10-L Fermenter Research Articles

Calcium malate overproduction by Penicillium viticola 152 using the medium containing corn steep liquor

In this study, after screening of eight fungal strains for their ability to produce calcium malate, it was found that Penicillium viticola 152 isolated from marine algae among them could produce the highest titer of calcium malate. At the same time, it was found that corn steep liquor (CSL) could stimulate calcium malate production and 0.5 % (v/v) CSL was the most suitable for calcium malate production. Under the optimal conditions, a titer of calcium malate in the supernatant was 132 g/l at flask level. During a 10-l fermentation, a titer of 168 g/l, a yield of 1.28 g/g of glucose, and a productivity of 1.75 g/l/h were reached within 96 h of the fermentation, and 93.4 % of the sugar was used for calcium malate production and cell growth, demonstrating that the titer, yield, and productivity of calcium malate by this fungal strain were very high and the fermentation period was very short. After analysis of the partially purified product with high-performance liquid chromatography, it was found that the main product was calcium malate. The results demonstrated that P. viticola 152 obtained in this study was the most suitable for developing a novel one-step fermentation process for calcium malate production from glucose on a large scale.

Antibacterial activity of the lipopetides produced by Bacillus amyloliquefaciens M1 against multidrug-resistant Vibrio spp. isolated from diseased marine animals

In this work, the antibacterial activity of the lipopeptides produced by Bacillus amyloliquefaciens M1 was examined against multidrug-resistant Vibrio spp. and Shewanella aquimarina isolated from diseased marine animals. A new and cheap medium which contained 1.0 % soybean powder, 1.5 % wheat flour, pH 7.0 was developed. A crude surfactant concentration of 0.28 mg/ml was obtained after 18 h of 10-l fermentation and diameter of the clear zone on the plate seeded with Vibrio anguillarum was 34 mm. A preliminary characterization suggested that the lipopeptide N3 produced by B. amyloliquefaciens M1 was the main product and contained the surfactin isoforms with amino acids (GLLVDLL) and hydroxy fatty acids (of 12-15 carbons in length). The evaluation of the antibacterial activity of the lipopeptide N3 was carried out against S. aquimarina and nine species of Vibrio spp.. It was found that all the Vibrio spp. and S. aquimarina showed resistance to several different antibiotics, suggesting that they were the multidrug resistance. It was also indicated that all the Vibrio spp. strains and S. aquimarina were sensitive to the surfactin N3, in particular V. anguillarum. The results demonstrated that the lipopeptides produced by B. amyloliquefaciens M1 had a broad spectrum of action, including antibacterial activity against the pathogenic Vibrio spp. with multidrug-resistant profiles. After the treatment with the lipopeptide N3, the cell membrane of V. anguillarum was damaged, and the whole cells of the bacterium were disrupted.

Efficient cloning and expression of a thermostable nitrile hydratase in Escherichia coli using an auto-induction fed-batch strategy

A nitrile hydratase (NHase) gene from Aurantimonas manganoxydans was cloned and expressed in Escherichia coli BL21 (DE3). A downstream gene adjacent to the β-subunit was necessary for the functional expression of the recombinant NHase. The structural gene order of the Co-type NHase was α-subunit beyond β-subunit, different from the order typically reported for Co-type NHase genes. The NHase exhibited adequate thermal stability, with a half-life of 1.5h at 50°C. The NHase efficiently hydrated 3-cyanopyridine to produce nicotinamide. In a 1-L reaction mixture, 3.6mol of 3-cyanopyridine was completely converted to nicotinamide in four feedings, exhibiting a productivity of 187g nicotinamide/g dry cell weight/h. An industrial auto-induction medium was applied to produce the recombinant NHase in 10-L fermenter. A glycerol-limited feeding method was performed, and a final activity of 2170U/mL culture was achieved. These results suggested that the recombinant NHase was efficiently cloned and produced in E. coli.

Genome Mining for New α-Amylase and Glucoamylase Encoding Sequences and High Level Expression of a Glucoamylase from Talaromyces stipitatus for Potential Raw Starch Hydrolysis

Mining fungal genomes for glucoamylase and α-amylase encoding sequences led to the selection of 23 candidates, two of which (designated TSgam-2 and NFamy-2) were advanced to testing for cooked or raw starch hydrolysis. TSgam-2 is a 66-kDa glucoamylase recombinantly produced in Pichia pastoris and originally derived for Talaromyces stipitatus. When harvested in a 20-L bioreactor at high cell density (OD600 > 200), the secreted TSgam-2 enzyme activity from P. pastoris strain GS115 reached 800 U/mL. In a 6-L working volume of a 10-L fermentation, the TSgam-2 protein yield was estimated to be ∼8 g with a specific activity of 360 U/mg. In contrast, the highest activity of NFamy-2, a 70-kDa α-amylase originally derived from Neosartorya fischeri, and expressed in P. pastoris KM71 only reached 8 U/mL. Both proteins were purified and characterized in terms of pH and temperature optima, kinetic parameters, and thermostability. TSgam-2 was more thermostable than NFamy-2 with a respective half-life (t1/2) of >300 min at 55 °C and >200 min at 40 °C. The kinetic parameters for raw starch adsorption of TSgam-2 and NFamy-2 were also determined. A combination of NFamy-2 and TSgam-2 hydrolyzed cooked potato and triticale starch into glucose with yields, 71-87 %, that are competitive with commercially available α-amylases. In the hydrolysis of raw starch, the best hydrolysis condition was seen with a sequential addition of 40 U of a thermostable Bacillus globigii amylase (BgAmy)/g starch at 80 °C for 16 h, and 40 U TSgam-2/g starch at 45 °C for 24 h. The glucose released was 8.7 g/10 g of triticale starch and 7.9 g/10 g of potato starch, representing 95 and 86 % of starch degradation rate, respectively.

Overproduction of poly(β-malic acid) (PMA) from glucose by a novel Aureobasidium sp. P6 strain isolated from mangrove system

After over 100 strains of Aureobasidium spp isolated from mangrove system were screened for their ability to produce poly(β-malic acid) (PMA), it was found that Aureobasidium sp. P6 strain among them could produce high level of Ca(2+)-PMA. Fourteen percent glucose and 6.5 % CaCO3 in the medium were the most suitable for Ca(2+)-PMA production. Then, 100.7 g/l of Ca(2+)-PMA was produced using Aureobasidium sp. P6 strain within 168 h at flask level. During 10-l batch fermentation, when the medium contained 12.0 % glucose, 98.7 g/l of Ca(2+)-PMA in the culture and 14.7 g/l of cell dry weight were obtained within 156 h, leaving 0.34 % reducing sugar in the fermented medium. When glucose concentration in the fermentation medium was 14.0 %, 118.3 g/l of Ca(2+)-PMA in the culture and 16.4 g/l of cell dry weight were obtained within 168 h, leaving 0.4 % reducing sugar in the fermented medium. After purification of Ca(2+)-PMA from the culture and acid hydrolysis of the pure Ca(2+)-PMA, analysis of HPLC showed that Aureobasidium sp. P6 strain only produced two main components of Ca(2+)-PMA and minor amount of calcium malate and that the hydrolysate of PMA was mainly composed of calcium malate. This is the first time to report that the novel yeast strain Aureobasidium sp. P6 strain isolated from the mangrove systems can produce such high amount of Ca(2+)-PMA.

Towards Industrially Feasible Treatment of Potato Starch Processing Waste by Mixed Cultures

The present study aimed at reducing the pollution of the waste generated by the potato starch industry to the environment and transform the potato pulp and wastewater into single-cell protein (SCP) to be used as animal feed. The chemical oxygen demand of the wastewater was reduced from 26,700 to 9,100 mg/L by batch fermentation with mixed cultures in an aerated 10-L fermenter. The SCP products, with a crude protein content of 46.09 % (higher than soybean meal), were found palatable and safe for mice. During the treatment process, the microbial community was analyzed using the terminal restriction fragment length polymorphism for bacterial 16S rRNA genes. The results of the analysis suggested that Curacaobacter/Pseudoalteromonas and Paenibacillus/Bacillus were the main microorganisms in treating potato starch processing wastes. The 150-m(3)-scale fermentation demonstrated a potential for treatment in industrial applications. Fermentation of potato pulp and wastewater without adding an extra nitrogen source was a novel approach in treating the potato starch processing waste.

Streptomycin resistance-aided genome shuffling to improve doramectin productivity of Streptomyces avermitilis NEAU1069

Genome shuffling is an efficient approach for the rapid engineering of microbial strains with desirable industrial phenotypes. In this study, a strategy of incorporating streptomycin resistance screening into genome shuffling (GS-SR) was applied for rapid improvement of doramectin production by Streptomyces avermitilis NEAU1069. The starting mutant population was generated through treatment of the spores with N-methyl-N'-nitro-N-nitrosoguanidine and ultraviolet (UV) irradiation, respectively, and five mutants with higher productivity of doramectin were selected as starting strains for GS-SR. Finally, a genetically stable strain F4-137 was obtained and characterized to be able to yield 992 ± 4.4 mg/l doramectin in a shake flask, which was 7.3-fold and 11.2-fold higher than that of the starting strain UV-45 and initial strain NEAU1069, respectively. The doramectin yield by F4-137 in a 50-l fermentor reached 930.3 ± 3.8 mg/l. Furthermore, the factors associated with the improved doramectin yield were investigated and the results suggested that mutations in ribosomal protein S12 and the enhanced production of cyclohexanecarboxylic coenzyme A may contribute to the improved performance of the shuffled strains. The random amplified polymorphic DNA analysis showed a genetic diversity among the shuffled strains, which confirmed the occurrence of genome shuffling. In conclusion, our results demonstrated that GS-SR is a powerful method for enhancing the production of secondary metabolites in Streptomyces.

Constitutive expression of a novel isoamylase from Bacillus lentus in Pichia pastoris for starch processing

Isoamylase is essential to saccharifying starch by cleavage of 1,6-glucoside linkages in starch molecules. In this study, a novel isoamylase gene from Bacillus lentus JNU3 was cloned. The open reading frame of the gene was 2412 base pairs long and encoded a polypeptide of 804 amino acids with a calculated molecular mass of 90kDa. The deduced amino acid sequence shared less than 40% homology with that of microbial isoamylase ever reported, which indicated it was a novel isoamylase. A constitutive GAP promoter was used to express the recombinant isoamylase in the yeast Pichia pastoris by continuous high cell-density fermentation to avoid the use of methanol, which resulted in 318U/mL extracellular isoamylase activity after 72h in a 10L fermenter. The recombinant enzyme was purified and characterized. It had an estimated molecular mass of 90kDa, with its optimal activity at 70°C, pH 6.5 and was quite stable between 30°C and 70°C. The recombinant isoamylase proves to be superior to pullulanase as an auxiliary enzyme in maltose production from starch. Therefore it will contribute significantly to the starch debranching process.

Combined strategies for improving the heterologous expression of an alkaline lipase from Acinetobacter radioresistens CMC-1 in Pichia pastoris

In this study, a series of strategies was developed to enhance the expression of an alkaline lipase from Acinetobacter radioresistens (ARL) in Pichia pastoris. Activity of the lipase from recombinant strain carrying a single copy of codon-optimized ARL gene was 65U/mL in shake flask culture with p-nitrophenyl caprylate as the substrate. The lipase yield was increased to 104U/mL by introducing a short N-extension spacer peptide coding for the 10 amino acids (EEAEAEAEPK) between α-factor signal peptide and ARL. The N-terminal extension spacer did not affect the pH or temperature properties of the recombinant ARL. After the multi-copy constructs were identified by Q-PCR assay, a higher lipase activity of 180U/mL was obtained. Further introduction of the spliced HAC1 gene into multi-copy integrants (>6 copies) extensively enhanced the ARL yield by 30–40%. As a result, the ARL yield reached 1.06×104U/mL in a 10-L scaled-up fed-batch fermenter as well as the lipase showed some better properties compared to that wild one from A. radioresistens.

High-level production of calcium malate from glucose by Penicillium sclerotiorum K302

In this study, after screening of 9 fungal strains for their ability to produce calcium malate, it was found that Penicillium sclerotiorum K302 among them could produce high-level of calcium malate. Under the optimal conditions, the titer of calcium malate in the supernatant was 88.6 g/l at flask level. During 10-l fermentation, the titer of 92.0 g/l, the yield of 0.88 g/g of glucose and the productivity of 1.23 g/l/h were reached within 72 h of the fermentation, demonstrating that the titer, yield and productivity of calcium malate by this strain were very high and the fermentation period was very short. After analysis of the partially purified product with HPLC, it was found that the main product was calcium malate. The results showed that P. sclerotiorum K302 obtained in this study was suitable for developing a novel one-step fermentation process for calcium malate production from glucose on large scale.

Scale-up of docosahexaenoic acid production in fed-batch fermentation by Schizochytrium sp. based on volumetric oxygen-transfer coefficient

The scale-up of fed-batch fermentation process for the DHA (docosahexaenoic acid)-rich microbial lipids production by Schizochytrium sp. from shake flasks to bench-scale (10l and 50l) and pilot-scale (1500l and 7000l) stirred fermentors was studied. The fermentation process was successfully scaled-up from experimental-scale to bench-scale and pilot-scale at matched KLa value (88.9h−1) estimated by the KLa model developed in this study and the KLa empirical equations. The fermentation performances of the bench-scale and pilot-scale (DHA content of 13.96, 14.44 and 14.16gl−1 in the 10l, 50l and 1500l fermentor) processes were similar to that of the experimental-scale process (DHA content of 13.84gl−1) and the fermentation results obtained in the 7000l stirred fermentors (DHA content of 19.72gl−1) were much higher. These results indicated that KLa was applicable for the scale-up of the fed-batch fermentation process of DHA production by Schizochytrium sp. and this scale-up strategy enabled a maximum of 150-fold quantitative scale-up of the fed-batch fermentation process.

Production of sophorolipids with enhanced volumetric productivity by means of high cell density fermentation

To achieve high time-space efficiency for sophorolipid production with yeast Candida bombicola, a strategy of high cell density fermentation was employed. The approach consisted of two sequential stages: (1) the optimization of the carbon source and the nutrient concentration to achieve the maximal cell density and (2) the computer-aided adjustment of physical parameters and the controlled feeding of substrates for enhanced volumetric productivity. Both stages have been successfully implemented in a 10-L fermenter, where up to 80g dry cell weight/L was obtained and a remarkably high volumetric productivity (> 200g isolated sophorolipids/L/day) was achieved. Both the biomass and volumetric productivity were markedly higher than previously reported. Specifically, the high productivity of sophorolipids could be attained on a very short time scale (24h), highlighting the industrial potential of the platform developed in this work.

Directed Evolution of Penicillium janczewskii zalesk α-Galactosidase Toward Enhanced Activity and Expression in Pichia pastoris

In this study, the activity of an α-galactosidase obtained from Penicillium janczewskii zalesk was improved via modifying its gene by error-prone PCR and DNA shuffling. The mutated DNA was ligated to pBGP1, an autonomous-replicating vector, which was subsequently transformed into Pichia pastoris X-33. The expressed enzyme activities were measured after single colonies were cultured in yeast-peptone-dextrose medium in deep-well plates. After two rounds of screening, two mutants with higher activity were obtained. By PCR analysis, four mutation sites (S167G, P455L, N637S, and P490L/P490H) were found in these two variants (mutant-59 and mutant-8). Mutant-59 showed the highest activity at pH 5.0 and 40 °C with an increased V(max) value of 769 μmol/min and the specific activity of 667 U/mg against p-nitrophenyl α-D-galactopyranoside. The two mutant enzymes also showed similar resistance to the metal ions of Cu(2+), Fe(2+), and Zn(2+). In a 10-L fermenter, the supernatant enzyme activity reached the maximum of 550.2 U/mL upon the methanol induction for 96 h. This fermentation activity of the mutant was improved approximately two more folds than the wild type α-galactosidase. This mutant of α-galactosidase is prospective in feed manufacturing as feed additives to improve nutrient digestibility in monogastric animals.

Deletion of the aroK gene is essential for high shikimic acid accumulation through the shikimate pathway in E. coli

Shikimic acid (SA) is an important metabolic intermediate with diverse commercial applications. In this work, antisense RNA interference and gene deletion were carried out to inactivate the aroK gene in an SA-producing Escherichia coli strain, DHPYA-T7. In this strain, the aroL, ptsHIcrr and ydiB genes are deleted, and the tktA, glk, aroE and aroB genes are overexpressed. Flask cultivations of the DHPYA-T7 derivative strains showed that the accumulation of SA increased 2.69-fold after aroK gene deletion (DHPYAAS-T7) and 1.29-fold after antisense RNA interference (DHPYAS-T7). Furthermore, the activity of shikimate kinase in DHPYAAS-T7 was 0.21-fold of that in strain DHPYAS-T7. In a 10-L fermentation, SA accumulation increased to 1850mgL−1 in strain DHPYAAS-T7, which is a 1.5-fold increase over that in strain DHPYAS-T7. These results demonstrate that aroK gene inactivation in DHPYA-T7 leads to high SA accumulation, especially when this inactivation is caused by chromosomal deletion.

Utilization of biodiesel waste as a feedstock for the production of polyhydroxybutyrate by Cupriavidus necator

This study aimed to investigate the potential of using wastewater and crude glycerol from biodiesel refinery to produce polyhydroxyalkanoates (PHAs) through fermentation of Cupriavidus necator TISTR 1095. The result indicates that crude glycerol yielded high cell growth (35 to 37 g/L) and poly-3-hydroxybutyrate (PHB; 17.85 to 19.98 g/L). However, no cell growth obtained from biodiesel-wastewater due to high Na + presented. Among medium and experimental factors influencing PHB accumulation, crude glycerol, (NH 4 ) 2 SO 4 and trace element concentration revealed significant effects (P<0.1). Their optimal values were 60 g/L crude glycerol, 1.32 g/L (NH 4 ) 2 SO 4 and 2.0 g/L trace element. Under these optimal conditions, the strain TISTR 1095 produced the highest biomass (46.25±2.10 g/L) and PHB concentration of 24.98±1.87 g/L with PHB content of 54.01% of DCW. Effect of experimental conditions including aeration rate and agitation speed as well as sterile condition on PHB accumulation was also studied. The optimal aeration rate (2 vvm) and agitation speed (150 rpm) under septic condition during cultivation gave slightly increase of biomass and PHB. The maximum biomass (46.96±0.28 g/L) and PHB concentration of 25.32±0.20 g/L (53.92% of DCW) was achieved in 20-L fermentor. Moreover, the purified PHB from C. necator TISTR 1095 was partially characterized; their properties were similar to commercial PHB. Key words : Biodiesel, Cupriavidus necator , glycerol, optimization, polyhydroxybutyrate, poly-3-hydroxybutyrate (PHB).

Heterologous expression of functional G‐protein‐coupled receptors inCaenorhabditis elegans

New strategies for expression, purification, functional characterization, and structural determination of membrane-spanning G-protein-coupled receptors (GPCRs) are constantly being developed because of their importance to human health. Here, we report a Caenorhabditis elegans heterologous expression system able to produce milligram amounts of functional native and engineered GPCRs. Both bovine opsin [(b)opsin] and human adenosine A(2A) subtype receptor [(h)A(2A)R] expressed in neurons or muscles of C. elegans were localized to cell membranes. Worms expressing these GPCRs manifested changes in motor behavior in response to light and ligands, respectively. With a newly devised protocol, 0.6-1 mg of purified homogenous 9-cis-retinal-bound bovine isorhodopsin [(b)isoRho] and ligand-bound (h)A(2A)R were obtained from C. elegans from one 10-L fermentation at low cost. Purified recombinant (b)isoRho exhibited its signature absorbance spectrum and activated its cognate G-protein transducin in vitro at a rate similar to native rhodopsin (Rho) obtained from bovine retina. Generally high expression levels of 11 native and mutant GPCRs demonstrated the potential of this C. elegans system to produce milligram quantities of high-quality GPCRs and possibly other membrane proteins suitable for detailed characterization.

STATISTICAL MEDIA OPTIMIZATION FOR THE BIOMASS PRODUCTION OF POSTHARVEST BIOCONTROL YEAST Rhodosporidium paludigenum

A cane molasses-based medium for the biomass production of biocontrol agent Rhodosporidium paludigenum was statistically optimized. Molasses concentration (after pretreatment), yeast extract, and initial pH were identified by the Plackett–Burman design to show significant influence on the biomass production. The three factors were further optimized by central composite design and response-surface methodology. The statistical analysis indicated the optimum values of the variables were 89.98 g/L for cane molasses, 2.35 g/L for yeast extract and an initial pH of 8.48. The biomass yield at the optimal culture achieved 15.89 g/L in flask fermentation, which was 2.1 times higher than that at the initial NYDB medium. In a 10-L fermenter, 18.97 g/L of biomass was obtained after 36 hr of cultivation. Moreover, the biocontrol efficacy of the yeast was investigated after culture optimization. The results showed the yeast harvested in the optimal medium maintained its initial biocontrol properties by reducing the percentage of decayed apples to below 20%.

Large-scale expression, purification, and glucose uptake activity of recombinant human FGF21 in Escherichia coli

As a novel important regulator of glucose and lipid metabolism homeostasis, human fibroblast growth factor 21 (hFGF21) has become a potential drug candidate for the treatment of metabolic diseases including obesity, and type 2 diabetes, as well as non-alcoholic fatty liver disease. To improve the production of recombinant hFGF21 to meet the increasing demand in clinical applications, an artificial gene encoding its mature peptide sequence was constructed, cloned into vector pET-3c and then expressed in Escherichia coli Origami B (DE3). Under optimal conditions in a 50-L fermentor, the average bacterial yield and the soluble expression level of recombinant hFGF21 of six batches attained 1750 ± 185 g and 32 ± 1.5%, respectively. The target protein was purified by the combination of nickel-nitrilotriacetic acid affinity chromatography and Sephadex S-100 resin. 5% (w/v) trehalose solution was able to prevent rhFGF21 from degradation effectively. The purity of rhFGF21 was higher than 97%, and the yield was 213 ± 17 mg/L. The preliminary biochemical characterization of rhFGF21 was confirmed using Western blot and peptide map finger analysis. Based on the glucose oxidase-peroxidase assay, the EC50 of glucose uptake activity of the purified rhFGF21 was 22.1 nM.

Rendered-protein hydrolysates for microbial synthesis of cyanophycin biopolymer

Cyanophycin is a poly(arginyl-aspartate) biopolymer produced and stored intracellularly by bacteria. Cyanophycin has been proposed as a renewable replacement for petrochemical-based industrial products. An abundant source of amino acids and nitrogen such as in the form of protein hydrolysates is needed for the biosynthesis of cyanophycin. Rendered proteins are largely used as a feed supplement in animal husbandry and aquaculture. New uses would expand the market size of this class of protein coproducts. We prepared and thoroughly characterized the hydrolysates of meat and bone meal, and proceeded to demonstrate for the first time that these hydrolysates could be used in the fermentative production of cyanophycin. Using the enzyme-hydrolyzed meat and bone meal preparation, we obtained crude cyanophycin product at 33-35% level of that produced using the reference casamino acids in both shake-flask and 10-L bioreactor fermentation studies. Polyacrylamide-gel electrophoresis of the cyanophycin under denaturing conditions showed the molecular weight of the isolated polyamide at 24kDa. Our results open a new avenue for the utilization of rendered protein coproducts to produce the cyanophycin biopolymer.

Development of a mutant strain of Bacillus subtilis showing enhanced production of acetoin

This paper described the screening process of a mutant strain of Bacillus subtilis TH-49 showing specific characteristic and enhanced production of acetoin. The mutant was obtained by treating B. subtilis N-12 with ultraviolet ray (ultraviolet ray (UV)) or nitrosoguanidine (NTG) or compound mutation and subjecting it to a shake flask fermentation selection procedure. The acetoin production rate reached the highest value of 43.8 and 46.9 g/l in flask and 10-l fermenter fermentations, respectively. It was almost 4 times higher than that of the starting strain B. subtilis N-12. Through fermentation experiments, it was confirmed that the mutant strain, TH-49, was not capable of using acetoin accumulated in broth as its energy sources for growth after glucose was consumed. This phenomenon was inconsistent with that the majorities of bacteria accumulate acetoin as stored energy sources and could continue to use acetoin as energy sources after the exhaustion of glucose. By gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) analysis, it was proved that the main metabolite produced by mutant strain TH-49 was acetoin and there were no other metabolites commonly found as byproducts of acetoin fermentation, such as 2,3-butanediol and 2,3butanedione. The above characteristic of the mutant strain, TH-49, may be related to its specific composition of acetoin metabolism-related enzymes system. These results indicated that, the mutant strain TH-49 is conspicuously different from other acetoin producing strains reported previously in metabolic mechanism of acetoin.