Repeated Batch Fermentation Research Articles

Bioflavour production from orange peel hydrolysate using immobilized Saccharomyces cerevisiae

The rising trend of bioflavour synthesis by microorganisms is hindered by the high manufacturing costs, partially attributed to the cost of the starting material. To overcome this limitation, in the present study, dilute-acid hydrolysate of orange peel was employed as a low-cost, rich in fermentable sugars substrate for the production of flavour-active compounds by Saccharomyces cerevisiae. With this purpose, the use of immobilized cell technology to protect cells against the various inhibitory compounds present in the hydrolysate was evaluated with regard to yeast viability, carbon and nitrogen consumption and cell ability to produce flavour active compounds. For cell immobilization the encapsulation in Ca alginate beads was used. The results were compared with those obtained using free-cell system. Based on the data obtained immobilized cells showed better growth performance and increased ability for de novo synthesis of volatile esters of "fruity" aroma (phenylethyl acetate, ethyl hexanoate, octanoate, decanoate and dodecanoate) than those of free cells. The potential for in situ production of new formulations containing flavour-active compounds derive from yeast cells and also from essential oil of orange peel (limonene, α-terpineol) was demonstrated by the fact that bioflavour mixture was found to accumulate within the beads. Furthermore, the ability of the immobilized yeast to perform efficiently repeated batch fermentations of orange peel hydrolysate for bioflavour production was successfully maintained after six consecutive cycles of a total period of 240 h.

Aeration strategy for repeated-batch ethanol fermentation by Saccharomyces cerevisiae and its effects on yeast intracellular compositions

Aeration strategy for repeated-batch ethanol fermentation by Saccharomyces cerevisiae and its effects on yeast intracellular compositions

Efficient production of l-sorbose from d-sorbitol by whole cell immobilization of Gluconobacter oxydans WSH-003

The effect of immobilizing Gluconobacter oxydans WSH-003 on a novel combination carrier material to produce l-sorbose was investigated. A combination carrier of sodium alginate and diatomite was shown to be biocompatible and durable. Our results showed that the carrier material concentration and bead diameter of immobilized cells significantly affected l-sorbose yield. Optimal medium conditions for fermenting l-sorbose were determined to be concentrations of 2.5% (w/v) sodium and 0.5% (w/v) diatomite as well as 2.0-mm bead diameter. Under optimal conditions, fermentation time was 1.3 times lower compared to previous experiments involving free cells. Additionally, the immobilized cells showed excellent stability and were maintained for more than 15 days in a bioreactor. l-sorbose yield was more than 81.0% in the previous nine runs of repeated-batch fermentation. These results indicate that maintaining highly active immobilized cells is feasible for the biosynthesis of l-sorbose.

Production of butanol from glucose and xylose with immobilized cells of Clostridium acetobutylicum

Pretreated cotton towels were used as carriers to immobilize Clostridium acetobutylicum CGMCC 5234 cells for butanol or ABE production from glucose and xylose. Results showed that cell immobilization was a promising method to increase butanol concentration, yield and productivity regardless of the sugar sources compared with cell suspension. In this study, a high butanol concentration of 10.02 g/L with a yield of 0.20 g/g was obtained from 60 g/L xylose with 9.9 g/L residual xylose using immobilized cells compared with 8.48 g/L butanol and a yield of 0.141 g/g with 20.2 g/L residual xylose from 60 g/L xylose using suspended cells. In mixed-sugar fermentation (30 g/L glucose plus 30 g/L xylose), the immobilized cultures produced 11.1 g/L butanol with a yield of 0.190 g/g, which were 28.3% higher than with suspended cells (8.65 g/L) during which 30 g/L glucose was utilized completely using both immobilized and suspended cells while 3.46 and 13.1 g/L xylose maintained untilized for immobilized and suspended cells, respectively. Based on the results, we speculated that immobilized cells showed enhanced tolerance to butanol toxicity and the cultures preferred glucose to xylose during ABE fermentation. Moreover, the cultures showed obvious difference when grown between high initial concentrations of glucose and those of xylose. Repeated-batch fermentations from glucose with immobilized cells showed better long-term stability than from xylose. At last, the morphologies of free and immobilized cells adsorbed on pretreated cotton towels during the growth cycle were examined by SEM.

Coproduction of acetic acid and electricity by application of microbial fuel cell technology to vinegar fermentation

The coproduction of a useful material and electricity via a novel application of microbial fuel cell (MFC) technology to oxidative fermentation was investigated. We focused on vinegar production, i.e., acetic acid fermentation, as an initial and model useful material that can be produced by oxidative fermentation in combination with MFC technology. The coproduction of acetic acid and electricity by applying MFC technology was successfully demonstrated by the simultaneous progress of acetic acid fermentation and electricity generation through a series of repeated batch fermentations. Although the production rate of acetic acid was very small, it increased with the number of repeated batch fermentations that were conducted. We obtained nearly identical (73.1%) or larger (89.9%) acetic acid yields than that typically achieved by aerated fermentation (75.8%). The open-cycle voltages measured before and after fermentation increased with the total fermentation time and reached a maximum value of 0.521 V prior to the third batch fermentation. The maximum current and power densities measured in this study (19.1 μA/cm² and 2.47 μW/cm², respectively) were obtained after the second batch fermentation.

Dried Spent Yeast and Its Hydrolysate as Nitrogen Supplements for Single Batch and Repeated-Batch Ethanol Fermentation from Sweet Sorghum Juice

Dried spent yeast (DSY) and its hydrolysate (DSYH) were used as low-cost nitrogen supplements to improve ethanol production from sweet sorghum juice by Saccharomyces cerevisiae NP01 under very high gravity (VHG) fermentation (280 g·L−1 of total sugar) conditions. The supplemented DSY and DSYH concentrations were 11, 16 and 21 g·L−1, corresponding to a yeast extract nitrogen content of 6, 9 and 12 g·L−1, respectively. The initial yeast cell concentration for ethanol fermentation was approximately 5 × 107 cells·mL−1. The fermentation was carried out in single batch mode at 30 °C in 1-L air-locked bottles with an agitation rate of 100 rpm. Ethanol production from the juice with and without yeast extract (9 g·L−1) was also performed as control treatments. The results showed that DSY at 21 g·L−1gave the highest ethanol concentration (PE, 107 g·L−1) and yield (Yp/s, 0.47 g·g−1). The use of DSYH at the same DSY concentration improved ethanol productivity (Qp), but not PE and Yp/s. The ethanol production efficiencies of the juice under DSY and DSYH supplementations were markedly higher than those without nutrient supplementation. However, the PE and Qp values of the juice containing 21 g·L−1 of DSY was approximately 7 g·L−1 and 0.62 g·L−1·h−1 lower than those under the presence of yeast extract (9 g·L−1), respectively. At the end of the single batch fermentation under the optimum DSY concentration, the sugar consumption was approximately 80%. Therefore in the repeated-batch fermentation, the initial total sugar was reduced to 240 g·L−1. The results showed that the system could be carried out at least 20 successive batches with the average PE, Yp/s and Qp of 95 g·L−1, 0.46 g·g−1 and 1.45 g·L−1·h−1, respectively.

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/Lh 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.

Enhancement of chitosanase production by cell immobilization of Gongronella sp. JG

Chitosanase production of Gongronella sp. JG cells immobilized in calcium alginate gel and polyurethane foam was compared with that of the free cells, there was a 60% increase in the enzyme yield (2429 U/L) compared to the highest yield obtained from free cells (1513 U/L). The optimal immobilization parameters (concentrations of sodium alginate, calcium chloride, bead inoculums, bead diameter, etc) for the enhanced production of chitosanase were determined as: sodium alginate 2% (w/v), 0.1 M calcium chloride, inoculum 10 mL beads to 100 mL production media and 2.7 mm bead diameter. Maximum chitosanase production was achieved with initial pH of 5.5 and temperature of 30 °C. The alginate beads had well stability, retained 85% ability of enzyme production even after 7 cycles of repeated batch fermentation. These results showed the immobilization technique was a feasible and economical method for chitosansase production by Gongronella sp. JG.

Ethanol Production by Repeated Batch and Continuous Fermentations by Saccharomyces cerevisiae Immobilized in a Fibrous Bed Bioreactor

In this work, a fibrous bed bioreactor with high specific surface area and good adsorption efficacy for S. cerevisiae cells was used as the immobilization matrix in the production of ethanol. In batch fermentation, an optimal ethanol concentration of 91.36 g/l and productivity of 4.57 g l(-1) h(-1) were obtained at an initial sugar concentration of 200 g/l. The ethanol productivity achieved by the immobilized cells was 41.93% higher than that obtained from free cells. Ethanol production in a 22-cycle repeated batch fermentation demonstrated the enhanced stability of the immobilized yeast cells. Under continuous fermentation in packed-bed reactors, a maximum ethanol concentration of 108.14 g/l and a productivity of 14.71 g l(-1) h(-1) were attained at 35 degrees C, and a dilution rate of 0.136 h(-1) with 250 g/l glucose.

Improved lactic acid productivity by an open repeated batch fermentation system using Enterococcus mundtii QU 25

Decreasing the production cost of polymer-grade lactic acid is a challenge that the polylactic acid industry must surmount to be competitive with producers of petrochemical-derived plastics. In this study, we aimed to improve lactic acid productivity by investigating different fermentation systems. Enterococcus mundtii QU 25, previously reported by our group as a thermotolerant lactic acid bacterium, homofermentatively metabolizes glucose with the production of high optically pure L-lactic acid. The strain showed high lactic acid concentration and yield by controlled pH (7.0) and at a high temperature (43 °C). In a batch fermentation system, the optimal initial glucose concentration was 100 g L−1, at which high cell mass was achieved with production of 82.4 g L−1 of lactic acid at yield of 0.858 g g−1 glucose consumed. However, the productivity was approximately 2.0 g L−1 h−1. Subsequently, open repeated batch or fed-batch fermentation with recycled cells of the previous fermentation was conducted for 11 runs to enhance the productivity. As a result, lactic acid productivity was enhanced-up to 5.5-fold higher than that of the conventional batch culture, and fermentation was completed within 6 h with almost the same yield (0.761–0.832 g g−1 glucose consumed) and lactic acid concentration (81.6–84.5 g L−1). Up to 132 g L−1 lactic acid at yield of 0.853 g g−1 glucose consumed and productivity of 6.99 g L−1 h−1 were obtained in open repeated fed-batch fermentation. This indicates that strain QU 25 has the potential for industrial-scale production of lactic acid due to its high productivity, high yield of substrate conversion, and high final concentration of lactic acid produced under non-sterile conditions.

Preparation of mango (Mangifera indica L.) wine using a new yeast-mango-peel immobilised biocatalyst system

The preparation of mango wine by yeast-mango peel immobilised biocatalyst system by repeated batch fermentation was conducted and compared to free cells fermentation at 15, 20, 25, and 30°C. The operational stability of the biocatalyst was good as the ethanol concentrations (76.0–96.0 g/l) and productivities (1.53–3.29 g/l/h) were high, showing the suitability of the biocatalyst for even low temperature winemaking. The concentration of ethyl acetate was not above 40 mg/l in all cases, and higher alcohols were low (< 330 mg/l) in wine with immobilised cells indicating an improvement in the product compared to free cells fermentation. Amyl alcohols were proved to be temperature dependent and decreased with the decrease in temperature (262.48–146.83 and 239.74–184.34 mg/l) in the case of fermentation batches with immobilised and free cells, respectively, from 30°C to 15°C. Sensory evaluation revealed fruity aroma (7.9 ± 0.73), fine taste (7.7 ± 0.24), and the overall improved quality of the wines produced by the immobilised system.

Bioethanol from fermentation of cassava pulp in a fibrous-bed bioreactor using immobilized Δldh, a genetically engineered Thermoanaerobacterium aotearoense

There is an increasing worldwide interest in bioethanol production from agricultural, industrial, and urban residues for both ecological and economic reasons. The acid hydrolysis of cassava pulp to reducing sugars and their fermentation to ethanol were evaluated in a fibrousbed bioreactor with immobilized Δldh, a genetically engineered Thermoanaerobacterium aotearoense. A maximum yield of total reducing sugars of 53.5% was obtained after 8 h of hydrolysis at 85oC in 0.4 mol/L hydrochloric acid with a solid-to-liquid ratio of 1:20, which was optimized by using an orthogonal design based on preliminary experiments. In the FBB, the fed-batch fermentation, using glucose as the sole carbon source, gave a maximum ethanol production of 38.3 g/L with a yield of 0.364 g/g in 100 h; whereas the fed-batch fermentation, using xylose as the sole carbon source, gave 34.1 g/L ethanol with a yield of 0.342 g/g in 135 h. When cassava pulp hydrolysate was used as a carbon source, 39.1 g/L ethanol with a yield of 0.123 g/g cassava pulp in185 h was observed, using the fed-batch fermentation model. In addition, for repeated batch fermentation of cassava pulp hydrolysate carried out in the fibrous-bed bioreactor, long-term operation with high ethanol yield and volumetric productivity were achieved. The above results show that the acid hydrolysate of cassava pulp can be used for ethanol production in a fibrous-bed bioreactor, although some inhibition phenomena were observed during the process of fermentation.

Enhanced butanol production by modulation of electron flow in Clostridium acetobutylicum B3 immobilized by surface adsorption

The objective of this study was to improve butanol yield and productivity by redox modulation and immobilization of Clostridium acetobutylicum B3 cells. Stoichiometric network analysis revealed that NAD(P)H that had escaped from the fermentation as H2 limited the butanol yield and led to the accumulation of oxidation byproducts, e.g., acetone. Methyl viologen was used as an electron carrier to divert the electron flow away from H2 production and to reinforce the NAD(P)H supply. Butanol yield was increased by 37.8% with severely diminished acetone production. Immobilization of the cells by adsorption onto a fibrous matrix improved their butanol tolerance and production rate. An average of 15.6g/L butanol was achieved within 12h with a solvent productivity of 1.88g/L/h in repeated batch fermentation. To our knowledge, this is the highest solvent productivity with a relatively high butanol titer produced by a Clostridium strain in batch fermentation.

Evaluation for Rock Phosphate Solubilization in Fermentation and Soil–Plant System Using A Stress-Tolerant Phosphate-Solubilizing Aspergillus niger WHAK1

A strain WHAK1, identified as Aspergillus niger, was isolated from Yichang phosphate mines in Hubei province of China. The fungus developed a phosphate solubilization zone on modified National Botanical Research Institute's phosphate growth (NBRIP) agar medium, supplemented with tricalcium phosphate. The fungus was applied in a repeated-batch fermentation process in order to test its effect on solubilization of rock phosphate (RP). The results showed that A. niger WHAK1 could effectively solubilize RP in NBRIP liquid medium and released soluble phosphate in the broth, which can be illustrated by the observation of scanning electron microscope, energy-dispersive X-ray microanalysis, and Fourier transform infrared spectroscopy. Acidification of the broth seemed to be the major mechanism for RP solubilization by the fungus. Indeed, multiple organic acids (mainly gluconic acid) were detected in the broth by high-performance liquid chromatography analysis. These organic acids caused a significant drop of pH and an obvious rise of titratable acidity in the broth. The fungus also exhibited high levels of tolerance against temperature, pH, salinity, and desiccation stresses, although a significant decline in the fungal growth and release of soluble phosphate was marked under increasing intensity of stress parameters. Further, the fungus was introduced into the soil supplemented with RP to analyze its effect on plant growth and phosphate uptake of wheat plants. The result revealed that inoculation of A. niger WHAK1 significantly increased the growth and phosphate uptake of wheat plants in the RP-amended soil compared to the control soil.

Lactic acid production on liquid distillery stillage by Lactobacillus rhamnosus immobilized onto zeolite

In this study, lactic acid and biomass production on liquid distillery stillage from bioethanol production with Lactobacillus rhamnosus ATCC 7469 was studied. The cells were immobilized onto zeolite, a microporous aluminosilicate mineral and the lactic acid production with free and immobilized cells was compared. The immobilization allowed simple cell separation from the fermentation media and their reuse in repeated batch cycles. A number of viable cells of over 1010 CFUg−1 of zeolite was achieved at the end of fourth fermentation cycle. A maximal process productivity of 1.69gL−1, maximal lactic acid concentration of 42.19gL−1 and average yield coefficient of 0.96gg−1 were achieved in repeated batch fermentation on the liquid stillage without mineral or nitrogen supplementation.

Application of palm pressed fiber as a carrier for ethanol production by Candida shehatae TISTR5843

Palm pressed fiber (PPF) is a clean and renewable lignocellulosic material. The PPF and delignified PPF (DPPF) were used as a carrier for immobilization of Candida shehatae TISTR5843 in bioethanol production. PPF was pre-treated by milling to obtain small particles, whereas DPPF was the delignification of PPF using NaClO 2 . C. shehatae TISTR5843 was grown in modified yeast extract- malt (YM) medium at 30 ± 2oC on an orbital shaker at 150 rpm for batch and repeated batch fermentation. In the batch system, immobilized cells on a small size, less than 0.5 mm, of DPPF (sDPPF) gave the maximum ethanol production of 11.5 g L -1 at 24 hrs cultivation period. The ethanol concentration and ethanol yield of sDPPF were 6.2% and 6.8% higher (ethanol production 11.5 g L -1 , ethanol yield 0.47 g g -1 ) than those of free cells (ethanol production 10.8 g L -1 , ethanol yield 0.44 g g -1 ) after 36 hrs of cultivation. In contrast, the small size of PPF (sPPF) was selected as a carrier in repeated batch fermentation for cost effectiveness. The ethanol productivity of immobilized yeast cells in repeated batch fermentations was 45.2-51.6% greater than that obtained from batch fermentations. The immobilized cells on sPPF improved the ethanol production and could be reused 4 times with retaining the activity of 93%. In conclusion, PPF is a potential carrier in the immobilization system. The pre-treatment of PPF increases the surface area that enhances cell adsorption and ethanol production by C. shehatae TISTR5843.

Repeated batch fermentation with water recycling and cell separation for microbial lipid production

Large waste water disposal was the major problem in microbial lipid fermentation because of low yield of lipid. In this study, the repeated batch fermentation was investigated for reducing waste water generated in the lipid fermentation of an oleaginous yeast Trichosporon cutaneum CX1 strain. The waste fermentation broth was recycled in the next batch operation after the cells were separated using two different methods, centrifugation and flocculation. Two different sugar substrates, glucose and inulin, were applied to the proposed operation. The result showed that at least 70% of the waste water was reduced, while lipid production maintained satisfactory in the initial four cycles. Furthermore, it is suggested that T. cutaneum CX1 cells might produce certain naturally occurring inulin hydrolyzing enzyme(s) for obtaining fructose and glucose from inulin directly. Our method provided a practical option for reducing the waste water generated from microbial lipid fermentation.

Repeated-batch production of hydrogen using Rhodobacter sphaeroides S10

Abstract Photofermentation of acid hydrolyzed oil palm empty fruit bunch is reported for hydrogen production in repeated-batch fermentations using the bacterium Rhodobacter sphaeroides S10. Photofermentations were carried out at 35 °C at an incident light level of 10 klux. At specified times, different specified volumes of the culture broth were removed and replaced with an equal volume of the fresh medium. The initial mixed carbon (glucose, xylose, acetic acid) content in the medium of the repeated-batch reactors was adjusted to 20 mM. The kinetics of hydrogen production were evaluated in repeated-batch fermentations carried out in various ways: different volume exchange levels, different switch times from batch to repeated-batch operation, and different cycle times. A 90% replacement of the broth volume with the fresh medium proved to be the best, resulting in a maximum hydrogen production rate of 27.7 ± 1.7 mL H 2 /L h with a conversion efficiency of 33.7 ± 0.9%. At a fixed broth exchange volume of 90%, experiments with varying intervals between volume exchanges, showed an exchange interval of 84 h to be the best. At this exchange interval, the maximum hydrogen production rate was 35.9 ± 1.6 mL H 2 /L h and the conversion efficiency was 42.6 ± 1.0%. With the broth exchange volume fixed at 90%, the batch to repeated-batch switch time fixed at 84 h, four repeated-batch cycles were monitored, each with a cycle time of 60 h. For the five cycles (including the first batch phase), the average conversion efficiency was 51.8 ± 4.8%; the average substrate specific hydrogen production rate was 13.8 ± 2.4 mL H 2 /g h; and the average maximum hydrogen production rate was 32.3 ± 1.1 mL H 2 /L h. Thus, the kinetics of hydrogen production for the five cycles were highly consistent.

Animal Bone Char Solubilization with Itaconic Acid Produced by Free and Immobilized Aspergillus terreus Grown on Glycerol-Based Medium

Cells of Aspergillus terreus, free and immobilized in polyurethane foam, were employed in itaconic acid fermentation processes on glycerol-based media. The purpose was to assess their suitability for animal bone char solubilization and the development of a biotechnological alternative to P fertilizers chemically produced from rock phosphate. Animal bones constitute a renewable source of P that can replace the traditionally used finite, nonrenewable rock phosphate as a P source. Glycerol was an excellent substrate for growth (10.2g biomass L(-1)) and itaconic acid production (26.9g L(-1)) by free fungal cells after 120-h fermentation. Simultaneously, A. terreus solubilized the insoluble phosphate to a yield of 23 to 50%, depending on the particle size and concentration. Polyurethane foam cut into cubes of 0.5-0.6cm per side, with 0.3mm pore size and applied at 2.0g L(-1) proved to be an excellent cell carrier. In repeated batch fermentation, the immobilized mycelium showed a high capacity to solubilize animal bone char, which resulted on average in 168.8mg L(-1) soluble phosphate per 48-h cycle and 59.4% yield (percent of total phosphate) registered in the fourth batch.

Comparative study on ethanol production from pretreated sugarcane bagasse using immobilized Saccharomyces cerevisiae on various matrices

Abstract Microwave alkali pretreated sugarcane bagasse was used as a substrate for production of cellulolytic enzymes, needed for biomass hydrolysis. The pretreated sugarcane bagasse was enzymatic hydrolyzed by crude unprocessed enzymes cellulase (Filter paper activity 9.4 FPU/g), endoglucanase (carboxymethylcellulase, 148 IU/g), β-glucosidase (116 IU/g) and xylanase (201 IU/g) produced by Aspergillus flavus using pretreated sugarcane bagasse as substrate under solid state fermentation. Concentrated enzymatic hydrolyzate was used for ethanol production using Saccharomyces cerevisiae immobilized on various matrices. The yield of ethanol was 0.44 gp/gs in case of yeast immobilized sugarcane bagasse, 0.38 gp/gs using Ca-alginate and 0.33 gp/gs using agar-agar as immobilization matrices. The immobilized yeast studied up to 10 cycles in case of immobilized sugarcane bagasse and up to 4 cycles in case of agar-agar and calcium alginate for ethanol production under repeated batch fermentation study.