Optimization of l-(+)-lactic acid production using pelletized filamentous Rhizopus oryzae NRRL 395

Lactic acid is used as a food additive for flavor and preservation and a precursor in the development of poly-lactic acid, a product used to make biodegradable plastics and textiles. Rhizopus oryzae NRRL 395 is known to be a strain that produces optically pure L: -(+)-lactic acid. The morphology of Rhizopus cultures is complex, forming filamentous, clumps, and pellet mycelia. Different morphology growth has significant effects on lactic acid production. In bioreactors, the filamentous or clump mycelia increase the viscosity of the medium, wrap around impellers, and block the nutrient transportation, leading to a decrease in production efficiency and bioreactor performance. Growing fungi in pellet form can significantly improve these problems. In this study, factors that affect lactic acid production in pelletized flask cultures using R. oryzae NRRL 395 were investigated in detail. Completely randomized designs were used to determine the influence of culture temperature, time, concentration of glucose, and inoculum size. Lactic acid fermentation using clump and pellet morphologies were performed in a 5 L fermentor at the optimal values obtained from flask culture. Finally, fed-batch culture was used to enhance the lactate concentration in broth. The final lactate concentration of fed-batch culture reached 92 g/L. The data presented in the article can provide useful information on optimizing lactic acid production using alternative source materials.     

Enhanced <Emphasis Type="SmallCaps">l</Emphasis><Emphasis Type="Italic">-</Emphasis>(+)-Lactic Acid Production by an Adapted Strain of <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> using Corncob Hydrolysate

Corncob is an economic feedstock and more than 20 million tons of corncobs are produced annually in China. Abundant xylose can be potentially converted from the large amount of hemicellulosic materials in corncobs, which makes the crop residue an attractive alternative substrate for a value-added production of a variety of bioproducts. Lactic acid can be used as a precursor for poly-lactic acid production. Although current industrial lactic acid is produced by lactic acid bacteria using enriched medium, production by Rhizopus oryzae is preferred due to its exclusive formation of the l-isomer and a simple nutrition requirement by the fungus. Production of l-(+)-lactic acid by R. oryzae using xylose has been reported; however, its yield and conversion rate are poor compared with that of using glucose. In this study, we report an adapted R. oryzae strain HZS6 that significantly improved efficiency of substrate utilization and enhanced production of l-(+)-lactic acid from corncob hydrolysate. It increased l-(+)-lactic acid final concentration, yield, and volumetric productivity more than twofold compared with its parental strain. The optimized growth and fermentation conditions for Strain HZS6 were defined.     

In Vivo Regulation of Alcohol Dehydrogenase and Lactate Dehydrogenase in <Emphasis Type="Italic">Rhizopus Oryzae</Emphasis> to Improve <Emphasis Type="SmallCaps">l</Emphasis>-Lactic Acid Fermentation

Rhizopus oryzae is becoming more important due to its ability to produce an optically pure l-lactic acid. However, fermentation by Rhizopus usually suffers from low yield because of production of ethanol as a byproduct. Limiting ethanol production in living immobilized R. oryzae by inhibition of alcohol dehydrogenase (ADH) was observed in shake flask fermentation. The effects of ADH inhibitors added into the medium on the regulation of ADH and lactate dehydrogenase (LDH) as well as the production of cell biomass, lactic acid, and ethanol were elucidated. 1,2-diazole and 2,2,2-trifluroethanol were found to be the effective inhibitors used in this study. The highest lactic acid yield of 0.47 g/g glucose was obtained when 0.01 mM 2,2,2-trifluoroethanol was present during the production phase of the pregrown R. oryzae. This represents about 38% increase in yield as compared with that from the simple glucose fermentation. Fungal metabolism was suppressed when iodoacetic acid, N-ethylmaleimide, 4,4′-dithiodipyridine, or 4-hydroxymercury benzoic acid were present. Dramatic increase in ADH and LDH activities but slight change in product yields might be explained by the inhibitors controlling enzyme activities at the pyruvate branch point. This showed that in living R. oryzae, the inhibitors regulated the flux through the related pathways.     

Production of α-amylase with Aspergillus oryzae on spent brewing grain by solid substrate fermentation

Ten Aspergillus oryzae strains were screened in solid substrate fermentation for α-amylase production on spent brewing grain (SBG) and on corn fiber. SBG proved to be a better substrate for enzyme production than corn fiber. A Plackett-Burman experimental design was used to optimize the medium composition for the best strain. Solid substrate fermentation on optimized medium with A. oryzae NRRL 1808 (=ATCC 12892) strain in stationary 500-mL Erlenmeyer flask culture yielded 4519 U of α-amylase/g of dry matter substrate in 3 d. The whole solid substrate fermentation material (crude enzyme, in situ enzyme) may be considered a cheap biocatalytic material for animal feed rations and for bioalcohol production from starchy materials.     

Studying pellet formation of a filamentous fungus Rhizopus oryzae to enhance organic acid production

Using pelletized fungal biomass can effectively improve the fermentation performance for most of fugal strains. This article studied the effects of inoculum and medium compositions such as potato dextrose broth (PDB) as carbon source, soybean peptone, calcium carbonate, and metal ions on pellet formation of Rhizopus oryzae. It has been found that metal ions had significantly negative effects on pellet formation whereas soybean peptone had positive effects. In addition PDB and calcium carbonate were beneficial to R. oryzae for growing small smooth pellets during the culture. The study also demonstrated that an inoculum size of less than 1.5x10(9) spores/L had no significant influence on pellet formation. Thus, a new approach to form pellets has been developed using only PDB, soybean peptone, and calcium carbonate. Meanwhile, palletized fungal fermentation significantly enhanced organic acid production. Lactic acid concentration reached 65.0 g/L in 30 h using pelletized R. oryzae NRRL 395, and fumeric acid concentration reached 31.0 g/L in 96 h using pelletized R. oryzae ATCC 20344.     

Enhanced Fumaric Acid Production from Brewery Wastewater and Insight into the Morphology of Rhizopus oryzae 1526

The present work explores brewery wastewater as a novel substrate for fumaric acid production employing the filamentous fungal strain Rhizopus oryzae 1526 through submerged fermentation. The effects of different parameters such as substrate total solid concentrations, fermentation pH, incubation temperature, flask shaking speed, and inoculum size on the fungal morphologies were investigated. Different morphological forms (mycelium clumps, suspended mycelium, and solid/hairy pellets) of R. oryzae 1526 were obtained at different applied fermentation pH, incubation temperature, flask shaking speed, and inoculum size. Among all the obtained morphologies, pellet morphology was found to be the most favorable for enhanced production of fumaric acid for different studied parameters. Scanning electron microscopic investigation was done to reveal the detailed morphologies of the pellets formed under all optimized conditions. With all the optimized growth conditions (pH 6, 25 °C, 200 rpm, 5 % (v/v) inoculum size, 25 g/L total solid concentration, and pellet diameter of 0.465?±?0.04 mm), the highest concentration of fumaric acid achieved was 31.3?±?2.77 g/L. The results demonstrated that brewery wastewater could be used as a good substrate for the fungal strain R. oryzae 1526 in submerged fermentation for the production of fumaric acid.     

Production of l-lactic acid byRhizopus oryzae in a bubble column fermenter

Two distinctive forms of growth (mycelial filamentous and mycelial pellets) ofRhizopus oryzae were obtained by manipulating the initial pH of the medium with the controlled addition of CaCO₃ in a bubble fermenter. In the presence of CaCO₃, diffused filamentous growth was obtained when the initial pH of the substrate was 5.5. In the absence of CaCO3, mycelial pellet growth was obtained when the initial pH was 2.0. The fermentation study indicated that the mycelial growth has a shorter lag period before the onset of acid formation. Both physical forms of growth ofRhizopus exhibited a high yield of L-lactic acid in the bubble fermenter when the initial glucose concentration exceeded 70 g/L. A final lactic acid concentration of 62 g/L was produced by the filamentous form ofRhizopus from 78 g/L glucose after 27 h. This showed a weight yield of 80% of glucose consumed, with an average specific productivity of 1.46 g/h/g. Similarly, the pellet form ofRhizopus produced a final lactic acid concentration of 66 g/L from 76 g/L glucose after 43 h, with a weight yield of 86% and an average specific productivity of 1.53 g/h/g.     

Mycelial pellet formation by Rhizopus oryzae ATCC 20344

Factors in a cultivation medium affecting fungal growth morphology and funmaric acid production by Rhizopus oryzae ATCC 20344 were investigated. These factors included the initial pH value and trace metals such as zinc, magnesium, iron, and manganese in the cultivation medium. It was found that a significant change in the growth morphology of R. oryzae ATCC 20344 occurs when the initial pH value is varied. A lower initial pH value in the cultivation medium was inhibitory to fungal growth, and fast growth in the cultivation medium at a higher initial pH value promoted, the formation of large pellets or filamentous forms. Trace metals in the cultivation media also had significant effects on pellet formation and fumaric acid fermentation.     

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.     

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.     

Membrane-mediated extractive fermentation for lactic acid production from cellulosic biomass

Lactic acid production from cellulosic biomass by cellulase andLactobacillus delbrueckii was studied in a fermenter-extractor employing a microporous hollow fiber membrane (MHF). This bioreactor system was operated under a fed-batch mode with continuous removal of lactic acid by anin situ extraction. A tertiary amine (Alamine 336) was used as an extractant for lactic acid. The extraction capacity of Alamine 336 is greatly enhanced by addition of alcohol. Long-chain alcohols serve well for this purpose since they are less toxic to micro-organism. Addition of kerosene, a diluent, was necessary to reduce the solvent viscosity. A solvent mixture of 20% Alamine 336, 40% oleyl alcohol, and 40% kerosene was found to be most effective in the extraction of lactic acid. Progressive change of pH from an initial value of 5.0 down to 4.3 has significantly improved the overall performance of the simultaneous saccharification and extractive fermentation over that of constant pH operation. The change of pH was applied to promote cell growth in the early phase, and extraction in the latter phase.     

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.     

Optimization of Effect Factors for Mycelial Growth and Exopolysaccharide Production by Schizophyllum commune

This paper is concerned with the optimization of effect factors for mycelial growth and exopolysaccharide production by Schizophyllum commune by one-factor-at-a-time and orthogonal methods. The one-factor-at-a-time method was adopted to investigate the effects of six different compounds (sodium carboxymethylcellulose, l-glutamic acid, V_B1, naphthalene acetic acid, oleic acid, and Tween 80) on mycelial growth and exopolysaccharide production. Among these factors, oleic acid, V_B1 and Tween 80 were identified to be the most important factors. Subsequently, the concentration of oleic acid, V_B1 and Tween 80 were optimized using the orthogonal matrix method. The effects of the factors on the mycelial growth of S. commune were in the order of oleic acid > V_B1 > Tween 80, and those on exopolysaccharide production were in the same order. The optimal concentration for mycelia and exopolysaccharide were determined as oleic acid 0.1% (v/v), V_B1 0.5 mg/L, and Tween 80 6 mg/L. The subsequent verification experiments confirmed the validity of the models. Under this optimized conditions in shake flask culture, the mycelial yield and exo-biopolymer production were 25.93 and 2.79 g/L, respectively, which were considerably higher than those obtained in the preliminary studies. The result was further confirmed in a 7-L fermentor experiments.     

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.     

Production of lactic acid from pulp mill solid waste and xylose using Lactobacillus delbrueckii (NRRL B445)

Using the simultaneoussaccharification and fermentation (SSF) technique, pulp mill solid waste cellulose was converted into glucose using cellulase enzyme and glucose into lacticacid using NRRL B445. SSF experiments were conducted at various pH levels, temperatures, and nutrient concentrations, and the lactic acid yield ranged from 86 to 97%. The depletion of xylose in SSF was further investigated by inoculating NRRL B445 into a xylose-only medium. On prolonged incubation, depletion of xylose with lactic acid production was observed. An experimental procedure with a nonglucose medium was developed to eliminate the lag phase. From xylose fermentation, Lactobacillus delbrueckii yielded 88–92% lactic acid and 2–12% acetic acid.     

Efficient 1,3-propanediol Production by Fed-Batch Culture of Klebsiella Pneumoniae: The Role of pH Fluctuation

The fermentative production of 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae under different fed-batch strategies was investigated. pH-stat fed-batch strategies proved to be not effective for economical 1,3-PD production for the existence of relatively high concentration of byproducts and residual glycerol at the end of the fermentation. However, in the pH-stat fed-batch strategy, an important phenomenon was observed that the yields of two main byproducts, 2,3-butanediol and lactic acid, were closely related to pH value. The dominant byproduct was 2,3-butanediol at a pH value of 5.0 to 6.5 but changed to be lactic acid at a pH value of 7.1 to 8.0. Based on the analysis of the phenomenon, a self-protection mechanism in K. pneumoniae, namely that the growing K. pneumoniae cells switch the metabolic pathways responding to environmental pH changes, was proposed. Thus a kind of feeding strategy was further applied during which the pH value was fluctuated between 6.3 and 7.3 periodically by feeding glycerol–ammonia mixture and sulphuric acid to make the metabolic pathways of 2,3-butanediol and lactic acid sub-active under the periodical low or high pH stress. At last, efficient 1,3-PD production was fulfilled under this fed-batch strategy, and the best results were achieved leading to 70 g/l 1,3-PD with a yield of 0.70 mol/mol glycerol and productivity of 0.97 g/l/h, while the two main byproducts and residual glycerol were under low concentrations.     

Effects of Pellet Characteristics on L-Lactic Acid Fermentation by R. oryzae: Pellet Morphology,Diameter, Density,and Interior Structure

The effects of pellet morphology, diameter, density, and interior structure on L-lactic acid fermentation by Rhizopus oryzae were characterized for different inoculum sizes and concentrations of peptone and CaCO₃. Inoculum size was the most important factor determining pellet formation and diameter. The diameter decreased with increasing inoculum size, and larger pellets were observed for lower inoculum sizes. Peptone concentration had the greatest effect on pellet density, which increased with increasing peptone concentration. L-lactic acid production depended heavily on pellet density but not on pellet diameter. Low-density pellets formed easily under conditions of low peptone concentration and often had a relatively hollow structure, with a thin condensed layer surrounding the pellet and an extraordinarily loose biomass or hollow center. As expected, this structure greatly decreased production. The production of L-lactic acid increased until the density reached a certain level (50–60 kg/m³), in which the compact part was distributed homogeneously in the thick outer layer of the pellet and loose in the central layer. Homogeneously structured, denser pellets had limited mass transfer, causing a lower overall turnover rate. However, the interior structure remained nearly constant throughout all fermentation phases for pellets with the same density. CaCO₃ concentration only had a slight influence on pellet diameter and density, probably because it increases spore germination and filamentous hypha extension. This work also provides a new analysis method to quantify the interior structure of pellets, thus giving insight into pellet structure and its relationship with productivity.     

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.     

Steady-state measurements of lactic acid production in a wild-type and a putative d-lactic acid dehydrogenase-negative mutant of zymomonas mobilis

This work represents a continuation of our investigation into environmental conditions that promote lactic acid synthesis by Zymomonas mobilis. The characteristic near theoretical yield of ethanol from glucose by Z. mobilis can be compromised by the synthesis of d- and l-lactic acid. The production of lactic acid is exacerbated by the following conditions: pH 6.0, yeast extract, and reduced growth rate. At a specific growth rate of 0.048/h, the average yield of dl-lactate from glucose in a yeast extract-based medium at pH 6.0 was 0.15 g/g. This represents a reduction in ethanol yield of about 10% relative to the yield at a growth rate of 0.15/h. Very little lactic acid was produced at pH 5.0 or using a defined salts medium (without yeast extract) Under permissive and comparable culture conditions, a tetracycline-resistant, d-ldh negative mutant produced about 50% less lactic acid than its parent strain Zm ATCC 39676. d-lactic acid was detected in the cell-free spent fermentation medium of the mutant, but this could be owing to the presence of a racemase enzyme. Under the steady-state growth conditions provided by the chemostat, the specific rate of glucose consumption was altered at a constant growth rate of 0.075/h. Shifting from glucose-limited to nitrogen-limited growth, or increasing the temperature, caused an increase in the specific rate of glucose catabolism. There was good correlation between an increase in glycolytic flux and a decrease in lactic acid yield from glucose. This study points to a mechanistic link between the glycolytic flux and the control of end-product glucose metabolism. Implications of reduced glycolytic flux in pentose-fermenting recombinant Z. mobilis strains, relative to increased byproduct synthesis, is discussed.     

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