Lactic acid production by Lactobacillus delbrueckii in a dual reactor system using packed bed biofilm reactor

Aims:  An integrated dual reactor system for continuous production of lactic acid by Lactobacillus delbrueckii using biofilms developed on reticulated polyurethane foam (PUF) is demonstrated.
Methods and Results:  Lactobacillus delbrueckii was immobilized on PUF, packed in a bioreactor and used in lactic acid fermentation. The rate of lactic acid production was significantly high with a volumetric productivity of 5 g l⁻¹ h⁻¹ over extended period of time. When coupled to a bioreactor, the system could be operated as dual reactor for over 1000 h continuously without augmentation of inoculum and no compromise on productivity.
Conclusions:  Polyurethane foams offer an excellent support for biofilm formation.
Significance and Impact of the Study:  The system was very robust and could be operated for prolonged period at a volumetric productivity of 4–6 g l⁻¹ h⁻¹.     

Biosorption of Malathion using dry cells of Bacillus species S14 in a packed bed column reactor

Abstract

The ability of dried bacterial strain Bacillus sp. S₁₄ to adsorb Malathion in a packed bed column reactor was studied. The effects of important design parameters such as bed height, flow rate and influent Malathion concentration on Malathion removal from an aqueous solution was studied using a packed bed column reactor. The optimised conditions for maximum Malathion removal were found to be: flow rate: 5 mL min^-1, bed height: 6.0 cm and influent Malathion concentration: 25 mg L^-1. The Adams-Bohart model, Wolborska model, Thomas model, Yoon and Nelson Model were employed to determine characteristic parameters such as N₀ (saturation concentration, mg L^-1), β_o (external mass transfer coefficient, min^-1), k _Th(Thomas rate constant, mL min^-1mg^-1), q₀ (maximum solid phase concentration of the solute, mg L^-1), k_YN (rate constant, min^-1) and τ (time required for 50 % adsorbate breakthrough time, min) which are useful for process design. Data were fitted with Adams-Bohart model at lower region of (C/C₀) values but more accurately fitted with Wolborska and Thomas model.     

R-mandelic acid production with immobilized recombinant Escherichia coli cells in a recirculating packed bed reactor

A recirculating packed bed reactor (RPBR) was used for efficient production of R-mandelic acid (R-MA) by kinetic resolution of racemic R,S-mandelonitrile (R,S-MN) using the recombinant E. coli cells crosslinked with diatomite (DA)/glutaraldehyde (GA)/polyethyleneimine (PEI). The performance and productivity of RPBR were evaluated by several parameters, including cell load, substrate feeding rate, height diameter (H/D) ratio, reactor structures, and operation stability. The kinetic resolution process showed higher initial reaction rate (1.52?mM/min) and yield (100%) by recycling 100?mL of substrate solution (70?mM) through RPBR packed with 6.0?g immobilized cells at a substrate-feeding rate of 19?mL/min while the H/D ratio was 2.8. The immobilized cells were successfully applied into kinetic resolution of R,S-MN in the RPBR for 50 batches with an average productivity of 4.12?g/L/h for R-MA with >99% of enantiomeric excess.     

Continuous removal of malathion by immobilised biomass of Bacillus species S14 using a packed bed column reactor

Abstract

Biosorption of malathion from aqueous solution was studied using Bacillus sp. S₁₄ immobilised on calcium alginate (3%) using a packed bed column reactor at a temperature of 25 °C and a pH of 7.0. The experiments were conducted to study the effect of important design parameters such as bed height, flow rate and influent malathion concentration. Maximum removal capacity (57%) was found at 4 mL min^-1 flow rate, 6.0 cm bed height and 25 mg L^-1 influent malathion concentration. The Adam-Bohart model, Wolborska model, Thomas model, Yoon-Nelson model were employed to determine characteristic parameters such as saturation concentration, external mass transfer coefficient, Thomas rate constant, the maximum solid phase concentration of the solute, rate constant, and the time required for 50% adsorbate breakthrough time, which are all useful for process design. Experimental data were well fitted with Adam–Bohart model at the lower region of effluent/influent malathion concentration values but at higher region values data fitted well with the Thomas and Yoon-Nelson models.     

Continuous enzymatic production and adsorption of laminaribiose in packed bed reactors

Bienzymatic production of laminaribiose from sucrose and glucose was combined with adsorption on zeolite BEA to introduce a first capture and purification step. Downstream processing including washing and desorption steps was characterized and optimized on a milliliter scale in batch mode. Results were then transferred to a packed bed system for enzymatic production and adsorption where the influence of adsorbent particle diameter on purity and productivity was evaluated. Finally, a continuous enzymatic production of laminaribiose was conducted over 10 days. The subsequent downstream processing of the loaded zeolites led to purities of over 0.5 g_{Laminaribiose} g_sugar^?1 in the desorbate with a total productivity of 5.6 mg_{Laminaribiose} L_{enzyme bed}^?1 h^?1 without the use of recycles.     

Continuous acetone-butanol-ethanol (ABE) fermentation using immobilized cells of Clostridium acetobutylicum in a packed bed reactor and integration with product removal by pervaporation

An integrated solvent (ABE) fermentation and product removal process was investigated. A stable solvent productivity of 3.5 g/L h was achieved by using cells of Clostridium acetobutylicum immobilized onto a packed bed of bonechar, coupled with continuous product removal by pervaporation. Using a concentrated feed solution containing lactose at 130g/L, a lactose value of 97.9% was observed. The integrated fermentation and product removal system, with recycling of the treated fermentor effluent containing only low amount of solvents (/but lactose and acids), leads to only low acid losses. Therefore, most of the acids are converted to solvents, and this results in a high solvent yield of 0.39 g solvents/g lactose utilized. The pervaporation system provided a high product removal rate even at low solvent concentrations. A solvent membrane flux of 7.1 g/m(2) h with a selectivity of 5 was achieved during these investigations. The system proved to be very reliable.     

Synthesis of 2-ethyl hexanol fatty acid esters in a packed bed bioreactor using a lipase immobilized on a textile membrane

An enzymatic process using a packed bed bioreactor with recirculation was developed for the scale-up synthesis of 2-ethylhexyl palmitate with a lipase from Candida sp. 99–125 immobilized on a fabric membrane by natural attachment to the membrane surface. Esterification was effectively performed by circulating the reaction mixture between a packed bed column and a substrate container. A maximum esterification yield of 98% was obtained. Adding molecular sieves and drying the immobilized lipase both decreased the water content at the reactor outlet and around the enzyme, which led to an increase in the rate of esterification. The long-term stability of the reactor was tested by continuing the reaction for 30 batches (over 300 h) with an average esterification yield of about 95%. This immobilized lipase bioreactor is scalable and is thus suitable for industrial production of 2-ethylhexyl palmitate.     

Performance of a pilot-scale packed bed reactor for perchlorate reduction using a sulfur oxidizing bacterial consortium

A novel sulfur‐utilizing perchlorate reducing bacterial consortium successfully treated perchlorate (ClO) in prior batch and bench‐scale packed bed reactor (PBR) studies. This study examined the scale up of this process for treatment of water from a ClO and RDX contaminated aquifer in Cape Cod Massachusetts. A pilot‐scale upflow PBR (～250‐L) was constructed with elemental sulfur and crushed oyster shell packing media. The reactor was inoculated with sulfur oxidizing ClO reducing cultures enriched from a wastewater seed. Sodium sulfite provided a good method of dissolved oxygen removal in batch cultures, but was found to promote the growth of bacteria that carry out sulfur disproportionation and sulfate reduction, which inhibited ClO reduction in the pilot system. After terminating sulfite addition, the PBR successfully removed 96% of the influent ClO in the groundwater at an empty bed contact time (EBCT) of 12 h (effluent ClO of 4.2 µg L^?1). Simultaneous ClO and NO reduction was observed in the lower half of the reactor before reactions shifted to sulfur disproportionation and sulfate reduction. Analyses of water quality profiles were supported by molecular analysis, which showed distinct groupings of ClO and NO degrading organisms at the inlet of the PBR, while sulfur disproportionation was the primary biological process occurring in the top potion of the reactor. Biotechnol. Bioeng. 2012; 109:637–646. © 2011 Wiley Periodicals, Inc.     

Lipase-mediated acidolysis of butteroil with free conjugated linoleic acid in a packed bed reactor

Acidolysis of butteroil with free conjugated linoleic acid (CLA) was studied in a packed bed reactor containing an immobilized Candida antarctica (fraction B) lipase. Kinetic data were used to develop quantitative reversible rate expressions of the general Michaelis-Menten form that also incorporate a term for first-order deactivation of the enzyme. The extent of incorporation of CLA in the triacylglycerols of butteroil was characterized for reactions carried out at several temperatures (namely 45 degrees, 50 degrees, and 55 degrees C) with different weight ratios of butteroil to CLA (namely 10:1 and 2:1). At the optimum operating temperature of 50 degrees C, similar levels of incorporation of CLA (60% to 85%) were achieved at low space times (<3 h) for both 10:1 and 2:1 (w/w) ratios of butteroil to CLA.     

解淀粉芽胞杆菌PC2产抑菌物质培养基及发酵条件优化

【目的】优化解淀粉芽胞杆菌PC2产抑菌活性物质发酵培养基及发酵条件。【方法】以马铃薯葡萄糖液体培养基为基础,依据发酵液对金黄色葡萄球菌抑菌圈的单因素试验结果,采用Box-Behnken响应面法优化发酵培养基,二次通用旋转组合设计,频率分析法优化发酵条件。【结果】影响发酵液抑菌活性的培养基主要组分为马铃薯、蔗糖和L-谷氨酸钠,最优发酵培养基配方为:马铃薯188.0 g/L,蔗糖22.0 g/L,L-谷氨酸钠1.80 g/L,培养基成本为0.81元/L;最佳发酵条件为:接种量6%、发酵温度30°C、装液量40 mL/250 mL、摇床转速185 r/min、发酵时间24 h、初始pH 7.0。优化后发酵液对金黄色葡萄球菌抑菌圈直径为30.82 mm,较优化前的18.22 mm增加了12.60 mm。【结论】优化后的培养基和发酵条件提高了解淀粉芽胞杆菌PC2发酵液的抑菌活性,为该菌株的工业化生产应用提供了依据。     

Efficient production of S-(+)-2-chlorophenylglycine by immobilized penicillin G acylase in a recirculating packed bed reactor

(S)-(+)-2-Chlorophenylglycine 1 is an important intermediate in the synthesis of Clopidogrel. A recirculating packed bed reactor (RPBR) was constructed for efficient production of (S)-1 by kinetic resolution of racemic N-phenylacetyl-2- chlorophenylglycine 2 using immobilized penicillin G acylase (PGA). The immobilized PGA exhibited maximum activity at 50 °C and pH 8.0 with (R,S)-2 as substrate. The kinetic constants (K_m and v_max) of immobilized PGA were calculated to be 20.61 mM and 83.2 mM/min/g, respectively. The substrate displayed inhibitory effect on immobilized PGA with inhibition constant of 221.23 mM. The immobilized PGA showed a strict enantiospecificity for substrate at different temperature, pH and substrate concentration examined. The performance and productivity of RPBR were evaluated by several critical parameters, including immobilized PGA load, substrate feeding rate, height to diameter ratio and so on. The kinetic resolution process shows higher initial reaction rate and conversion by recycling 100 mL of substrate solution (80 mM) through RPBRs packed with 6.0 g immobilized PGA with a feeding rate of 1.5 mL/min while the H/D ratio was 4.0. The immobilized PGA-catalyzed kinetic resolution of (R,S)-2 was successfully operated in the RPBR for 60 batches, with an average productivity of 1.2 g/L/h for (S)-1 in high optical purity (>97% enantiomeric excess) in semi-continuous operation. The residual (R)-2 can be easily racemized and then used as substrate.     

Efficient preparation of (R)-alpha-monobenzoyl glycerol by lipase catalyzed asymmetric esterification: optimization and operation in packed bed reactor

Optically active (R)-alpha-monobenzoyl glycerol (MBG) was synthesized by Candida antarctica lipase B (CHIRAZYME L-2) catalyzed asymmetric esterification of glycerol with benzoic anhydride in organic solvents. Various conditions, such as the type and composition of the organic solvent, water content of the system, reaction temperature, and concentrations of the substrates were systematically examined and optimized in screw-capped test tubes with respect to both the reaction rate and the enzyme selectivity. 1,4-Dioxane was found to be the best solvent and no additional water was needed for the system. The optimum temperature was around 30 degrees C, while the most suitable substrate concentrations were 100 mM each for glycerol and benzoic anhydride, respectively. However, when excessive anhydride (e.g., 200 mM) was used, the produced MBG could be further transformed into 1,3-dibenzoyl glycerol (DBG) by the same enzyme with a priority to (S)-MBG, resulting in a significant improvement of the product optical purity from ca. 50-70% e.e. Under optimal conditions (100 mM glycerol, 100-200 mM benzoic anhydride, dioxane, 25-30 degrees C), the enzymatic synthesis of (R)-MBG was successfully operated in a packed-bed reactor for about 1 week, with an average productivity of 0.79 g MBG/day/g biocatalyst in the case of continuous operation and 0.94 g MBG/day/g biocatalyst in the case of semicontinuous operation. After refinement and preferential crystallization of the crude product, (R)-MBG could be obtained in an almost optically pure form (>98% e.e.).     

A statistical approach for the enhanced production of thermostable alkaline protease showing detergent compatibility activity from Bacillus circulans

Abstract

Response surface methodology (RSM) was employed to enhance the production of a thermostable alkaline protease from Bacillus circulans. Significant influences of peptone, yeast extract, and glucose on protease production were noted with a one-variable-at-a-time optimization strategy. Then, a full factorial central composite design (CCD) was applied to study the effects of glucose, peptone, and yeast extract to determine the optimal concentrations of these compounds for protease production by B. circulans under shake flask fermentation conditions. The statistical reliability and significance of the model was validated by an F-test for analysis of variance (ANOVA); enzyme production was improved significantly under optimized conditions. The enzyme was purified by ammonium sulphate fractionation, and gel filtration chromatography. Maximum enzyme activity was observed at 60°C temperature, and at pH 10. Alkaline protease from B. circulans showed excellent compatibility and stability in the presence of commercial detergents like Ariel, Surf Excel, Tide, Rin, Nirma, Wheel, and Doctor and showed excellent blood destaining effectiveness with commercial detergents.     

Response-surface methodology for the production and the purification of a new H2O2-tolerant alkaline protease from Bacillus invictae AH1 strain

This work deals with the optimization of the culture conditions of Bacillus invictae AH1 in order to increase the production level of the proteolytic activity. Response-surface methodology (RSM) was applied for the most significant fermentation parameters (concentration of wheat bran and K₂HPO₄/KH₂PO₄) that were earlier identified by Plackett–Burman Design from seven possible factors. A central composite design was used and the quadratic regression model of producing active protease was built. A maximum protease activity was reached and validated experimentally, using a maximum wheat bran concentration (50 g/L) with increased K₂HPO₄/KH₂PO₄ concentration (2.275 g/L). Protease production obtained experimentally coincident with the predicted value and the model was proven to be adequate. Interestingly, the use of RSM increased the protease production by four times (7,000 U/mL) using a low-cost substrate and a culture time of 40 hr, as compared to the standard culture conditions. In the second part of this study, a H₂O₂-tolerant alkaline protease produced from B. invictae AH1 with a molecular mass of about 41 kDa, noted P3, was purified by successive steps of ultrafiltration, gel filtration and ion exchange chromatography. The K _m and V_max values of the purified protease using casein, as substrate, were about 4 mg/mL and 27 μM/min, respectively. The highest enzyme activity was found at pH 9.0 and a temperature of 60°C. In addition, the enzyme showed a quasi-total stability against H₂O₂ (5% for 1 hr) and against most of the tested solid and liquid detergents, suggesting its eventual use in bio-detergent formulations.     

Dimethylsulfoxide and sodium butyrate enhance the production of recombinant cyclooxygenase 2 in stably transformed Drosophila melanogaster S2 cells

Recombinant human cyclooxygenase 2 (Cox 2) was expressed in stably transformed Drosophila melanogaster S2 cells, and was present primarily in the cellular fraction at a molecular weight of 70 to 74 kDa. Recombinant Cox 2 was purified using Ni²⁺-affinity fractionation to a specific activity of 24 800 U mg^–1 protein. The peak level of recombinant Cox 2 production was 1.6 g (10⁷ cells)^–1, seven days after induction with 0.5 mM CuSO₄. Supplementing the cultures with dimethylsulfoxide or sodium butyrate increased recombinant Cox 2 production by 170% and 86%, respectively.     

Cyclin A2/cyclin-dependent kinase 1-dependent phosphorylation of Top2a is required for S phase entry during retinal development in zebrafish

Cyclin-dependent kinase 1 (CDK1) plays an essential role in cell cycle regulation.However,as mouse Cdk1embryos die early,the role of CDK1 in regulating the cell cycle and embryo development remains unclear.Here,we showed that zebrafish cdk1~(-/-)embryos exhibit severe microphthalmia accompanied by multiple defects in S phase entry,M phase progression,and cell differentiation but not in interkinetic nuclear migration.We identified Top2a as a potential downstream target and cyclin A2 and cyclin B1 as partners of Cdk1 in cell cycle regulation via an in silico analysis.While depletion of either cyclin A2 or Top2a led to the decreased S phase entry in zebrafish retinal cells,the depletion of cyclin B1 led to M phase arrest.Moreover,phosphorylation of Top2a at serine 1213 (S1213) was nearly abolished in both cdk1 and ccna2mutants,but not in ccnb1 mutants.Furthermore,overexpression of TOP2A~(S1213D),the phosphomimetic form of human TOP2A,rescued S phase entry and alleviated the microphthalmia defects in both cdk1~(-/-)and ccna2~(-/-)embryos.Taken together,our data suggest that Cdk1 interacts with cyclin A2 to regulate S phase entry partially through Top2a phosphorylation and interacts with cyclin B1 to regulate M phase progression.     

GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone 4-phosphate synthase are rate-limiting enzymes in riboflavin synthesis of an industrial Bacillus subtilis strain used for riboflavin production

One of the proteins encoded by the riboflavin operon of Bacillus subtilis, RibA, was identified as the rate limiting enzyme in an industrial riboflavin producing strain. An additional single copy of the ribA gene was introduced into the sacB locus of the riboflavin production strain and was expressed constitutively from the medium strength vegI promoter. This led to improved riboflavin titers and yields of riboflavin on glucose of up to 25%. Both enzymatic activities of RibA, the 3,4-dihydroxy-2-butanone 4-phosphate synthase activity located in the N-terminal half of the protein and the GTP cyclohydrolase II activity of the C-terminal domain, are necessary for the improved riboflavin productivity. Received 16 June 1998/ Accepted in revised form 30 October 1998     

Optimization of media for β-fructofuranosidase production by Aspergillus niger in submerged and solid state fermentation

The kinetics of β-fructofuranosidase (Ffase) production by Aspergillus niger in submerged (SmF) and solid-state fermentation (SSF) systems was investigated. The maximum productivity of Ffase (81.8 U/l per h) was obtained in SSF for 72 h while it was 18.3 U/l per h in SmF for 120 h. The productivity of extra cellular Ffase produced in SSF was 5-fold higher than in SmF. Optimization of fermentation medium for Ffase production was carried out using De Meo's fractional factorial design with seven components such as (NH₄)₂SO₄, KH₂PO₄, FeSO₄, MgSO₄ · 7H₂O, sucrose, urea and yeast extract. The media designed for SmF after two steps of optimization supported the growth of A. niger and higher productivity of Ffase (58.3 U/l per h) than with the medium before optimization. The optimized medium of SmF when used in SSF, did not improve the Ffase productivity and therefore medium for SSF was optimized independent of SmF. After two optimization steps, the media was defined for SSF which supported the growth and high level of Ffase productivity (149.1 U/l per h) in SSF compared to the medium before optimization (81.8 U/l per h) and optimized medium for SmF (58.3 U/l per h). Our results suggested that the optimized media for SmF and SSF for the production of Ffase have to be different.     

Evaluation of L-Cystine-amended H2S test for the detection of faecal pollution in potable water: comparison with standard multiple tube fermentation method

L-Cystine-amended H₂S strip test was compared with conventional multiple tube fermentation tests for detecting faecal pollution in 200 drinking water samples drawn from different water sources. Of these, 134 samples were positive for total coliforms (TC), whereas the cystine-amended H₂S test was positive with 116 samples. Faecal coliforms were present in 99 samples. Seventy-seven samples were also tested with presence–absence (P–A) strip test. By taking only the colour change (acid production) as positive indication for the presence of coliforms, 12 (47%) out of 26 water samples were graded as polluted in the absence of detectable coliforms. This indicated the critical importance of observing P–A bottle for gas production along with the colour. This aspect is important when commercially available P–A bottles are used in rural areas by unskilled personnel. Among different unpiped water sources tested, 94% samples drawn from India Mark II hand-pumps had TC counts within WHO guidelines. This revised version was published online in July 2006 with corrections to the Cover Date.