5L Bioreactor Research Articles

Improving the production of human‐like collagen by pulse‐feeding glucose during the fed‐batch culture of recombinant Escherichia coli

To increase the target protein production and reduce acetic acid accumulation during fed-batch cultivation of recombinant Escherichia coli BL21 in a 30-L bioreactor, 12 different models of pulse feeding were performed to evaluate the effect of pulse feeding at different cultivation phases and pulse frequency on cell growth, acetic acid accumulation, and human-like collagen (HLC) synthesis. The results showed that the acetate concentration was kept at a low level (below 0.5 g/L) in all cases when pulse feeding was introduced before induction, whereas the pulse frequency affected cytoactivity significantly through cell growth rate, oxygen uptake rate, carbon dioxide evolution rate, and the synthesis of the target protein. The final biomass and HLC reached 75.46 and 7.26 g/L, respectively, in the model of 8-Sec feedings per 188 Sec. After induction, the pulse frequency had a great effect on HLC synthesis after high-temperature induction; low frequency was adverse to microorganisms. The model of 3-Sec feeding per 27 Sec was best and resulted in the highest biomass and HLC production. Compared to the pseudo-exponential feeding, pulse feeding reduced acetic acid accumulation effectively.

Efficient production of l-asparaginase from Bacillus licheniformis with low-glutaminase activity: Optimization, scale up and acrylamide degradation studies

l-Asparaginase has potential as an anti-cancer drug and for prevention of acrylamide formation in fried and baked foods. Production of the enzyme by Bacillus licheniformis (RAM-8) was optimized by process engineering using a statistical modeling approach and a maximum yield of 32.26IU/ml was achieved. The l-asparaginase exhibited glutaminase activity of only 0.8IU/ml and would therefore be less prone to cause the side effects associated with asparaginase therapy compared to enzyme preparations with higher glutaminase activities. When production was carried out in a 30-L bioreactor, enzyme production reached 29.94IU/ml in 15h. The enzyme inhibited poly-acrylamide formation in 10% acrylamide solution and reduced acrylamide formation in fried potatoes by 80%.

Effect of Additives on Freeze-Drying and Storage of Yarrowia lipolytica Lipase

The extracellular lipase of Yarrowia lipolytica presents numerous potentialities for biotechnological applications. This work describes the development and storage of powders obtained from supernatants containing Y. lipolytica lipase by freeze-drying as downstream process that is important in obtaining a stable lipase powder with high enzymatic activity. Lipase was produced by Y. lipolytica U6 mutant strain in 20-L bioreactor. Non-concentrated cell-free culture supernatant samples were supplemented with different concentrations (0.5-1%) of maltodextrin and glycerol as additives to freeze-drying. Effects of additives, temperature, pH, and storage time on lipase powders were determined. After addition of additives, freeze-drying yield increased 3.5-fold compared to supernatant without additive. Maltodextrin with 0.5% concentration gave the best protection of lipase during dehydration treatment and its freeze-drying yield (77%) is better than other formulations. Lipase powders were stored at 4 and 25°C for 46weeks without loss of lipase activity. A common impediment to the production of commercial enzyme is their low-stability aqueous solutions. The present study shows that freeze-dried lipase powders of Y. lipolytica have good stability for storage and various applications.

Optimization of Xylanase production from Penicillium sp.WX-Z1 by a two-step statistical strategy: Plackett-Burman and Box-Behnken experimental design.

The objective of the study was to optimize the nutrition sources in a culture medium for the production of xylanase from Penicillium sp.WX-Z1 using Plackett-Burman design and Box-Behnken design. The Plackett-Burman multifactorial design was first employed to screen the important nutrient sources in the medium for xylanase production by Penicillium sp.WX-Z1 and subsequent use of the response surface methodology (RSM) was further optimized for xylanase production by Box-Behnken design. The important nutrient sources in the culture medium, identified by the initial screening method of Placket-Burman, were wheat bran, yeast extract, NaNO3, MgSO4, and CaCl2. The optimal amounts (in g/L) for maximum production of xylanase were: wheat bran, 32.8; yeast extract, 1.02; NaNO3, 12.71; MgSO4, 0.96; and CaCl2, 1.04. Using this statistical experimental design, the xylanase production under optimal condition reached 46.50 U/mL and an increase in xylanase activity of 1.34-fold was obtained compared with the original medium for fermentation carried out in a 30-L bioreactor.

Abstract 153: Creation of Cell Sheet-Based Bioengineered Cardiac Tissue Using Pluripotent Stem Cell-Derived Cells

Recent evidences have suggested that current cardiac cell therapy contributes to the improved cardiac function through mainly the paracrine effects. Thereby the bioengineered functional heart tissue is expected to function for repairing the broad injured heart. We have developed the cell sheet-based bioengineered vascularized cardiac tissue, however the system to collect the enough amount of cells from ES/iPS cells and the function of ES-derived cardiac tissue remain elusive. Recently we have established the cultivation system with the suitable conditions for expansion and cardiac differentiation of mouse ES cells and human iPS cells via embryoid body formation using three-dimensional bioreactor with the continuous perfusion system. For the cardiac differentiation experiments, we used several mouse ES cells that express EGFP or neomycin resistant gene under the control of αMHC promoter. At 10 days of differentiation, mouse ES cells increased up to 300 times (6.0×10 6 cells/mL). After the further 8 days of cultivation with the purification step, we collected around 5.0×10 8 cells in the 1L bioreactor culture and 99% of cells were positive for myosin heavy chain. The co-culture of ES-derived cardiomyocytes with the appropriate number of primary cultured fibroblasts on the temperature-responsive culture dishes enabled to form the cardiac cell sheets. Furthermore, when ES-derived cardiomyocytes were co-cultured with ES-derived endothelial cells, robust endothelial cell network was observed in the cardiac cell sheets. Mouse ES- or human iPS-derived cardiomyocytes in cell sheets beat spontaneously and synchronously and connexin43 was expressed at the edge of the adjacent cardiomyocytes. Furthermore the action potential propagation was observed between ES/iPS-derived cardiac cell sheets. These findings suggest that pluripotent stem cell-derived cardiomyocytes and endothelial cells might be useful for creating cell-sheet-based functional cardiac tissue and the layered stem cell-derived cardiac tissue might promote not only the cardiac regenerative medicine but also the understanding the molecular mechanisms of heart diseases.

Production and separation of dipeptidyl peptidase IV from Lactococcus lactis: scale up for industrial production

Lactococcus lactis spp. lactis and Lactococcus lactis spp. cremoris are widely used in the manufacture of fermented milk. These strains were compared for production of Dipeptidyl Peptidase IV (DPP IV) enzyme in terms of enzyme activity, specific growth rates and productivity. Lactococcus lactis spp. lactis was produced in 3L bioreactor and scaled up to 30 and 150L stirred tank bioreactors, and the enzyme activities were found as 110, 110 and 122mUmL(-1), respectively. After 8h of production, separation steps were performed. While purification fold was 127 and yield was 2.69%, the molecular weight of the enzyme was estimated as 68kDa. Partially purified enzyme was enteric coated with capsules and a 95.5% of DPP IV enzyme passed into the artificial intestine. Results show that production of DPP IV enzyme by Lactococcus lactis spp. lactis strain in submerged culture is comparable with the productions by commercial strains, mostly Aspergillus, in solid state fermentations based on productivity.

Cluster analysis of ginseng tissue cultures, dynamic change of growth, total saponins, specific oxygen uptake rate in bioreactor and immuno-regulative effect of ginseng adventitious root

In order to identify ginsenoside composition in native ginseng, cell, hairy root and adventitious root, the ESI-MSn analysis was performed. Rg1, Re, Ro, Malonyl-Rb1, Rb1, Rc, Rb2 and Rd were identified from ginseng tissue cultures on the basis of LC–MSn analysis. The content of total saponins in adventitious root was much higher than that in other tissue cultures. Cluster analysis showed that the quality of adventitious root was mostly similar with native ginseng. During the 5l bioreactor culture of ginseng adventitious root, the maximum dry weight and total saponin were achieved on day 40 and 30, respectively. Specific oxygen uptake rate (SOUR) increased quickly within the first 10 days. After day 10, SOUR showed a sharp drop and was almost zero after 20 days. Delayed-type hypersensitivity reaction to dinitrofluorobenzene (DNFB) and the proliferation of splenocytes from mice in response to concanavalin A (ConA) were used to evaluate the effects of native ginseng root (NGR) and ginseng adventitious root (GAR) on cellular immune response. As the result, GAR possessed a similar cellular immuno-regulation as NGR had.

Production and characterisation of recombinant α-l-arabinofuranosidase for production of xylan hydrogels

A recombinant strain of the protease-deficient, non-acidifying pH mutant Aspergillus niger D15 (A. niger D15 [abfB]) strain was developed to secrete α-L-arabinofuranosidase (AbfB) free of endo-1,4-β-xylanases for selective hydrolysis of xylan into hydrogels. The A. niger D15 [abfB] strain expressed the α-L-arabinofuranosidase abfB gene under the transcriptional control of the glyceraldehyde-3-phosphate dehydrogenase promoter (gpd(P)) and glucoamylase terminator (glaA(T)) in fermentation cultures containing 10 % glucose. The yield, activity, purity, kinetics and ability of the recombinant AbfB to selectively hydrolyse xylans into hydrogels were assessed. The recombinant AbfB secreted in 125-mL shake flasks and 10-L bioreactor fermentation cultures had specific activities against ρ-nitrophenyl-α-arabinofuranoside of up to 4.4 and 2.7 U g⁻¹ (dry weight), respectively. In addition, the recombinant AbfB was present as a single protein species on silver-stained 10 % sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The recombinant AbfB had optimal activity at 40-55 °C and pH 3.0 to pH 5.0 and was stable at temperature and pH of up to 60 °C and pH 6.0, respectively. About 20 % of the available arabinose in the xylan was released by the recombinant AbfB from the hydrolysis of low viscosity wheat and oat spelt arabinoxylans and about 9 and 5 % from bagasse and bamboo arabinoglucuronoxylans, respectively, that led to the formation of the hydrogels. Therefore, the constructed A. niger D15 [abfB] strain presented a microbial system for the production of recombinant AbfB with the required purity for the modification of xylans into hydrogels.

Is quorum sensing involved in lovastatin production in the filamentous fungus Aspergillus terreus?

A novel role for butyrolactone I as a quorum sensing molecule in the filamentous fungus Aspergillus terreus is proposed based on its ability to affect both lovastatin and its own production.In this work, the exogenous addition of 100nM butyrolactone I at 96h post-inoculation to submerged cultures of A. terreus in 5L bioreactors resulted in 2.5-fold increase in lovastatin production as compared to control cultures at 168h. An increase in endogenous butyrolactone I levels (2.5-fold) is also detected in cultures supplemented with butyrolactone I, thus suggesting an auto-stimulatory function. Molecular analysis of butyrolactone I-mediated enhancement of lovastatin production revealed induction of lovastatin biosynthetic genes, lovB and lovF, at the transcriptional level. Microarray analysis of A. terreus transcriptome depicted a growth phase-specific response to butyrolactone I addition as the majority of the expressed genes showed differential expression during the specific growth phase (p<0.01). This study demonstrates for the first time the potential of butyrolactone I as a growth phase-specific inducer of the secondary metabolite lovastatin production and shows the auto-stimulatory effect of this molecule on its own production in the filamentous fungus A. terreus.

Modular Pathway Engineering of Diterpenoid Synthases and the Mevalonic Acid Pathway for Miltiradiene Production

Microbial production can be advantageous over the extraction of phytoterpenoids from natural plant sources, but it remains challenging to rationally and rapidly access efficient pathway variants. Previous engineering attempts mainly focused on the mevalonic acid (MVA) or methyl-d-erythritol phosphate (MEP) pathways responsible for the generation of precursors for terpenoids biosynthesis, and potential interactions between diterpenoids synthases were unexplored. Miltiradiene, the product of the stepwise conversion of (E,E,E)-geranylgeranyl diphosphate (GGPP) catalyzed by diterpene synthases SmCPS and SmKSL, has recently been identified as the precursor to tanshionones, a group of abietane-type norditerpenoids rich in the Chinese medicinal herb Salvia miltiorrhiza . Here, we present the modular pathway engineering (MOPE) strategy and its application for rapid assembling synthetic miltiradiene pathways in the yeast Saccharomyces cerevisiae . We predicted and analyzed the molecular interactions between SmCPS and SmKSL, and engineered their active sites into close proximity for enhanced metabolic flux channeling to miltiradiene biosynthesis by constructing protein fusions. We show that the fusion of SmCPS and SmKSL, as well as the fusion of BTS1 (GGPP synthase) and ERG20 (farnesyl diphosphate synthase), led to significantly improved miltiradiene production and reduced byproduct accumulation. The MOPE strategy facilitated a comprehensive evaluation of pathway variants involving multiple genes, and, as a result, our best pathway with the diploid strain YJ2X reached miltiradiene titer of 365 mg/L in a 15-L bioreactor culture. These results suggest that terpenoids synthases and the precursor supplying enzymes should be engineered systematically to enable an efficient microbial production of phytoterpenoids.

Simultaneous hexadecane and oxygen transfer rate on the production of an oil-degrading consortium in a three-phase airlift bioreactor

The hexadecane (HXD) transfer rate (HTR) and the oxygen transfer rate (OTR) were simultaneously evaluated as a single engineering criterion to enhance the production of an oil-degrading bacterial consortium. In order to accomplish the task, a 10-L three-phase airlift bioreactor (ALB) was used. Oxygen transfer volumetric coefficient (kLaO2) values of 25–49h−1 were found for several superficial gas velocities (Ug 0.15–2.7cms−1). HXD transfer volumetric coefficient (kLaHXD) values of 0.005–0.041h−1 were obtained for the same range of Ug values. OTR and HTR were calculated as the product of kLaO2 or kLaHXD and the respective concentration gradient. HXD transfer parameters were evaluated by using an early reported novel technique. During 14 days culture, using constant Ug values, the ratio of HTR to OTR (HTR/OTR) never reached the stoichiometric ratio (0.25±0.05g HXD (gO2)−1). However, the productivity at the higher assayed constant Ug (2.7cms−1) was as good as 1.02±0.03g SS (Ld)−1. We propose a variable Ug strategy that consumes 33% less energy than a higher constant Ug strategy to achieve the same yield and productivity. In this study, the oil-degrading consortium, able to remediate contaminated soils and water, productivity was successfully enhanced with simultaneous HXD and oxygen transfer considerations.

An Integrated Control Strategy for the Fermentation of the Marine-Derived Fungus Aspergillus glaucus for the Production of Anti-cancer Polyketide

An integrated control strategy of pH, shear stress, and dissolved oxygen tension (DOT) for fermentation scale-up of the marine-derived fungus Aspergillus glaucus HB 1–19 for the production of the anti-cancer compound aspergiolide A was studied. Keeping initial pH of 6.5 and shifting pH from 6.0 to 7.0 intermittently during the production phase greatly facilitated biosynthesis of aspergiolide A in shake flask cultures. Thus, a pH-shift strategy was proposed that shifting pH to 7.0 once it went lower than 6.0 by pulsed feeding NaOH solution during the production phase in bioreactor fermentation of A. glaucus HB 1–19. As a result, aspergiolide A production in a 30-L bioreactor was increased to 37.6 mg/L, which was 48.6% higher than that in 5-L bioreactor without pH shift. Fermentation scale-up was then performed in a 500-L bioreactor on the basis of an integrated criterion of near-same impeller tip velocity of early phase, DOT levels, and pH shift. The production of aspergiolide A was successfully obtained as 32.0 mg/L, which was well maintained during the process scale-up. This work offers useful information for process development of large-scale production of marine microbial metabolites.

Constitutive Expression of Yarrowia lipolytica Lipase LIP2 in Pichia pastoris Using GAP as Promoter

A gene encoding Yarrowia lipolytica lipase LIP2 (YlLIP2) was cloned into a constitutive expression vector pGAPZαA and electrotransformed into the Pichia pastoris X-33 strain. The high-yield clones obtained by high copy and enzyme activity screening were chosen as the host strains for shaking flask and fermentor culture. The results showed that glucose was the optimum carbon source for YlLIP2 production, and the maximum hydrolytic activity of recombinant YlLIP2 reached 1,315U/ml under the flask culture at 28°C, pH7.0, for 48h. The fed-batch fermentation was carried out in 3- and 10-l bioreactors by continuously feeding glucose into the growing medium for achieving high cell density and YlLIP2 yields. The maximum hydrolytic activity of YlLIP2 and cell density obtained in the 3-l bioreactor were 10,300U/ml and 116g dry cell weight (DCW)/l, respectively. The peak hydrolytic activity of YlLIP2 and cell density were further improved in the 10-l fermentor where the values respectively attained were 13,500U/ml and 120g DCW/l. The total protein concentration in the supernatant reached 3.3g/l and the cell viability remained approximately 99% after 80h of culture. Furthermore, the recombinant YlLIP2 produced in P. pastoris pGAP and pAOX1 systems have similar content of sugar (about 12%) and biochemical characteristics. The above results suggest that the GAP promoter-derived expression system of P. pastoris is effective for the expression of YlLIP2 by high cell density culture and is probably an alternative to the conventional AOX1 promoter expression system in large-scale production of industrial lipases.

Production and characterization of milk-clotting enzyme from Bacillus amyloliquefaciens JNU002 by submerged fermentation

Milk-clotting enzymes produced by microorganisms have been developed to replace calf rennet, yet the enzymatic level and the ratio of milk-clotting activity to proteolytic activity still need further improvement. This work described a strain Bacillus amyloliquefaciens JNU002 that screened from wheat bran that has a promising characterization. After optimization, B. amyloliquefaciens JNU002 showed a high milk-clotting activity (4969 SU/mL) and low proteolytic activity (4.02 U/mL) at 48 h with an inoculum size of 0.2% (v/v) at initial pH 6.0 in 15-L bioreactor. After purification, the purified enzyme gave a single protein band on SDS–PAGE, corresponding to 28 kDa. The purified enzyme showed a high ratio (2,575) of milk-clotting activity to proteolytic activity at 35 °C, and the ratio would be even higher (22,992) at 70 °C. The milk-clotting reaction and proteolytic reaction were prevented at 75 °C. This enzyme was stable at pH 4–6 and below 40 °C, and this was convenient for storage and transportation.

Combined strategies for the improvement of heterologous expression of a His-tagged Yarrowia lipolytica lipase Lip2 in Pichia pastoris

Yarrowia lipolytica lipase Lip2 (YlLip2) is an important biocatalyst for ester synthesis, biodiesel production and enantiomer resolution. The YlLip2 with an N-terminal histidine-tag (His 6-YlLip2) was successfully expressed in Pichia pastoris . Three different cultivation strategies had been compared for the production of His 6-YlLip2 by P. pastoris using a 10-l bioreactor. The results showed that His 6-YlLip2 activity and cell viability could be greatly improved by employing the combined strategies. Using a low salt medium (LSM) instead of the basal salt medium (BSM) and lowering the temperature from 28 to 25°C, the maximum His 6-YlLip2 activity and volumetric productivity were respectively increased by 55.3 and 79.8%. The production of His 6-YlLip2 and cell viability was further improved by combining sorbitol co-feeding with methanol. In this culture strategy, the maximum activity of His 6-YlLip2 reached 15,600 U/ml after 114 h of induction. The cell mortality decreased by 11.2% (while the control decreased about 27.6%) after 120 h methanol induction. The N-terminal histidine-tag brought convenience to purification. The molecular weight of His 6-YlLip2 was about 38 kDa. The pure His 6-YlLip2 presented a specific activity of 4,830 U/mg when olive oil was used as the substrate.

Process development of ATROSAB, an anti TNFR1 Monoclonal Antibody: in three steps from research to GMP

The humanized monoclonal antibody ATROSAB is targeting specifically the TNF receptor 1(TNFR1). TNF is a central mediator of inflammation and key target for intervention in inflammatory diseases such as rheumatoid arthritis, psoriasis and Crohn’s Diseases. Notably, blockade of the second TNF receptor, TNFR2, has been associated with increased sensitivity to viral infections or increased susceptibility to demyelinating disorders and lymphomas. In this context, a selective inhibition of TNF-induced TNFR1 but not TNFR2 signaling activity holds great promises to overcome undesired effects observed with less specific TNF antagonists currently used in clinic. The recently humanized antibody was used to establish a rCHO-K1 cell line under serum-free, chemically defined conditions (1). The process development was based on two sets of shake flask experiments, three 10L bioreactor runs and finally 2 GMP production runs in 300 L scale. The scale up strategy was based on quality by design considerations specified in the design space for the scale-up parameters. The main parameter for the scale-up development was mixing time as a function of the bioreactor geometry, tip-speed, Reynolds number, and the power input of the systems. Those parameters are generally considered as important parameters during up-stream process development. Protein characteristics were specified based on several considerations. A major part was the ICH-Q8 guideline as well as antibody and other drug guidelines. The other part is based on experience with antibody development and the specific protein in special. The specifications are described in Table ​Table1.1. The analytical methods used to characterize the antibody were SDS-PAGE, western blot, HPLC-SEC, protein A HPLC, HPAEC-PAD, Isoelectric focusing and a cell based potency assay. As the antibody is declined to bind to the TNF-R1 on target cells, it was engineered not to elicit ADCC or CDC. Therefore those parameters were not checked during process development. Table 1 specified scale-up parameters and defined protein characteristics The basal medium was fixed to a commercial available chemically defined medium during clone screening. In a batch process, performed in a fully equipped 1L bioreactor, a titer of approximately 180 mg/L was obtained. At this stage the antibody was characterized and specifications were partly narrowed and some were newly set. In the first step of process development different feeding solutions and concentrations thereof were tested in shake flask experiments. Metabolite data were monitored on a daily basis comprising glucose, lactate, ammonia, and amino acid levels. Additionally cell density and protein concentration were measured at the same time interval. For in-depth protein characterization, supernatant was purified after 6 days of fermentation and at harvest, in order to check the quality attributes already early at the development stage. This step led to an increase of about 2.5 fold in product concentration, while the protein characteristics stayed within specifications at both sampling time points. The second step was the optimization of the feeding strategy as well as the fortification of the solutions with certain chemically defined supplements. This led to another increase of about 2.5 fold regarding final product concentration. Product quality changed slightly, but stayed within specifications. Especially the glycosylation structure changed towards less glycosylated versions. However, no impact on the bioassay was observable. The process was finally scaled up to the 300L stirred tank bioreactor in the GMP facility. An engineering run was performed to test the process and product parameters. Some influence was measured, thus the process was slightly changed prior the first GMP run. The obtained product quality was well within specifications, the achieved titer was more than 1.2 g/L, which was a more than 7 fold increase in comparison to the initial batch process. Although the cell density profile of the process changed between run 1 and 2, the volumetric product concentration was only slightly below 1.2 g/L and the product characteristics were identical to the first run. This indicates the robustness of the developed process and the validity to define and specify design spaces for process development especially during scale-up development. The analysis of protein characteristics employing SDS-PAGE, IEF, HP-SEC, potency assay and glycosylation pattern with HPAEC-PAD ensures the quality of the protein. Figure 1 The left diagram shows the cell density of two GMP runs in the 300L bioreactor. The right diagram focuses on the achieved product concentrations during the different process development steps. The batch experiment was performed in 1L bioreactors, the ...

Production of Yarrowia lipolytica lipase LIP2 in Pichia pastoris using the nitrogen source-regulated FLD1 promoter

BACKGROUND:Yarrowia lipolytica lipase LIP2 (YlLIP2) is an important industrial enzyme that has many potential applications. Although it has been successfully expressed in Pichia pastoris under the control of the AOX1 promoter (pAOX1), there have been many efforts to develop new alternative promoters to pAOX1 in order to avoid using methanol in the fermentation. Investigation of YlLIP2 production in P. pastoris using the formaldehyde dehydrogenase 1 promoter (pFLD1) is especially attractive, since little is known about its application in methanol-free culture strategies. RESULTS: Three fed-batch cultivations were performed to investigate the production of YlLIP2 in a pFLD1-based system. When methanol was used as the fed-batch feeding substrate, the maximum YlLIP2 activity obtained in a 10-L bioreactor was 30 000 U mL−1 after 143 h of culture, whereas the maximum YlLIP2 activity was further increased to 35 000 U mL−1 by adopting a co-induction strategy with methanol and methylamine as a mixed fed-batch substrate. Furthermore, the maximum YlLIP2 activity reached 13 000 U mL−1 after 80 h of cultivation in a methanol-free culture. CONCLUSION: The expression levels of YlLIP2 in the pFLD1-based system were comparable with those in a pAOX1-based system. The results suggest that pFLD1 is an attractive alternative to pAOX1, and may make it feasible to induce high yields of protein expression. Copyright © 2011 Society of Chemical Industry

Artificial intelligence based modeling and optimization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production process by using Azohydromonas lata MTCC 2311 from cane molasses supplemented with volatile fatty acids: A genetic algorithm paradigm

The present work describes the optimization of medium variables for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] by Azohydromonas lata MTCC 2311 using cane molasses supplemented with propionic acid. Genetic algorithm (GA) has been used for the optimization of P(3HB-co-3HV) production through the simulation of artificial neural network (ANN) and response surface methodology (RSM). The predictions by ANN are better than those of RSM and in good agreement with experimental findings. The highest P(3HB-co-3HV) concentration and 3HV content have been reported as 7.35g/l and 16.84mol%, respectively by hybrid ANN–GA. Upon validation, 7.20g/l and 16.30mol% of P(3HB-co-3HV) concentration and 3HV content have been found in the shake flask, whereas 6.70g/l and 16.35mol%, have been observed in a 3l bioreactor, respectively. The specific growth rate and P(3HB-co-3HV) accumulation rate of 0.29 per h and 0.16g/lh determined with cane molasses are comparable to those observed on pure substrates.

Production of succinic acid from sucrose and sugarcane molasses by metabolically engineered Escherichia coli

Sucrose-utilizing genes (cscKB and cscA) from Escherichia coli KO11 were cloned and expressed in a metabolically engineered E. coli KJ122 to enhance succinate production from sucrose. KJ122 harboring a recombinant plasmid, pKJSUC, was screened for the efficient sucrose utilization by growth-based selection and adaptation. KJ122-pKJSUC-24T efficiently utilized sucrose in a low-cost medium to produce high succinate concentration with less accumulation of by-products. Succinate concentrations of 51g/L (productivity equal to 1.05g/L/h) were produced from sucrose in anaerobic bottles, and concentrations of 47g/L were produced in 10L bioreactor within 48h. Antibiotics had no effect on the succinate production by KJ122-pKJSUC-24T. In addition, succinate concentrations of 62g/L were produced from sugarcane molasses in anaerobic bottles, and concentrations of 56g/L in 10L bioreactor within 72h. These results demonstrated that KJ122-pKJSUC-24T would be a potential strain for bio-based succinate production from sucrose and sugarcane molasses.

Deep-bed solid state fermentation of sweet sorghum stalk to ethanol by thermotolerant Issatchenkia orientalis IPE 100

A solid state fermentation (SSF) of sweet sorghum stalk to ethanol was conducted in 250-mL flask using thermotolerant Issatchenkia orientalis IPE 100, and the optimal operation parameters were determined as 42 °C fermentation temperature, 75% (w/w) water content, 2 mm particle size and 3% (w/w) inoculation rate in 250-mL conical flask. When the SSF was scaled up from the flask to a 10-L bioreactor, temperature gradient in the substrate bed was observed due to heat accumulation in the bioreactor. The temperature gradient was dependent on both substrate depth and operation temperature. Due to high thermotolerance of the strain IPE 100, a deep-bed SSF of sweet sorghum stalk was developed in the bioreactor. The highest ethanol yield of 0.25 g-ethanol/g-dry stalk was obtained at 37 °C with 15–20 cm substrate depth in the bioreactor. These results provided a great potential for large-scale deep-bed SSF in practice.