Studies on the overproduction of indole-containing metabolites by a methanol-utilizing yeast

Production of indole-containing metabolites (“indoles”) from methanol has been studied using a mutant ofHansenula polymorpha resistant to 5-fluorotryptophan. Whereas the wild-type culture produces only a small amount of indoles, the mutant is partially deregulated and overproduces indoles. Indoles production was studied in batch and continuous culture and in a washed-cell system. When the pH was above 4.0, indoles production was growth-associated, in both minimal and complex media, and batch or continuous culture. When the pH was below or equal to 4.0, a low phosphate concentration was found to improve production. In a phosphate-deficient washed-cell suspension system, the addition of an amino acid such as methionine at 5 mM increased specific productivity by more than 60%. Addition of cycloheximide at 50 mg/L decreased residual growth and increased maximum productivity of indoles by more than 60%. When the antibiotic was added at 1000 mg/L, growth was completely inhibited and indoles production continued for about 35 h.     

Continuous production of butanol from starch-based packing peanuts

Acetone, butanol, ethanol (ABE, or solvents) were produced from starch-based packing peanuts in batch and continuous reactors. In a batch reactor, 18.9 g/L of total ABE was produced from 80 g/L packing peanuts in 110 h of fermentation. The initial and final starch concentrations were 69.6 and 11.1 g/L, respectively. In this fermentation, ABE yield and productivity of 0.32 and 0.17 g/(L·h) were obtained, respectively. Compared to the batch fermentation, continuous fermentation of 40 g/L of starch-based packing peanuts in P2 medium resulted in a maximum solvent production of 8.4 g/L at a dilution rate of 0.033 h⁻¹. This resulted in a productivity of 0.27 g/(L·h). However, the reactor was not stable and fermentation deteriorated with time. Continuous fermentation of 35 g/L of starch solution resulted in a similar performance. These studies were performed in a vertical column reactor using Clostridium beijerinckii BA101 and P2 medium. It is anticipated that prolonged exposure of culture to acrylamide, which is formed during boiling/autoclaving of starch, affects the fermentation negatively.     

Continuous fermentation for the production of citric acid from glucose

Citric acid is finding new areas of use each year and the demand for the acid is constantly increasing. Being a bulk chemical, the continuous production of citric acid would be advantageous. The paper presents the results from ammonia limited batch and continuous fermentations using the yeast strainSaccharomycopsis (Candida) lipolytica (NRRL Y-7576). Mathematical models were developed for growth and glucose utilization in batch and continuous culture. Cell and acid yields appeared to be almost the same in batch and continuous culture. The specific production rates were found to be constant, equal to 0.053 g/g h, in the batch fermentations but varied in the continuous experiments from 0 to 0.11 g/g h depending on the fermentation conditions. Continuous production in a single stage CSTR was studied for over 1,000 hours without shutdown.     

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).     

Continuous production of extracellular l-glutaminase by ca-alginate-immobilized marine Beauveria bassiana BTMF S-10 in packed-bed reactor

l-Glutamine amidohydrolase (l-glutaminase, EC 3.5.1.2) is a therapeutically and industrially important enzyme. Because it is a potent antileukemic agent and a flavor-enhancing agent used in the food industry, many researchers have focused their attention on l-glutaminase. In this article, we report the continuous production of extracellular l-glutaminase by the marine fungus Beauveria bassiana BTMF S-10 in a packed-bed reactor. Parameters influencing bead production and performance under batch mode were optimized in the order-support (Na-alginate) concentration, concentration of CaCl₂ for bead preparation, curing time of beads, spore inoculum concentration, activation time, initial pH of enzyme production medium, temperature of incubation, and retention time. Parameters optimized under batch mode for l-glutaminase production were incorporated into the continuous production studies. Beads with 12×10⁸ spores/g of beads were activated in a solution of 1% glutamine in seawater for 15 h, and the activated beads were packed into a packed-bed reactor. Enzyme production medium (pH 9.0) was pumped through the bed, and the effluent was collected from the top of the column. The effect of flow rate of the medium, substrate concentration, aeration, and bed height on continuous production of l-glutaminase was studied. Production was monitored for 5 h in each case, and the volumetric productivity was calculated. Under the optimized conditions for continuous production, the reactor gave a volumetric productivity of 4.048 U/(mL·h), which indicates that continuous production of the enzyme by Ca-alginate-immobilized spores is well suited for B. bassiana and results in a higher yield of enzyme within a shorter time. The results indicate the scope of utilizing immobilized B. bassiana for continuous commercial production of l-glutaminase.     

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.     

Butanol production by Clostridium beijerinckii BA101 in an immobilized cell biofilm reactor

Acetone butanol ethanol was produced in a continuous immobilized cell (biofilm) plug-flow reactor inoculated with Clostridium beijerinckii BA101. To achieve high reactor productivity, C. beijerinckii BA101 cells were immobilized by adsorption onto clay brick. The continuous plug-flow reactor offers high productivities owing to reduced butanol inhibition and increased cell concentration. Although high productivity was achieved, it was at the expense of low sugar utilization (30.3%). To increase sugar utilization, the reactor effluent was recycled. However, this approach is complicated by butanol toxicity. The effluent was recycled after removal of butanol by pervaporation to reduce butanol toxicity in the reactor. Recycling of butanolfree effluent resulted in a sugar utilization of 100.7% in addition to high productivity of 10.2g/(L·h) at a dilution rate of 1.5 h⁻¹. A dilution rate of 2.0h⁻¹ resulted in a reactor productivity of 16.2g/(L·h) and sugar utilization of 101.4%. It is anticipated that this reactor-recovery system would be economical for butanol production when using C. beijerinckii BA101.     

Continuous production of butanol with immobilized cells ofClostridium acetobutylicum

Spores ofClostridium acetobutylicum were immobilized in calcium alginate. An active gel preparation was obtained after outgrowth of the spores to vegetative cells within the gel matrix. A 100 mL column containing the immobilized cells was used for continuous production. At steady-state conditions the productivity of butanol was 67 g/L reactor volume/day.     

Effect of nitrogen sources in batch and continuous cultures to lipase production byCandida rugosa

The interaction between medium components, microorganisms and the operational mode to produce lipase byCandida rugosa was studied. It has been observed as an emulsion formation between oleic acid and urea during continuous culture. Because of this emulsion, the nitrogen source cannot be assimilated by the cells. The lack of nitrogen source provoked a decrease in protein synthesis, being lipase synthesis suppressed. The change of the nitrogen source from urea to ammonium sulfate avoided the emulsion formation, allowing to reach biomass and lipase steady states in continuous cultures. In batch cultures, no effect of nitrogen source on lipase production and biomass was observed. The specific productivity in continuous culture was slightly higher than in batch cultures.     

Immobilization of Xylanase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> Strain MK001 and its Application in Production of Xylo-oligosaccharides

Xylanase from Bacillus pumilus strain MK001 was immobilized on different matrices following varied immobilization methods. Entrapment using gelatin (GE) (40.0%), physical adsorption on chitin (CH) (35.0%), ionic binding with Q-sepharose (Q-S) (45.0%), and covalent binding with HP-20 beads (42.0%) showed the maximum xylanase immobilization efficiency. The optimum pH of immobilized xylanase shifted up to 1.0 unit (pH 7.0) as compared to free enzyme (pH 6.0). The immobilized xylanase exhibited higher pH stability (up to 28.0%) in the alkaline pH range (7.0–10.0) as compared to free enzyme. Optimum temperature of immobilized xylanase was observed to be 8 °C higher (68.0 °C) than free enzyme (60.0 °C). The free xylanase retained 50.0% activity, whereas xylanase immobilized on HP-20, Q-S, CH, and GE retained 68.0, 64.0, 58.0, and 57.0% residual activity, respectively, after 3 h of incubation at 80.0 °C. The immobilized xylanase registered marginal increase and decrease in K _m and V _max values, respectively, as compared to free enzyme. The immobilized xylanase retained up to 70.0% of its initial hydrolysis activity after seven enzyme reaction cycles. The immobilized xylanase was found to produce higher levels of high-quality xylo-oligosaccharides from birchwood xylan, indicating its potential in the nutraceutical industry.     

Enhanced production of xylanase from locally isolated fungal strain using agro-industrial residues under solid-state fermentation

This study is related to the isolation of fungal strain for xylanase production using agro-industrial residues. Forty fungal strains with xylanolytic potential were isolated by using xylan agar plates and quantitatively screened in solid-state fermentation. Of all the tested isolates, the strain showing highest ability to produce xylanase was assigned the code Aspergillus niger LCBT-14. For the enhanced production of the enzyme, five different fermentation media were evaluated. Out of all media, M4 containing wheat bran gave maximum enzyme production. Effect of different variables including incubation time, temperature, pH, carbon and nitrogen sources has been investigated. The optimum enzyme production was obtained after 72 h at 30°C and pH 4. Glucose as a carbon source while ammonium sulphate and yeast extract as nitrogen sources gave maximum xylanase production (946 U/mL/min). This study was successful in producing xylanase by A. niger LCBT-14 economically by utilising cheap indigenous substrate.     

Lipase production by acinetobacter radioresistens in a batch fill- and-draw culture

Alkaline lipase production byAcinetobacter radioresistens was performed in a batch fill- and-draw culture, and the results were compared with a batch culture. In the batch culture, the lipase yield was 18 U/mL, which was restricted by the occurrence of protease forming and excessive foaming as cell growth ceased. Because the formation of lipase was found to be chiefly growth-associated, the fill- and-draw culture, which provided an environment for continuous growth, could surpass the limitation encountered in a batch culture and increase the lipase yield to 30 U/mL. The improvement in the lipase yield was suggested to be caused by an adaptation of the cells to the medium during the repeated culture. Although the increase in the lipase yield was accompanied by a decrease in lipase productivity, the fill- and-draw culture could be a better mode for lipase production.     

Improvement of inulin hydrolysis yeast cell reactor by mutants selection

In a previous publication, we described a continuous production ofd-fructose from enzymatic hydrolysis of inulin with immobilized permeabilized cells (1).Kluyveromyces fragilis ATCC 12424 have been shown to possess inulase activity. The half life of the reactor was at least 1300 h, but productivity was relatively low (around 40 g/L/day). A selection of 50 mutants was tested on liquid medium for a possible increase of productivity. In relation to the improvement of the reactor, the most important factor is intracellular inulase activity, and this activity was increased with the KF 28 mutant. Productivity reached 2000 g/L/day with the increase (of the productivity), proportional to the increase of intracellular inulase activity.     

Influence of a new axial impeller on K L a and xylanase production by Penicillium canescens 10-10c

The effects of a new axial impeller (HTPG4) on oxygen volumetric transfer coefficient, K _L a, and xylanase production by Penicillium canescens 10-10c were studied and compared for dual-impeller systems, one with one DT4 impeller below and one HTPG4 above (DT4-HTPG4) and one with two DT4 (DT4-DT4) impellers, in a 5-L bioreactor. The volumetric coefficient of oxygen transfer was measured in culture medium using a gassing-out method at different gassing rates and agitation speeds. We observed that the DT4-HTPG4 combination provided better K _L a performance than the DT4-DT4 combination. The two combinations were also tested for their influence on xylanase production by a filamentous microorganism; P. canescens 10-10c. These experiments demonstrated that the DT4-HTPG4 combination impeller enhanced enzyme production up to 23% compared with the DT4-DT4 combination at an aeration rate of 1 vvm and an agitation speed of 600 rpm. The main cause for this difference is thought to be a higher shear stress generated by the DT4-DT4 combination, which damages the mycelium of P. canescens and decreases xylanase production.     

Continuous Production of Thermostable α-Amylase by Immobilized Bacillus Cells in a Fluidized-Bed Reactor

The exponential cells ofBacillus cereus immobilized in calcium alginate and spun into microcylindrical particles were used in a fluidized-bed reactor for continuous synthesis of thermostable α-amylase. The reactor was operated over a period of 30 d with a dilution rate of 0.33 h^-1, producing 1000–1200 U/mL of enzyme. The productivity of the reactor was in the range of 330–396 kU/h. A 20-fold increase in the productivity with respect to batch fermentation with free cells was attained.     

Continuous Production of Ethanol from Starch Using Glucoamylase and Yeast Co-Immobilized in Pectin Gel

This work presents a continuous simultaneous saccharification and fermentation (SSF) process to produce ethanol from starch using glucoamylase and Saccharomyces cerevisiae co-immobilized in pectin gel. The enzyme was immobilized on macroporous silica, after silanization and activation of the support with glutaraldehyde. The silica–enzyme derivative was co-immobilized with yeast in pectin gel. This biocatalyst was used to produce ethanol from liquefied manioc root flour syrup, in three fixed bed reactors. The initial reactor yeast load was 0.05 g wet yeast/ml of reactor (0.1 g wet yeast/g gel), used in all SSF experiments. The enzyme concentration in the reactor was defined by running SSF batch assays, using different amount of silica–enzyme derivative, co-immobilized with yeast in pectin gel. The chosen reactor enzyme concentration, 3.77 U/ml, allowed fermentation to be the rate-limiting step in the batch experiment. In this condition, using initial substrate concentration of 166.0 g/l of total reducing sugars (TRS), 1 ml gel/1 ml of medium, ethanol productivity of 8.3 g/l/h was achieved, for total conversion of starch to ethanol and 91% of the theoretical yield. In the continuous runs, feeding 163.0 g/l of TRS and using the same enzyme and yeast concentrations used in the batch run, ethanol productivity was 5.9 g ethanol/l/h, with 97% of substrate conversion and 81% of the ethanol theoretical yield. Diffusion effects in the extra-biocatalyst film seemed to be reduced when operating at superficial velocities above 3.7 × 10⁻⁴ cm/s.     

Repeated Batch Cell-Immobilized System for the Biotechnological Production of Xylitol as a Renewable Green Sweetener

The present paper studies the biotechnological production of xylitol using sugarcane bagasse hydrolysate in a repeated batch fermentation system with immobilized cells of Candida guilliermondii FTI20037. Immobilized cell system is considered as an attractive alternative to reuse the well-grown and adapted yeast cells in a new fresh fermentation media, without the need of the inoculum stage. In this work, seven repeated batches were performed in a fluidized bed bioreactor using immobilized cells in calcium alginate beads. According to the obtained results it was observed that the immobilized cells of C. guilliermondii can be reused for six successive batches maintaining an average xylitol yield (Y _p/s) of 0.7 g/L and a volumetric productivity (Q _p) of 0.42 g/L?h at the end of 432 h of fermentation. On the other hand, in the seventh batch (504 h), a decrease of 44 % in the final concentration of xylitol was observed. This reduction can be explained by the possible diffusion and accumulation of insoluble substances, found in the hemicellulosic hydrolysate, in the interior of the immobilization support resulting in substrate mass transfer limitations.     

The Effects of Wheat Bran Composition on the Production of Biomass-Hydrolyzing Enzymes by Penicillium decumbens

The effects of the starch, protein, and soluble oligosaccharides contents in wheat bran on the extracellular biomass-hydrolyzing enzymes activities released by Penicillium decumbens mycelia grown in batch fermentations have been examined. The results showed increased starch content correlated directly with an increase in released amylase activity but inversely with the levels of secreted cellulase and xylanase. High amounts of protein in wheat bran also reduced the activities of cellulase, xylanase and protease in the culture medium. The effects of the soluble and insoluble components of wheat bran and cello-oligosaccharides supplements on production of extracellular cellulase and xylanase were compared. The soluble cello-oligosaccharides compositions in wheat bran were proved to be one of the most significant factors for cellulase production. According to the results of this research, determining and regulating the composition of wheat bran used as a fermentation supplement may allow for improved induction of cellulase and xylanase production.     

Biological Hydrogen Production Using Chloroform-treated Methanogenic Granules

In fermentative hydrogen production, the low-hydrogen-producing bacteria retention rate limits the suspended growth reactor productivity because of the long hydraulic retention time (HRT) required to maintain adequate bacteria population. Traditional bacteria immobilization methods such as calcium alginate entrapment have many application limitations in hydrogen fermentation, including limited duration time, bacteria leakage, cost, and so on. The use of chloroform-treated anaerobic granular sludge as immobilized hydrogen-producing bacteria in an immobilized hydrogen culture may be able to overcome the limitations of traditional immobilization methods. This paper reports the findings on the performance of fed-batch cultures and continuous cultures inoculated with chloroform-treated granules. The chloroform-treated granules were able to be reused over four fed-batch cultures, with pH adjustment. The upflow reactor packed with chloroform-treated granules was studied, and the HRT of the upflow reactor was found to be as low as 4 h without any decrease in hydrogen production yield. Initial pH and glucose concentration of the culture medium significantly influenced the performance of the reactor. The optimum initial pH of the culture medium was neutral, and the optimum glucose concentration of the culture medium was below 20 g chemical oxygen demand/L at HRT 4 h. This study also investigated the possibility of integrating immobilized hydrogen fermentation using chloroform-treated granules with immobilized methane production using untreated granular sludge. The results showed that the integrated batch cultures produced 1.01 mol hydrogen and 2 mol methane per mol glucose. Treating the methanogenic granules with chloroform and then using the treated granules as immobilized hydrogen-producing sludge demonstrated advantages over other immobilization methods because the treated granules provide hydrogen-producing bacteria with a protective niche, a long duration of an active culture, and excellent settling velocity. This integrated two-stage design for immobilized hydrogen fermentation and methane production offers a promising approach for modifying current anaerobic wastewater treatment processes to harvest hydrogen from the existing systems.     

Production of cellulase on mixtures of xylose and cellulose

Cellulase production by the RUT-C30 mutant of the fungusTrichoderma reesei was studied on mixtures of xylose and cellulose. In mixed substrates, the lag phase of the growth cycle was shorter and reached the maximum of total productivity in a shorter time compared to growth on the single substrate, cellulose. A diauxic pattern of utilization of the two carbon sources was observed as well: Xylose was utilized first to support growth, followed by cellulose to induce the cellulase enzyme production and provide an additional carbon source for cellular metabolism. Of the various mixtures of xylose and cellulose used in batch enzyme production, a ratio of 30∶30 g/L of xylose to cellulose was optimal. This mixture produced the highest maximal enzyme productivity of 122 IFPU/L h, and its total productivity reached a maximum value of 55 IFPU/L h in less time than others. However, similar total productivities and higher enzyme titers were observed for growth on cellulose alone.