Effect of operating conditions on biomass growth during citric acid production by solid-state fermentation

The effect of aeration, agitation, and bed loading on biomass and product concentration during citric acid biosynthesis by solid-state fermentation was investigated. For this purpose, Aspergillus niger S was cultivated on sugar beet pulp in a 4.5 dm³ horizontal rotating drum bioreactor. The results suggest that the parameters examined have a remarkable effect on the quantity of biomass being formed and on the product concentration. The maximum citric acid production (about 150 g per kg of the substrate dry matter) was obtained under the following conditions: aeration rate 0.2 dm³ kg⁻¹ min⁻¹, mixing (periodical) 1 min once an hour, and bed loading 30 % of the bioreactor working volume. However, these values did not favour biomass formation. Moreover, it was found that accumulation of the product reached its maximum when the amount of biomass was minimal (approximately 252-29 g per kg of the substrate dry matter) under the conditions involved. Presented at the 34th International Conference of the Slovak Society of Chemical Engineering, Tatranské Matliare, 21–25 May 2007.     

High-yield bacillus subtilis protease production by solid-state fermentation

A Bacillus subtilis isolate was shown to be able to produce extracellular protease in solid-state fermentations (SSF) using soy cake as culture medium. A significant effect of inoculum concentration and physiological age on protease production was observed. Maximum activities were obtained for inocula consisting of exponentially growing cells at inoculum concentrations in the range of 0.7–2.0 mg g⁻¹. A comparative study on the influence of cultivation temperature and initial medium pH on protease production in SSF and in submerged fermentation (SF) revealed that in SSF a broader pH range (5–10), but the same optimum temperature (37°C), is obtained when compared to SF. A kinetic study showed that enzyme production is associated with bacterial growth and that enzyme inactivation begins before biomass reaches a maximum level for both SF and SSF. Maximum protease activity and productivity were 960 U g⁻¹ and 15.4 U g⁻¹ h⁻¹ for SSF, and 12 U mL⁻¹ and 1.3 U mL⁻¹ h⁻¹ for SF. When SSF protease activity was expressed by volume of enzyme extract, the enzyme level was 10-fold higher and the enzyme productivity 45% higher than in SF. These results indicate that this bacterial strain shows a high biotechnological potential for protease production in solid-state fermentation.     

Liquid fluidized bed starter culture ofTrichoderma reesei for cellulase production

A starter culture ofTrichoderma reesei (Rut-C30) prepared in a liquid fluidized bed reactor (LFBR) gave better growth and greater cellulase production in submerged fermentation than a conventional shake flask inoculum. The LFBR starter was prepared by first coatingT. reesei spores to 0.25 mm size corncob (1.0x10⁸g^-1) in a medium containing 1.0% corncob, 0.5 gL^-1 xylose and 0.1 gL^-1 lactose in a balanced salt solution, then fluidizing the particles in the LFBR for 36 h to allow germination of the spores, and covering the particles with an approx 30 μm thick biofilm. This biofilm that developed in constant adherence to the lignocellulosic carrier, apparently became well adapted to grow rapidly on insoluble cellulose substrates (Solca Floc), and had the enzymes of the cellulase complex induced for increased cellulase production. The LFBR starter used in a stirred tank reactor (STR) gave 15 gL^-1 biomass production and 6.5 IU mL^-1 overall cellulase activity with a volumetric productivity of 64 IU L^-1h^-1 in a 5 d fermentation, compared with a 7 d shake flask inoculum that gave 11 gL^-1 biomass and 3.2 IU mL^-1 cellulase activity, with a volumetric productivity of 31IU L^-1h^-1. The LFBR starter culture retained its viability in dry storage for 6–9 mo.     

Production of lipase by a newly isolated <Emphasis Type="Italic">Bacillus coagulans</Emphasis> under solid-state fermentation using melon wastes

Summary An extracellular lipase was produced by Bacillus coagulans by solid-state fermentation. Solid waste from melon was used as the basic nutrient source and was supplemented with olive oil. The highest lipase production (78,069 U/g) was achieved after 24h of cultivation with 1% olive oil enrichment. Enzyme had an optimal activity at 37°C and pH 7.0, and sodium dodecyl sulfate increased lipase activity. NH ₄NO₃ increased enzyme production, whereas organic nitrogen had no effect. The effect of the type of carbon sources on lipolytic enzyme production was also studied. The best results were obtained with starch and maltose (148,932 and 141,629 U/g, respectively), whereas a rather low enzyme activity was found in cultures grown on glucose and galactose (approx 118,769 and 123,622 U/g, respectively). Enzyme was inhibited with Mn⁺² and Ni⁺² by 68 and 74%, respectively. By contrast, Ca⁺² enhanced enzyme production by 5%.     

Succinic Acid Production from Cheese Whey using Actinobacillus succinogenes 130 Z

Actinobacillus succinogenes 130 Z was used to produce succinic acid from cheese whey in this study. At the presence of external CO₂ supply, the effects of initial cheese whey concentration, pH, and inoculum size on the succinic acid production were studied. The by-product formation during the fermentation process was also analyzed. The highest succinic acid yield of 0.57 was obtained at initial cheese whey concentration of 50 g/L, while the highest succinic acid productivity of 0.58 g h⁻¹ L⁻¹ was obtained at initial cheese whey concentration of 100 g/L. Increase in pH and inoculum size caused higher succinic acid yield and productivity. At the preferred fermentation condition of pH 6.8, inoculum size of 5% and initial cheese whey concentration of 50 g/L, succinic acid yield of 0.57, and productivity of 0.44 g h⁻¹ L⁻¹ were obtained. Acetic acid and formic acid were the main by-products throughout the fermentation run of 48 h. It is feasible to produce succinic acid using lactose from cheese whey as carbon resource by A. succinogenes 130 Z.     

Production of α-keto acids with alginate-entrapped whole cells of the yeastTrigonopsis variabilis

The yeast,Trigonopsis variabilis, was immobilized by entrapment in alginate. The immobilized cells containing high amounts of D-amino acid oxidase were used to convert D-amino acids to their corresponding α-keto acids.     

Citric acid production from raw glycerol by acetate mutants of Yarrowia lipolytica

Three acetate mutants of the yeast species Yarrowia lipolytica were screened using batch cultivation. The strain Y. lipolytica 1.31 was found to be the most suitable for citric acid production from raw glycerol, a by-product of biodiesel production from rapeseed oil. At the initial concentration of glycerol of 200 g dm⁻³, the citric acid production of 124.5 g dm⁻³, yield of 0.62 g g⁻¹, and productivity of 0.88 g dm⁻³ h⁻¹ were achieved. Presented at the 33rd International Conference of the Slovak Society of Chemical Engineering, Tatranské Matliare, 22–26 May 2006.     

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.     

Diminished Respirative Growth and Enhanced Assimilative Sugar Uptake Result in Higher Specific Fermentation Rates by the MutantPichia stipitis FPL-061

A mutant strain ofPichia stipitis, FPL-061, was obtained by selecting for growth on L-xylose in the presence of respiratory inhibitors. The specific fermentation rate of FPL-061, was higher than that of the parent,Pichia stipitis CBS 6054, because of its lower cell yield and growth rate and higher specific substrate uptake rate. With a mixture of glucose and xylose, the mutant strain FPL-061 produced 29.4 g ethanol/L with a yield of 0.42 g ethanol/g sugar consumed. By comparison, CBS 6054 produced 25.7 g ethanol/L with a yield of 0.35 gJg. The fermentation was most efficient at an aeration rate of 9.2 mmoles O₂ L^-1 h^-1. At high aeration rates (22 mmoles O₂ L^-1 h^-1), the mutant cell yield was less than that of the parent. At low aeration rates, (1.1 to 2.5 O₂ L^-1 h^-1), cell yields were similar, the ethanol formation rates were low, and xylitol accumulation was observed in both the strains. Both strains respired the ethanol once sugar was exhausted. We infer from the results that the mutant, P.stipitis FPL-061, diverts a larger fraction of its metabolic energy from cell growth into ethanol production.     

The Potential of Immobilized Biocatalysts for Production of Industrial Chemicals

The successful translation from conception to practice of processes based on immobilized biocatalyst technology has been slower than anticipated. There are severe barriers, both technical and economic, limiting the introduction of immobilized biocatalyst technology to replace conventional processing procedures and processes for the production of chemicals by synthetic or fermentative routes. A small number of immobilized enzyme processes are in operation commercially, the most noteworthy being in food-related processes and in the pharmaceutical industry, where they are used for carbohydrate conversions and antibiotic transformations, respectively. There does not, as yet, appear to be any large-scale industrial application of immobilized cell technology. Examples from our laboratory—immobilized yeast for ethanol production andAspergillus niger for citric acid synthesis—illustrate the problems that have to be overcome.     

Denitrification and removal of heavy metals from waste water by immobilized microorganisms

Pseudomonas fluorescens, immobilized on soft polyvinyl chloride granules containing up to 35% softeners as carbon source, was used for simultaneous removal of nitrate and heavy metals. In typical continuous column operation, a 100 mg/L nitrate input solution was reduced to a 20 mg/L output at a feeding rate of 1500 mL/h, with a capacity of 14 kg/day/m³, and with an efficiency of 79%. In the same column, Pb(NO₃)₂ concentration was reduced from 1.0 to 0.05−0.1 mg/L and ZnSO₄ concentration was reduced from 10 to 5 mg/L.Pseudomonas aeruginosa immobilized on an O₂ plasma-treated melt blown polypropylene web was used for removing 95% of a 1.7 nCi PuCl₄ activity from a nuclear plant waste water in a batch operation.     

Production of citric acid using immobilized conidia of Aspergillus niger

Conidia of Aspergillus niger were immobilized in calcium alginate gel for the production of citric acid. First, the type of the preactivation medium, together with the preactivation period, was investigated. It was found that A. niger requires a 2-d preactivation period at a 0.05 g/L NH₄NO₃ concentration. Second, preactivated cells were used to determine the effects of nitrogen concentration and the flow rate of oxygen and air on the production of citric acid. Maximum citric acid production was attained with medium containing 0.01 g/L of NH₄NO₃. The rate of citric acid production in the nitrogenous medium was 33% higher when oxygen was used instead of air during the production phase. This corresponds to an increase of 85% when compared to production when neither oxygen nor air was fed into the system. In the nonnitrogenous medium citric acid concentration remained similar regardless of the use of air or oxygen. However, in the nonnitrogenous production medium, citric acid production was not influenced considerably when oxygen was used instead of air. The advantage of using immobilized cells is that production is achieved easily in the continuous system. Therefore, citric acid production was also tested using a packed-bed bioreactor, and an increase in productivity by a factor of 22 was achieved compared to the batch system.     

Production of antitumoral retamycin during fed-batch fermentations of Streptomyces olindensis

Fed-batch runs were performed in order to correlate the production of retamycin, an anthracycline antibiotic produced by Streptomyces olindensis in submerged cultures, with the specific growth rate. Maximum retamycin production was achieved with an exponential feed rate, controlling the specific growth rate at a low value (0.03h⁻¹, about 10% of the maximum specific growth rate). Control of the specific growth rate at higher values (0.10 and 0.17 h⁻¹) caused a decrease in antibiotic production. Morphology, assessed by image analysis, was shown to be highly relevant in this process. Cell growth mainly in the form of clumps (90% clumps and 10% free filaments) led to better results than growth as clumps (75%) and free filaments (25%).     

Entrapment of living microbial cells in covalent polymeric networks

The kinetics of oxidative phenol degradation with microbial cellsCandida tropicalis, immobilized in a polyacrylamide and polymethacrylamide matrix, were mathematically simulated assuming zero-order and Michaelis-Menten rate equations. For zero-order kinetics an expanded equation for catalytic effectiveness as a function of the Thiele modulus, Biot number, and partition coefficients was derived and compared with numerical solutions for Michaelis-Menten kinetics. Errors with regard to the zero-order approximation become negligible ifc _o/K _M >2. Experimentally determined catalyst activities as a function of particle size and cell concentration were compared to calculated ones. Additional experiments to determine the diffusion and oxygen consumption ratios have been carried out in an effort to resolve the physical parameters to be used in the above mentioned calculations. Furthermore, experiments on cell growth during reincubation with nutrients and oxygen are reported; an increase in activity up to a factor of ten was observed. These experiments demonstrate that the microbial cells are entrapped in the polymer matrix in the living state.     

Continuous production of L-aspartic acid

For the continuous production ofl-aspartic acid from fumaric acid and ammonia by the action of aspartase, the enzyme extracted fromEscherichia coli orE. coli cells having high aspartase activity were immobilized by various methods. In 1973 we succeeded in the industrial production ofl-aspartic acid usingE. coli cells immobilized with polyacrylamide gel. For the improvement of this process, we developed a novel technique using κ-carrageenan as the immobilizing matrix forE. coli cells. Further, EAPc-7 strain, having higher aspartase activity, was contracted from the parentE. coli by continuous cultivation with a definite medium. The aspartase activity was about seven times higher than that of the parent cells. In 1982 we changed from the conventional method to the improved method, using EAPc-7 strain immobilized with κ-carrageenan.     

Optimization of Fermentation Parameters to Enhance the Production of Ethanol from Palmyra Jaggery Using <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in a Batch Fermentor

Application of statistical experimental designs for optimization of fermentation parameters to enhance ethanol production, which is an economical and renewable energy source using Saccharomyces cerevisiae NCIM 3090 from palmyra jaggery, was studied in a batch fermentor. Using Plackett–Burman design, impeller speed, concentrations of CoCl₂ and KH₂PO₄ were identified as significant variables, which highly influenced ethanol production, and these variables were further optimized using a central composite design (CCD). The ethanol production was adequately approximated with a full quadratic equation obtained from three factors and five levels of CCD. Maximum ethanol concentration of 132.56 g/l (16.8% [v/v]) was obtained for an impeller speed of 247.179 (≈250) rev/min, CoCl₂ of 0.263 g/l and KH₂PO₄ of 2.39 g/l. A second-order polynomial regression model was fitted and was found adequate with R ² of 0.8952. This combined statistical approach enables rapid identification and investigation of significant parameters for improving the ethanol production and could be very useful in optimizing processes.     

Properties of bacterial luciferase/NADH : FMN oxidoreductase and firefly luciferase immobilized onto sepharose

NADH : FMN oxidoreductase and bacterial luciferase have been efficiently coimmobilized onto Sepharose 4B. This luminescent immobilized enzyme system can be used to assay NADH. The assay is rapid and sensitive with a lower limit of detection of 0.2 pmol/assay tube. The intra-assay precision was 3.5% at 2 × 10^-5 M and 5.8% at 2 × 10^-6 M NADH. Light intensity was proportional to NADH concentration from 0.2 to 1000 pmol. Added serum and certain dehydrogenases were found to be inhibitory; however, inhibition could be eliminated by a combination of heat treatment and dilution. Firefly luciferase has also been immobilized onto both Sepharose 4B and CL 6B. The detection limit for ATP using this immobilized enzyme was 0.2 pmol and the assay was linear from 0.2 to 2000 pmol. The intra-assay precision was 4.8% at 2 × 10^-4 M and 3.2% at 1 × 10^-5 M ATP. The immobilized enzymes remained fully active when rapidly frozen in the presence of glycerol and DTT. Such preparations could be stored for at least two months with no loss of activity. A variety of different compounds were used to block any remaining reactive groups on the Sepharose following immobilization of the enzymes. Glycine, 2-aminoethanol, and ethylenediamine were examined. The preparations where ethylenediamine was used as a blocking agent exhibited better activity and stability than the others.     

<Emphasis Type="Italic">Rhizopus stolonifer</Emphasis> LAU 07: a novel source of fructosyltransferase

Increasing awareness of the importance of fructooligosaccharides (FOS) as ingredients of functional foods has led to intensive search of new sources of fructosyltransferases (FTase), enzymes responsible for the conversion of sucrose to fructooligosaccharides. A local strain of Rhizopus stolonifer isolated from spoilt orange fruit with high fructosyltransferase activity (U _t) of 12.31–45.70 U mL⁻¹ during a fermentation period of 24–120 h is herein reported. It showed low hydrolytic activity (U _h) in the range of 0.86–1.78 U mL⁻¹ during the same period. FOS yield of 34 % (1-kestose, GF₂, nystose, GF₃) was produced by FTase obtained from a 72 h-old culture using 60 g of sucrose per 100 mL of the substrate. When the isolate was grown in a defined submerged medium, its pH dropped sharply from the intial value of 5.5 to 1.0 within 24 h, and this value was maintained throughout the fermentation. The biomass content ranged from 8.8 g L⁻¹ at 24 h of fermentation to reach the maximum of 10 g L⁻¹ at 72 h. It was reduced to 5.6 g L⁻¹ at the end of 120 h of fermentation. This report represents the first reference to a strain of Rhizopus as a source of FTase for the production of FOS. The high U _t/U _h ratio shown by this isolate indicates that it may be a good strain for the industrial and commercial production of FOS. However, there is a need of further optimization of the bioprocess to increase the conversion efficiency of sucrose to FOS by the enzyme.     

Variable Volume Fed-Batch Fermentation for Nisin Production by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp<Emphasis Type="Italic">. lactis W28</Emphasis>

A feeding technology that was suitable for improving the nisin production by Lactococcus lactis subsp. lactis W28 was established. The effects of initial sucrose concentration (ISC) in the fermentation broth, feeding time, and feeding rate on the fermentation were studied. It was observed that a fed-batch culture (ISC = 10 g l⁻¹) with 100 ml sucrose solution (190 g l⁻¹) being evenly fed (9–10 ml h⁻¹) into the fermenter after 3-h fermentation gave the best performance in terms of biomass and nisin yield. Under these conditions, the total biomass and the total nisin yield were approximately 23% and 51% higher than those in batch fermentation, respectively. When the sucrose concentration was controlled at 5–10 g l⁻¹ in variable volume intermittent fed-batch fermentation (VVIF) with ISC = 10 g l⁻¹, the total biomass and the total nisin yield were 29% and 60% above those in batch fermentation, respectively. The VVIF proved to be effective to eliminate the substrate inhibition by maintaining sucrose at appropriate levels. It is also easy to be scaled up, since various parameters involved in industrial production were taken into account.     

Carob pod: A new substrate for citric acid production by Aspergillus niger

The production of citric acid from carob pod extract byA. niger in surface fermentation was investigated. A maximum citric acid concentration (85.5 g/L), citric acid productivity (4.07 g/L/d), specific citric acid production rate (0.18 g/g/d), and specific sugar uptake rate (0.358 g/g/d) was achieved at an initial sugar concentration of 200 g/L, pH of 6.5, and a temperature of 30°C. Other kinetic parameters, namely, citric acid yield, biomass yield, specific biomass production rate, and fermentation efficiency were maximum at pH 6.5, temperature 30°C, and initial sugar concentration 100 g/L. The external addition of methanol into the carob pod extract at a concentration up to 4% (v/v) improved the production of citric acid.