Repeated Batch Mode Research Articles

Enhanced production of butyric acid through immobilization of Clostridium tyrobutyricum in a novel inner disc-shaped matrix bioreactor

Cell immobilization coupled with the fermentation process and subsequent separation procedure has a great potential to improve the tolerance and efficiency of microorganisms, simplify the production process, and reduce production cost. A novel inner disc-shaped matrix (IDSM), composed of a stainless steel wire mesh and covered with fabric in a disc-shaped configuration, has been developed for butyric acid production by Clostridium tyrobutyricum. Our systematic investigation of the effects of fibrous materials, matrix size, and quantity on cell loading and butyric acid production resulted in a system, namely, fed-batch mode in an IDSMB, which achieved a high butyric acid concentration of 61.87 g/L, a productivity of 0.72 g/L·h productivity, and a yield of 0.42 g/g. The system was also evaluated for its long-term performance in a repeated-batch mode for 10 cycles. The highest concentration of butyric acid reached in 1 cycle was approximately 25.5 g/L, and the butyric yield varied from 0.41 to 0.47 g/g, with an average yield of 0.44 g/g per cycle. The volumetric productivity of butyric acid varied from 0.91 to 1.31 g/L·h, with an average of 1.16 g/L·h. The IDSMB system developed in this work for butyric acid production could be extremely applicable for industrial applications in organic acid biosynthesis.

Repeated-batch operation for the synthesis of lactulose with β-galactosidase immobilized by aggregation and crosslinking

Synthesis of lactulose under repeated-batch operation was done with cross-linked aggregates of Aspergillus oryzae β-galactosidase (CLAGs). The effect of the crosslinking agent to enzyme mass ratio and cross-linking time were first evaluated. Best results were obtained at 5.5gdeglutaraldehyde/g enzyme at 5h of cross-linking, obtaining a specific activity of 15,000IUg−1, with 30% immobilization yield. CLAG was more stable than the free enzyme under non-reactive conditions with a half-life of 123h at 50°C and when operated in repeated-batch mode, yield and productivity was 3.8 and 4.3 times higher. Maximum number of batches was determined considering biocatalyst replacement at 50% residual activity. 98 and 27 batches could be performed under such criterion at fructose/lactose molar ratio of 4 and 20 respectively, reflecting that enzyme stability is strongly affected by the sugars distribution in the reaction medium.

Effect of Complex Nutrients and Repeated-Batch Cultivation of Halobacterium salinarum on enhancing Bacteriorhodopsin Production

The extreme halophilic bacteria Halobacterium salinarum is known to produce bacteriorhodopsin (BR) protein in its purple membrane (PM) as a light-driven pump for the synthesis ATP. Its growth does not utilize simple carbon sources such as glucose, but relies on the complex carbon/nitrogen sources. The production yield of BR in the culture of H. salinarum also strongly depends on the complex nitrogen sources employed. From the various complex carbon/nitrogen sources employed in this work, tryptone rather than commonly used peptone was found to be the best complex nutrient for the growth of H. salinarum and its BR production. Bubble column photobioreactor operated in a repeated-batch mode was also employed to enhance the growth of H. salinarum by intermittent removing growth inhibitory metabolites. By employing 0.5% tryptone as carbon/nitrogen source in the culture medium, 201.8 mg/L of BR was obtained after 210 h repeated-batch cultivation which is about 50% higher than that obtained in shaker flask cultivation.

Process strategies for enhanced production of 1,3-propanediol by Lactobacillus reuteri using glycerol as a co-substrate

1,3-Propanediol (PDO) is a bulk chemical used in the synthesis of polymers for terephthalates, cosmetics and lubricants, among other things. PDO can be produced by microorganisms growing anaerobically on glycerol. The objective of this study was to analyze the behavior of Lactobacillus reuteri ATCC23272 when cultivated in batch, repeated batch and continuous modes during the conversion of glycerol into 1,3-propanediol, with regards to the main parameters for viable industrial production: productivity (QPDO), PDO concentration and yield on glycerol (YPDO/GLY). Cultures were performed in an MRS medium with glucose and glycerol as co-substrates, at 37°C. Besides the bioreactor operation mode, the following features were evaluated: anaerobiosis, limited oxygen respiration, pH 5.5 and 6.2. In batch mode, the best condition was anaerobiosis at pH 5.5, which resulted in YPDO/GLY and QPDO of 0.66 gPDOgGLY−1 and 1.42gL−1h−1, respectively. In repeated batch mode, the highest level of productivity was 4.12gL−1h−1, when cells were first decanted and 80% of the liquid phase was replaced with fresh medium. In chemostat mode, YPDO/GLY of 0.70gPDOgGLY−1 was achieved and productivity in the steady state was 20% higher (4.92gL−1h−1), compared to the best result in the repeated batch mode. The highest PDO productivity, QPDO, in chemostat mode, was due to the highest rate of glucose consumption, which can be directly related to PDO productivity.

Rapid bio-methanation of syngas in a reverse membrane bioreactor: Membrane encased microorganisms

The performance of a novel reverse membrane bioreactor (RMBR) with encased microorganisms for syngas bio-methanation as well as a co-digestion process of syngas and organic substances was examined. The sachets were placed in the reactors and examined in repeated batch mode. Different temperatures and short retention time were studied. The digesting sludge encased in the PVDF membranes was able to convert syngas into methane at a retention time of 1day and displayed a similar performance as the free cells in batch fermentation. The co-digestion of syngas and organic substances by the RMBR (the encased cells) showed a good performance without any observed negative effects. At thermophilic conditions, there was a higher conversion of pure syngas and co-digestion using the encased cells compared to at mesophilic conditions.

Butanol production by immobilised Clostridium acetobutylicum in repeated batch, fed-batch, and continuous modes of fermentation

Clostridium acetobutylicum immobilised in polyvinylalcohol, lens-shaped hydrogel capsules (LentiKats®) was studied for production of butanol and other products of acetone–butanol–ethanol fermentation. After optimising the immobilisation protocol for anaerobic bacteria, continuous, repeated batch, and fed-batch fermentations in repeated batch mode were performed. Using glucose as a substrate, butanol productivity of 0.41g/L/h and solvent productivity of 0.63g/L/h were observed at a dilution rate of 0.05h−1 during continuous fermentation with a concentrated substrate (60g/L). Through the process of repeated batch fermentation, the duration of fermentation was reduced from 27.8h (free-cell fermentation) to 3.3h (immobilised cells) with a solvent productivity of 0.77g/L/h (butanol 0.57g/L/h). The highest butanol and solvent productivities of 1.21 and 1.91g/L/h were observed during fed-batch fermentation operated in repeated batch mode with yields of butanol (0.15g/g) and solvents (0.24g/g), respectively, produced per gram of glucose.

Ethanol production from xerophilic and salt-resistant Tamarix jordanis biomass

Three different Tamarix species, namely Tamarix aphylla, T. aphylla “Erect”-type and Tamarix jordanis, were grown in an experimental field under extreme desert conditions and irrigated with either reclaimed sewage or brackish water. Depending on both species and source of irrigation, the above-ground biomass production ranged from 18 to 36 Mg ha−1 in the first year. Among the three chemically characterized Tamarix species, T. jordanis was selected due to its higher cellulose content, and lower hemicellulose and phenol contents so as to outline a preliminary process flow sheet for ethanol production. This included steam-injection heating under acidic conditions (200 °C; 90 s; 0.5% H2SO4; 160 g L−1 solid loading) using a novel lab-scale Direct Steam Injection Apparatus, enzymatic saccharification (50 °C; pH 5.0; 200 g L−1 solid loading; 20 FPU g−1 cellulose) and subsequent ethanolic fermentation (30 °C; Saccharomyces cerevisiae F-15 as the inoculum). Lab-scale fermentation runs were carried out in a 3-L stirred bioreactor in repeated-batch mode and showed an almost quantitative conversion of glucose into ethanol (0.507 ± 0.006 g g−1), thus leading to a satisfactory overall process ethanol yield of about 145 L Mg−1Tamarix biomass.

Laccase production by Trametes versicolor under limited-growth conditions using dyes as inducers

Laccase production by pre-growth pellets of Trametes versicolor using two types of textile dyes as inducers was studied. By decoupling the enzyme production phase from the growth phase, it is possible to reduce the time and nutrients required for laccase production. At the glucose maintenance level, the effect of the nitrogen source and textile dye was analysed using response surface methodology. Ammonium chloride was used as the inorganic nitrogen source. Two types of dyes were tested: Grey Lanaset G (GLG), a metal complex dye mixture containing nitrogen; and Alizarin Red (AR), an anthraquinonic dye with no nitrogen in its chemical structure. GLG induces laccase production at a higher extent than AR. Despite the limiting conditions required for the production of laccase, enzyme production increases with increasing ammonium chloride. When AR, the N-free dye, was used as an inducer, the optimal supply of N for laccase production was 1.2 mg/(g dry cell weight·d) as ammonium chloride. The reuse of fungal pellets in the repeated-batch mode under maintenance conditions was found to be a good strategy for improving laccase production, as enzyme production increased to up to seven times the production of the first cycle. It was demonstrated that GLG can be used as an inducer and as an N source and, thus, it is possible to decolorize the dye and to induce laccase production at the same time without adding an extra N source.

Changes in Microbial Community Structure During Anaerobic Repeated-Batch Treatment of Cheese-Processing Wastewater

This study investigated changes in microbial community structure, associated with changes in process performance, in an anaerobic bioreactor treating cheese-processing wastewater in repeated-batch mode. During the operation of three batch cycles, the overall reaction rate increased with the increase in starting microbial population size over cycles, indicating a positive effect of biomass accumulation on process performance. Bacterial community structure varied significantly over cycles in the reactor. In contrast, methanogen community structure was maintained almost unchanged (>90% similarity) during the experiment. This indicates that changes in bacterial community structure interestingly had little influence on the formation of methanogen community structure in the reactor. Another notable point is that the reactor methanogen community was greatly dominated by hydrogenotrophic methanogens (up to 90% of the total population), likely suggesting H2-utilizing pathway as the major methanogenesis route in the reactor. This is contrary to the conventional wisdom that aceticlastic methanogens are the main drivers of methanogenesis in anaerobic digesters under normal conditions.

Comparative study of changes in reaction profile and microbial community structure in two anaerobic repeated-batch reactors started up with different seed sludges

Microbial community structure and dynamics were examined in two anaerobic reactors run in repeated-batch mode to treat whey permeate. Despite being started up using different seeding sources, the reactors showed generally similar reaction patterns and performances. During the repeated-batch operation for three cycles, the overall reaction rate increased with the increase in the initial population size of both bacteria and methanogens over cycles. Clostridium- and Methanospirillum-related microorganisms were likely the main acidogenic and methanogenic populations, respectively, in both reactors. Bacterial community structure shifted dynamically over cycles, while little change was observed in methanogen community structure throughout the operation. This means that the changes in bacterial community structure changes had little influence on the formation and evolution of methanogen community structure in the reactors. The increased methanogenesis rate with cycles seemed therefore more likely due to the effect of the increase in methanogen abundance rather than the alteration of community structure.

Simple Strategy of Repeated Batch Cultivation for Enhanced Production of 1,3-Propanediol Using Clostridium diolis

Repeated batch cultivation (empty-and-fill protocol) using obligate anaerobe Clostridium diolis was attempted in the present study to improve the production of 1,3-propanediol (1,3-PD). In repeated batch operation, 20% (v/v) culture broth was removed from the bioreactor and supplemented with an equal volume of fresh nutrient medium when the residual glycerol concentration in the bioreactor decreased below 15g/L. Four cycles of culture broth withdrawal and subsequent replacement resulted in achieving a 1,3-PD concentration of 67.8g/L with a productivity of 1.04g/L/h at the end of 65h. This represented a 1,3-PD concentration and productivity enhancement by 2.6-fold and 1.5-fold, respectively, as compared to batch 1,3-PD fermentation. This is the first report on the use of repeated batch mode of bioreactor operation for enhanced 1,3-PD production.

Application of the aquatic fungus Phoma sp. (DSM22425) in bioreactors for the treatment of textile dye model effluents

AbstractBACKGROUND: Textile dyes are hardly removed from effluents by conventional wastewater treatment but can be degraded by a number of physicochemical processes, which nevertheless have specific limitations. The development of bioremediation processes may provide a viable alternative.RESULTS: Resting cells of three aquatic fungal strains, Alternaria sp. (Tt‐S1), Coniothyrium sp. (Kl‐S5) and Phoma sp. (DSM22425), were evaluated for their ability to decolorize model effluents of different dye application classes. Phoma sp., which decolorized all four applied model wastewaters (MWW), was immobilized on a polyester‐based fleece material originally designed for use as cooker‐hood filters, and applied in internal loop airlift and bubble column reactors in repeated batch mode under sterile and non‐sterile conditions. Depending on the applied MWW and bioreactor setup, a decolorization of more than 90% was achieved in three consecutive treatment cycles and total operating time of 16 days. In an upscaled reactor (10 L), Phoma sp. decolorized MWW containing acid dyes by 61%. Chemical oxygen demand of the MWW was reduced by 36%.CONCLUSION: The results presented underline the potential of aquatic fungi for the development of textile dyeing effluent treatment processes. Copyright © 2012 Society of Chemical Industry

Towards the integration of dark- and photo-fermentative waste treatment. 4. Repeated batch sequential dark- and photofermentation using starch as substrate

In this study we demonstrated the technical feasibility of a prolonged, sequential two-stage integrated process under a repeated batch mode of starch fermentation. In this durable scheme, the photobioreactor with purple bacteria in the second stage was fed directly with dark culture from the first stage without centrifugation, filtration, or sterilization (not demonstrated previously). After preliminary optimization, both the dark- and the photo-stages were performed under repeated batch modes with different process parameters. Continuous H2 production in this system was observed at a H2 yield of up to 1.4 and 3.9 mole mole−1 hexose during the dark- and photo-stage, respectively (for a total of 5.3 mole mole−1 hexose), and rates of 0.9 and 0.5 L L−1 d−1, respectively. Prolonged repeated batch H2 production was maintained for up to 90 days in each stage and was rather stable under non-aseptic conditions. Potential for improvements in these results are discussed.

Animal Bones Char Solubilization by Gel-EntrappedYarrowia lipolyticaon Glycerol-Based Media

Citric acid was produced with free and k-carrageenan-entrapped cells of the yeast Yarrowia lipolytica in single and repeated batch-shake-flask fermentations on glycerol-based media. Simultaneous solubilization of hydroxyapatite of animal bone origin (HABO) was tested in all experiments. The highest citric acid production by free yeast cells of 20.4 g/L and 18.7 g/L was reached after 96 h of fermentation in the absence and presence of 3 g/L HABO, respectively. The maximum values for the same parameter achieved by gel-entrapped cells in conditions of single batch and repeated-batch fermentation processes were 18.7 g/L and 28.1 g/L registered after 96 h and the 3d batch cycle, respectively. The highest citric acid productivity of 0.58 g L−1 h−1 was obtained with immobilized cells in repeated batch mode of fermentation when the added hydroxyapatite of 3 g/L was solubilized to 399 mg/L whereas the maximum efficiency of 89.0% was obtained with 1 g/L of HABO.

Production of Alkaline Protease by Entrapped Bacillus licheniformis Cells in Repeated Batch Process

In this study, Bacillus licheniformis cells were immobilized by entrapment in calcium alginate beads and were used for production of alkaline protease by repeated batch process. In order to increase the stability of the beads, the immobilization procedure was optimized by statistical full factorial method, by which three factors including alginate type, calcium chloride concentration, and agitation speed were studied. Optimization of the enzyme production medium, by the Taguchi method, was also studied. The obtained results showed that optimization of the cell immobilization procedure and medium constituents significantly enhanced the production of alkaline protease. In comparison with the free-cell culture in pre-optimized medium, about 7.3-fold higher productivity was resulted after optimization of the overall procedure. Repeated batch mode of operation, using optimized conditions, resulted in continuous production of the alkaline protease for 13 batches in 19 days.

Glycosylation of various flavonoids by recombinant oleandomycin glycosyltransferase from Streptomyces antibioticus in batch and repeated batch modes

An oleandomycin glycosyltransferase (OleD GT) gene from Streptomyces antibioticus was functionally expressed in Escherichia coli BL21 (DE3) with various molecular chaperones. The purified recombinant OleD GT catalyzed glycosylation of various flavonoids: apigenin, chrysin, daidzein, genistein, kaempferol, luteolin, 4-methylumbelliferone, naringenin, quercetin and resveratrol with UDP-glucose. 4.6 μg OleD GT was readily immobilized onto 1 mg hybrid nanoparticles of Fe(3)O(4)/silica/NiO on the basis of the affinity between His-tag and NiO nanoparticles with retention of 90% activity. In batch reaction, more than 90% naringenin (20 μM) was converted to its glycoside in 5 h. The immobilized OleD GT was efficiently reused for seven times whilst maintaining >60% of the residual activity in repeated glycosylation of naringenin.

Optimization of lab scale methanol production by Methylosinus trichosporium OB3b

Methylosinus trichosporium OB3b is a methanotrophic bacterium containing particulate methane monooxygenase (MMO), which catalyzes the hydroxylation of methane to methanol. The methanol is further oxidized to formaldehyde by methanol dehydrogenase (MDH). We developed a novel compulsory circulation diffusion system for cell cultivation. A methane/air mixture (1:1, v/v) was prepared in a tightly sealed gas reservoir and pumped into a nitrate mineral salt culture medium under optimal conditions (5 μM CuSO4, pH 7.0, 30°C). Cells were harvested, washed, and resuspended (0.6 mg dry cells/mL) in a 500 mL flask in 100 mL of 10 mM phosphate buffer (pH 7.0) containing 100 mM NaCl and 1 mM EDTA as MDH inhibitors, and 20 mM sodium formate. A single 12 h batch reaction at 25°C yielded a final concentration of 13.2 mM methanol. The use of a repeated batch mode, in which the accumulated methanol was removed after each of three 8 h cycles over a 24 h period, showed a productivity of 2.17 μmol methanol/h/mg dry cell wt. Finally, a lab-scale reaction performed using a 3 L cylindrical reactor with a working volume of 1 L produced 13.7 mM methanol after 16 h. Our results identify a simple process for improving the productivity of biologically derived methanol and, therefore the utility of methane as an energy source.

Improved methane fermentation of chicken manure via ammonia removal by biogas recycle

This study demonstrates methane fermentation that was carried out along with ammonia striping to avoid ammonia accumulation that significantly inhibited methane production. Ammonia was successfully removed by means of recycling of biogas followed by gas washing in sulfuric acid to capture ammonia, when chicken manure was anaerobically digested for 4 days at 55 °C and at an initial pH of 8–9. By using this method, 80% of total nitrogen in chicken manure was converted to ammonia and 82% of the produced ammonia was removed. A bench scale reactor equipped with an ammonia-stripping unit for methane production from chicken manure was developed and operated in repeated batch mode. At an initial pH of 8 and at 55 °C, 195 and 157 ml g-VS −1 of methane was successfully produced from the treated chicken manure and the mixture of treated chicken manure and raw chicken manure in the ratio of 1:1, respectively. In this method, ammonia concentration was maintained at a level lower than 2 g-N kg-wet sludge −1 in the reactor.

A novel honeycomb matrix for cell immobilization to enhance lactic acid production by Rhizopus oryzae

A new support matrix inspired by honeycomb was developed for cell immobilization to control fungal morphology and enhance mass transfer in bioreactor for lactic acid production by Rhizopus oryzae. The immobilization matrix composed of asterisk-shaped fibrous matrices in a honeycomb configuration provided high surface areas for cell attachment and biofilm growth. More than 90% of inoculated spores were adsorbed onto the matrices within 6–8 h and after 10 h there was no suspended cell in the fermentation broth, indicating a 100% immobilization efficiency. Compared to free-cell fermentation, lactic acid production increased ∼70% (49.5 g/L vs. 29.3 g/L) and fermentation time reduced 33% (48 h vs. 72 h) in shake-flasks with 80 g/L initial glucose. The immobilized-cell fermentation was evaluated for its long-term performance in a bubble-column bioreactor operated in a repeated batch mode for nine cycles in 36 days. The highest lactic acid production was 68.8 g/L, corresponding to a volumetric productivity of 0.72 g/L h and 93.4% (w/w) lactic acid yield from consumed glucose. The overall yield and productivity were 77.6% and 0.57 g/L h, respectively. The fermentation can be improved by increasing aeration and mixing in the bubble-column bioreactor.

Biodegradation of 4-Nitrophenol in a Two-Phase System Operating with Polymers as the Partitioning Phase

The present study has demonstrated the enhanced performance of a two-phase bioreactor, operating with polymers as a partitioning phase, as an alternative to both single phase biotreatment and to the use of an immiscible organic solvent partitioning phase, to deliver a toxic substrate (4-nitrophenol, or 4NP) to a microbial consortium in batch and repeated batch mode. Three commercial polymers were tested, Hytrel, Tone, and Elvax, and were shown to have superior properties related to the use of a consortium, including complete biocompatibility with the biomass and nonbiodegradability. Repeated kinetic tests performed with short reaction times demonstrated the accumulation of 4NP within the polymers in the range of 6-8 mg/g polymer, which reduced polymer performance in subsequent batch operations. Hytrel gave the best performance with residuals of up to 4 mg/g polymer showing no reduction in subsequent use, while for the other polymers a 4NP value lower than 2 mg/g polymer was required to have acceptable performance during repeated polymer use. Polymer reuse without affecting the process efficiency was confirmed with regeneration tests. A conventional methanol extraction method, as well as biological regeneration of the polymers by prolonged contact with the biomass, were assessed for their ability to remove the residual 4NP. Parallel kinetic tests performed with newand regenerated polymers showed a complete overlap of the 4NP concentration profiles indicating that a simple biological regeneration method provides a means of completely restoring polymer performance for repeated batch operation.