> and its Efficient Use as Starter for Whey Fermentation and Unsalted Cheese Making

Lactobacillus bulgaricus grown on whey was dried by a simple thermal drying method at the range 35–55°C and its efficiency for lactic acid fermentation of whey was evaluated. Drying of cells in whey suspension in the examined temperature range did not affect significantly their viability (82–87% survival), indicating a protective effect of whey as both growth and drying medium. The kinetics of fermentation of whey and mixtures of whey/molasses using the dried culture were comparable to those of non-dried cells, and only low pH had a detrimental effect on the fermentation ability of the dried cells. Furthermore, dried L. bulgaricus, free or immobilized on casein coagulates, was used as starter for the production of unsalted hard-type cheese. The effects of the amount of starter culture and the immobilization technique, the evolution of microbial counts, and the sensory properties of the produced cheeses were evaluated during ripening at various temperatures.     

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     

Production of Nisin Z by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> Isolated from Dahi

Lactococcus lactis CM1, an isolate from homemade “Dahi,” a traditional fermented milk from India, used maltose as carbon source to produce a high level of bacteriocin. The bacterial cell mass and the bacteriocin production correlated with the initial pH of the medium and were highest when the initial pH was 11.0. The level of bacteriocin reached its peak at the late log phase with concomitant reduction of culture pH to 4.2, regardless of the initial pH of the medium. A combination of maltose and an initial medium pH of 11 resulted in the highest bacteriocin production. The antibacterial spectrum of the bacteriocin was closely similar to that of nisin and it inhibited a number of food spoilage and pathogenic bacteria. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, the compound migrated close to the position of nisin (3.5 kDa). However, it had higher stability than nisin at a wide range of pH and temperature. PCR amplification using nisin gene-specific primers and sequencing of the amplified DNA revealed the structural gene for the bacteriocin to be identical to that of nisZ.     

Production of Lactic Acid from Raw Sweet Potato Powders by Rhizopus Oryzae Immobilized in Sodium Alginate Capsules

Rhizopus oryzae immobilized in calcium alginate was applied in lactic acid fermentation with unhydrolyzed raw sweet potato powders as the sole carbon source. The effects of sodium alginate concentration, calcium chloride concentration, and the immobilized bead diameter on lactic acid production were investigated. Increase in sodium alginate concentration during the gelation process would harden the immobilized capsule, which led to a decrease in lactic acid production. The increase in calcium chloride would increase the thickness of the immobilized capsule, which would increase the mass transfer resistance. Nevertheless, while the calcium chloride was lower than 15%, it would not have obvious effects on lactic acid production. A larger bead could have more space for cell growth, which led to the maximum lactic acid production observed at the 5-mm bead diameter. Moreover, results of repeated-batch operation suggested that immobilized cells could have higher stability in lactic acid production than free cells. The total cumulative lactic acid in immobilized-cell operation could increase by 55% as compared with free-cell operation after 216 h (seven repeated-batches), and no loss of amylolytic activity was observed. The results indicated that immobilized R. oryzae by Ca-alginate could be suitable for lactic acid production from unhydrolyzed raw potato powders.     

Influence of Osmotic Stress on Fermentative Production of Succinic Acid by <Emphasis Type="Italic">Actinobacillus succinogenes</Emphasis>

This study investigated the influence of osmotic stress on succinic acid production by Actinobacillus succinogenes NJ113. Both cell growth and succinic acid production were inhibited with the increase in osmotic stress of the medium. The use of three different osmoprotectants in the production of succinic acid was studied in order to decrease the inhibitory effects of osmotic stress during fermentation. Results indicated that proline offers optimal osmoprotection in the production of succinic acid by A. succinogenes NJ113. In tests of batch fermentation, the maximum cell concentration was observed to be 5.36 g DCW/L after the addition of 25 mmol/L proline to the fermentation medium. The cell concentration was 24% higher than that noted for the control. A total quantity of 56.2 g/L of succinic acid was produced, with a production rate of 1 g/L per hour, after 56 h of fermentation. The concentration and productivity of succinic acid was observed to be increased by 22.2% and 22%, respectively, as compared with the control. The specific activity levels of key enzymes in the metabolic network was noted to be higher following the addition of proline, particularly in the later stages of fermentation. This method of enhancing succinic acid production by the addition of an osmoprotectant may potentially provide an alternative approach for enhanced production of other organic acids.     

Stimulation of Nisin production from whey by a mixed culture of Lactococcus lactis and Saccharomyces cerevisiae

The production of nisin, a natural food preservative, by Lactococcus lactis subsp. lactis (ATCC 11454) is associated with the simultaneous formation of lactic acid during fermentation in a whey-based medium. As a result of the low concentration and high separation cost of lactic acid, recovering lactic acid as a product may not be economical, but its removal from the fermentation broth is important because the accumulation of lactic acid inhibits nisin biosynthesis. In this study, lactic acid removal was accomplished by biological means. A mixed culture of L. lactis and Saccharomyces cerevisiae was established in order to stimulate the production of nisin via the in situ consumption of lactic acid by the yeast strain, which is capable of utilizing lactic acid as carbon source. The S. cerevisiae in the mixed culture did not compete with the nisin-producing bacteria because the yeast does not utilize lactose, the major carbohydrate in whey for bacterial growth and nisin production. The results showed that lactic acid produced by the bacteria was almost totally utilized by the yeast and the pH of the mixed culture could be maintained at around 6.0. Nisin production by the mixed culture system reached 150.3 mg/L, which was 0.85 times higher than that by a pure culture of L. lactis.     

Lactic Acid Production from a Whole Slurry of Acid-Pretreated Spent Coffee Grounds by Engineered Saccharomyces cerevisiae

Spent coffee grounds (SCG) generated after coffee extraction are the main byproduct of the coffee industry. Valorization of the SCG has been increasingly focused following considerable attention in coffee consumption. Lactic acid bacteria fermentation is the primary source of generation of lactic acid, a monomer of polylactic acid that has various industrial applications; however, because of the low tolerance of lactic acid bacteria to toxic compounds, it is necessary to apply Saccharomyces cerevisiae to produce lactic acid whose tolerance to toxic compounds is higher. In this study, we evaluated the feasibility of using SCG as substrate for the production of lactic acid by S. cerevisiae strain expressing heterologous lactate dehydrogenase. The fermentation profiles of the engineered yeast showed that lactic acid production was promoted by xylose addition. From simultaneous saccharification and fermentation (SSF) using a whole slurry of acid-pretreated SCG, containing high amounts of hemicellulose fractions, lactic acid (0.11 g) and ethanol (0.10 g) per g SCG were obtained after 24 h of SSF, of which yields were 413% and 221% higher, respectively, than those of washed pretreated SCG. Thus, fermentation of whole slurry SCG by engineered S. cerevisiae is a suitable way of lactic acid production, selectively.

     

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.     

Characterization of Cellulolytic Extract from <Emphasis Type="Italic">Pycnoporus sanguineus</Emphasis> PF-2 and Its Application in Biomass Saccharification

The aim of this work was to evaluate the biochemical features of the white-rot fungi Pycnoporus sanguineus cellulolytic complex and its utilization to sugarcane bagasse hydrolysis. When cultivated under submerged fermentation using corn cobs as carbon source, P. sanguineus produced high FPase, endoglucanase, β-glucosidase, xylanase, mannanase, α-galactosidase, α-arabinofuranosidase, and polygalacturonase activities. Cellulase activities were characterized in relation to pH and temperature. β-Glucosidase and FPase activities were higher at 55 °C, pH 4.5, and endoglucanase activity was higher at 60 °C, in a pH range of 3.5–4.0. All cellulase activities were highly stable at 40 and 50 °C through 48 h of pre-incubation. Crude enzymatic extract from P. sanguineus was applied in a saccharification experiment using acid-treated and alkali-treated sugarcane bagasse as substrate, and the hydrolysis yields were compared to that obtained by a commercial cellulase preparation. Reducing sugar yields of 60.4% and 64.0% were reached when alkali-treated bagasse was hydrolyzed by P. sanguineus extract and commercial cellulase, respectively. Considering the glucose production, it was observed that P. sanguineus extract and commercial cellulase ensured yields of 22.6% and 36.5%, respectively. The saccharification of acid-treated bagasse was lower than that of alkali-treated bagasse regardless of the cellulolytic extract. The present work showed that P. sanguineus has a great potential as an enzyme producer for biomass saccharification.     

Lactic acid production through cell-recycle repeated-batch bioreactor

The effect of various nitrogen sources on cell growth and lactic acid production was investigated. The most effective nitrogen source was yeast extract; more yeast extract gave higher cell growth and lactic acid productivity. Yeast extract dosage and cell growth were proportional up to a yeast extract concentration of 30 g/L, and lactic acid productivity was linearly correlated up to a yeast extract dosage of 25 g/L. However, increasing the yeast extract content raises the total production cost of lactic acid. Therefore, we attempted to find the optimum yeast extract dosage for a repeated-batch operation with cell recycling. The results show that when using Enterococcus faecalis RKY1 only 26% of the yeast extract dosage for a conventional batch fermentation was sufficient to produce the same amount of lactic acid, whereas the lactic acid concentration in the product stream (92–94 g/L) and lactic acid productivity (6.03–6.20 g/[L·h]) were similar to those of a batch operation. Furthermore, long-term stability was established.