Bioprocess strategies for mass multiplication of and metabolite synthesis by plant growth promoting pseudomonads for agronomical applications

The exponential substrate feeding (open-loop) and automated feedback substrate feeding (closed loop) strategies were developed to obtain high cell densities of fluorescent pseudomonad strains R62 and R81 and enhanced production of antifungal compound 2,4-diacetylphloroglucinol (DAPG) from glycerol as a sole carbon source. The exponential feeding strategy resulted in increased glycerol accumulation during the fed-batch cultivation when the predetermined specific growth rate (μ) was set at 0.10 or 0.20 h^?1 (<μ_m = 0.29 h^?1). Automated feeding strategies using dissolved oxygen (DO) or pH as feedback signals resulted in minimal to zero accumulation of glycerol for both the strains. In case of DO-based feeding strategy, biomass productivity of 0.24 g/(L h) and 0.29 g/(L h) was obtained for R62 and R81, respectively. Using pH-based feeding strategy, biomass productivity could be increased to a maximum of 0.51 and 0.54 g/(L h), for the strains R62 and R81, respectively, whereas the DAPG concentration was enhanced to 298 mg/L for R62 and 342 mg/L for R81 strains. These yields of DAPG are thus far the highest reported from GRAS organisms.     

Fast conversion of glycerol to 1,3-propanediol by a new strain of Klebsiella pneumoniae

Five bacterial strains screened from a batch of 39 samples could convert glycerol anaerobically to 1,3-propanediol (1,3-PD). One of the strains, XJ-Li, which could synthesize 1,3-PD with a higher concentration, was identified and characterized. Phylogenetic analysis of the strain XJ-Li included the study of morphology, physiological and biochemical characteristics. In addition, 16SrDNA sequences were created. The results indicated that this strain is a member of Klebsiella pneumoniae. The optimal cultivation parameters for pH and temperature were determined as 8.0 and 40 °C, respectively. The optimized nitrogen source and carbon source were 6.0 g/L of (NH₄)₂SO₄ and 20 g/L of glycerol, respectively. After 8 h in batch fermentation, both the 1,3-PD concentration and glycerol consumption reached the maximum, with 12.2 g/L of 1,3-PD and 1.53 g/L h of productivity, and a molar yield of 1,3-PD to glycerol of 0.75. Fed-batch fermentation also indicated a higher molar yield of 0.70, and the concentration of 1,3-PD reached 38.1 g/L after 66.4 g/L of glycerol consumption. The results of batch and fed-batch fermentations demonstrated that K. pneumoniae XJ-Li would be an excellent 1,3-PD producer.     

Production of 1,3-propanediol, 2,3-butanediol and ethanol by a newly isolated Klebsiella oxytoca strain growing on biodiesel-derived glycerol based media

The production of 1,3-propanediol, 2,3-butanediol and ethanol was studied, during cultivations of strain Klebsiella oxytoca FMCC-197 on biodiesel-derived glycerol based media. Different kinds of glycerol feedstocks and experimental conditions had an important impact upon the distribution of metabolic products; production of 1,3-propanediol was positively influenced by stable pH conditions and by the absence of N₂ gas infusions throughout the fermentation. Thus, during batch bioreactor fermentations conducted at increasing glycerol concentrations, 1,3-propanediol at 41.3 g/L and yield ～47% (w/w) was achieved at initial glycerol concentration ～120 g/L. At even higher initial glycerol media (150 and 170 g/L), growth was not ceased, but 1,3-propanediol production declined. During fed-batch fermentation under optimal experimental conditions, 126 g/L of glycerol were converted into 50.1 g/L of 1,3-propanediol. In this experiment, also 25.2 g/L of ethanol (conversion yield ～20%, w/w) were formed. A batch-bioreactor culture was performed under non-sterilized conditions and the 1,3-propanediol production was almost equivalent to the sterilized process. Concerning 2,3-butanediol formation, the most detrimental parameter was the absence of N₂ sparging and as a result, no 2,3-butanediol was produced. The presence of glucose as co-substrate seriously enhanced 2,3-butanediol production; when commercial glucose was employed as sole substrate, 32.1 g/L of 2,3-butanediol were formed.     

Metabolic engineering of Clostridium tyrobutyricum for enhanced butyric acid production from glucose and xylose

Clostridium tyrobutyricum is a promising microorganism for butyric acid production. However, its ability to utilize xylose, the second most abundant sugar found in lignocellulosic biomass, is severely impaired by glucose-mediated carbon catabolite repression (CCR). In this study, CCR in C. tyrobutyricum was eliminated by overexpressing three heterologous xylose catabolism genes (xylT, xylA and xlyB) cloned from C. acetobutylicum. Compared to the parental strain, the engineered strain Ct-pTBA produced more butyric acid (37.8 g/L vs. 19.4 g/L) from glucose and xylose simultaneously, at a higher xylose utilization rate (1.28 g/L·h vs. 0.16 g/L·h) and efficiency (94.3% vs. 13.8%), resulting in a higher butyrate productivity (0.53 g/L·h vs. 0.26 g/L·h) and yield (0.32 g/g vs. 0.28 g/g). When the initial total sugar concentration was ~120 g/L, both glucose and xylose utilization rates increased with increasing their respective concentration or ratio in the co-substrates but the total sugar utilization rate remained almost unchanged in the fermentation at pH 6.0. Decreasing the pH to 5.0 significantly decreased sugar utilization rates and butyrate productivity, but the effect was more pronounced for xylose than glucose. The addition of benzyl viologen (BV) as an artificial electron carrier facilitated the re-assimilation of acetate and increased butyrate production to a final titer of 46.4 g/L, yield of 0.43 g/g sugar consumed, productivity of 0.87 g/L·h, and acid purity of 98.3% in free-cell batch fermentation, which were the highest ever reported for butyric acid fermentation. The engineered strain with BV addition thus can provide an economical process for butyric acid production from lignocellulosic biomass.     

Enhanced acetone/butanol/ethanol production by Clostridium beijerinckii IB4 using pH control strategy

PH is an essential factor for acetone/butanol/ethanol (ABE) production using Clostridium spp. In this study, batch fermentations by Clostridium beijerinckii IB4 at various pH values ranging from 4.9 to 6.0 were examined. At pH 5.5, the ABE production was dominant and maximum ABE concentration of 24.6 g/L (15.7 g/L of butanol, 8.63 g/L of acetone and 0.32 g/L of ethanol) was obtained with the consumption of 60 g/L of glucose within 36 h. However, in the control (without pH control), an ABE concentration of 14.1 g/L (11.0 g/L of butanol, 3.01 g/L of acetone and 0.16 g/L of ethanol) was achieved with the consumption of 41 g/L of glucose within 40 h. A considerable improvement in the productivity of up to 93.8% was recorded at controlled pH in comparison to the process without pH control. To better understand the influence of pH on butanol production, the reducing power capability and NADH-dependent butanol dehydrogenase activity were investigated, both of which were significantly improved at pH 5.5. Thus, the pH control technique is a convenient and efficient method for high-intensity ABE production.     

Fed-batch fermentation of recombinant Citrobacter freundii with expression of a violacein-synthesizing gene cluster for efficient violacein production from glycerol

The extensive prospects of violacein in the pharmaceutical industry have attracted increasing interest. However, the fermentation levels of violacein are currently inadequate to meet the demands of industrial production. This study was undertaken to develop an efficient process for the production of violacein by recombinant Citrobacter freundii. The effects of dissolved oxygen (DO) and pH on cell growth and violacein production in batch cultures were investigated first. When the DO and pH of the medium were controlled at around 25% and 7.0, respectively, the biomass and concentration of violacein were maximized. Based on the consumption of nutrients in the medium observed during batch culture, a fed-batch fermentation strategy with controlled DO and pH was implemented. By continuously feeding glycerol, NH₄Cl, and l-tryptophan at a constant feeding rate of 16 mL h⁻¹, the final concentration of violacein reached 4.13 g L⁻¹, which was 4.09-fold higher than the corresponding batch culture, and the maximal dry cell weight (DCW) and average violacein productivity obtained for the fed-batch culture were 3.34 g DCW L⁻¹ and 82.6 mg L⁻¹ h⁻¹, respectively. To date, this is the first report on the efficient production of violacein by genetically engineered strains in a fermentor.     

High-level production of erythritol by mutants of osmophilic Moniliella sp.

An erythritol-producing osmophilic yeast-like fungus, Moniliella sp. 440, was isolated from honey and then successively mutated with iterative rounds of N-methyl-N′-nitro-N-nitrosoguanidine (NTG) treatment and selection. Six generations of mutants, named N12115-6, N21105-6, N31074-3, N42208-2, N53199-9, and N61188-12, were selected for and produced erythritol at 151.0, 157.2, 177.8, 191.4, 196.6, and 237.8 g/L, respectively, while the wild type strain produced 113.0 g/L erythritol in media containing 40% glucose and 1% yeast extract. The mutant cells were found to have a short rod-like shape, while the wild type cells have a long rod-like shape. The most efficient erythritol producer, N61188-12, assimilated myo-inositol and weakly assimilated erythritol. However, the wild type strain did not assimilate myo-inositol and assimilated erythritol well. In 250-L and 2000-L pilot-scale fermentors, the erythritol production by N61188-12 was 151.4 g/L and 152.4 g/L, respectively. A simple fed-batch culture of strain N61188-12 in a 2000-L fermentor increased erythritol production to 189.4 g/L after 10 days fermentation.     

Continuous 2-keto-gluconic acid (2KGA) production from corn starch hydrolysate by Pseudomonas fluorescens AR4

A continuous fermentation process for 2-keto-gluconic acid (2KGA) production from cheap raw material corn starch hydrolysate was developed using the strain Pseudomonas fluorescens AR4. The dilution rate and feeding glucose concentration had a significant effect on the cell concentrations, glucose utilization and 2KGA production performance. The optimal operating factors were obtained as: 0.065 h⁻¹ of dilution rate, 180 g/L of feeding glucose concentration, and 16 h of batch fermentation time as the starting point. Under these conditions, the steady state had the 135.92 g/L of produced 2KGA concentration, 8.83 g/L.h of average volumetric productivity, and 0.9510 g/g of yield. In conclusion, the proposed efficient and stable continuous fermentation process for 2KGA production by the strain P. fluorescens AR4 is potentially competitive for industrial production from corn starch hydrolysate in terms of 2KGA productivity and yield.     

Fermentation process for continuous production of succinic acid in a fibrous bed bioreactor

Succinic acid (SA) was produced from Actinobacillus succinogenes with high cell density by continuous fermentation using fibrous bed bioreactor (FBB). The effects of feeding glucose concentration, dilution rate, and pH on continuous production of SA were examined to achieve an efficient and economical bioprocess. The optimum feeding glucose concentration, dilution rate, and pH were 80 g/L, 0.05 1/h, and 6.0–6.5, respectively. A SA concentration of 55.3 ± 0.8 g/L, productivity of 2.77 ± 0.04 g/L/h, and yield of 0.8 ± 0.02 g/g were obtained, and the continuous fermentation exhibited long-term stability for as long as 18 days (440 h) with no obvious fluctuations in both SA and biomass levels. The Jerusalimsky equation for the specific rate of SA production presented the inhibition phenomenon of the product, demonstrating that 60 g/L SA might be a critical concentration in this continuous FBB system. The results obtained could be beneficial for future fermentor designs and improvements in SA production.     

Use of different carbon sources in cultivation of recombinant Pichia pastoris for angiostatin production

To improve the growth of recombinant Pichia pastoris with a phenotype of Mut^S and expression of angiostatin, the effects of glycerol, sorbitol, acetate and lactic acid which were, respectively, added together with methanol in the expression phase, were studied in a 5-l fermentor. Methanol concentration was automatically controlled at 5 g/l by a methanol monitor and control system, while the feeding of the other carbon source was manually adjusted. The angiostatin production level was 108 mg/l when glycerol was added at an initial rate of 2.3 g/h and gradually increased to 9.9 g/h within an induction period of 96 h. The angiostatin concentration was 141 mg/l as sorbitol was used, while only 52 mg/l were obtained on acetate. The highest angiostatin production of 191 mg/l was achieved as lactic acid was used; whose feeding rate was gradually increased from 2.6 to 11.3 g/h. Lactic acid accumulated during the induction phase and reached 6.3 g/l at the end of fermentation. However, the accumulation of lactic acid did not interfere with angiostatin production, indicating that lactic acid to be a non-repressive carbon source. The average productivity and specific productivity of angiostatin obtained on lactic acid and methanol were, respectively, 2.96 and 0.044 mg/(g h), 1.7- and 2.5-fold of those obtained in the fermentation fed with glycerol and methanol.     

Rapid ethanol production at elevated temperatures by engineered thermotolerant Kluyveromyces marxianus via the NADP(H)-preferring xylose reductase-xylitol dehydrogenase pathway

Conversion of xylose to ethanol by yeasts is a challenge because of the redox imbalances under oxygen-limited conditions. The thermotolerant yeast Kluyveromyces marxianus grows well with xylose as a carbon source at elevated temperatures, but its xylose fermentation ability is weak. In this study, a combination of the NADPH-preferring xylose reductase (XR) from Neurospora crassa and the NADP⁺-preferring xylitol dehydrogenase (XDH) mutant from Scheffersomyces stipitis (Pichia stipitis) was constructed. The xylose fermentation ability and redox balance of the recombinant strains were improved significantly by over-expression of several downstream genes. The intracellular concentrations of coenzymes and the reduced coenzyme/oxidized coenzyme ratio increased significantly in these metabolic strains. The byproducts, such as glycerol and acetic acid, were significantly reduced by the disruption of glycerol-3-phosphate dehydrogenase (GPD1). The resulting engineered K. marxianus YZJ088 strain produced 44.95 g/L ethanol from 118.39 g/L xylose with a productivity of 2.49 g/L/h at 42 °C. Additionally, YZJ088 realized glucose and xylose co-fermentation and produced 51.43 g/L ethanol from a mixture of 103.97 g/L xylose and 40.96 g/L glucose with a productivity of 2.14 g/L/h at 42 °C. These promising results validate the YZJ088 strain as an excellent producer of ethanol from xylose through the synthetic xylose assimilation pathway.     

l-Lactic acid production from liquefied cassava starch by thermotolerant Rhizopus microsporus: Characterization and optimization

The thermotolerant Rhizopus microsporus DMKU 33 capable of producing l-lactic acid from liquefied cassava starch was isolated and characterized for its phylogenetic relationship and growth temperature and pH ranges. The concentrations of (NH₄)₂SO_4, KH₂PO₄, MgSO₄ and ZnSO₄·7H₂O in the fermentation medium was optimized for lactic acid production from liquefied cassava starch by Rhizopus microsporus DMKU 33 in shake-flasks at 40 °C. The fermentation was then studied in a stirred-tank bioreactor with aeration at 0.75 vvm and agitation at 200 rpm, achieving the highest lactic acid production of 84 g/L with a yield of 0.84 g/g at pH 5.5 in 3 days. Lactic acid production was further increased to 105–118 g/L with a yield of 0.93 g/g and productivity of 1.25 g/L/h in fed-batch fermentation. R. microsporus DMKU 33 is thus advantageous to use in simultaneous saccharification and fermentation for l-lactic acid production from low-cost starchy substrates.     

Newly developed medium and strategy for bacterial cellulose production

Bacterial cellulose (BC) has unique properties, such as high crystallinity, a high degree of polymerisation, high tensile strength and high purity, compared with native cellulose. In this study, a previously determined BC production medium was improved in static culture, and the production cost was evaluated and compared with molasses and with other defined media, such as Hestrin–Schramm, Zhou, Yamanaka and Park, using Gluconacetobacter xylinus. In addition to this analysis, because the surface area/volume ratio is an important parameter in static culture, different surface area/volume ratios were analysed in the range of 0.2–1.46. The defined medium (M1A05P5) and culture type contained glucose (10 g/L), yeast extract (10 g/L), peptone (7 g/L), acetic acid (1.5 ml/L), and ethanol (5 ml/L), and the pH was adjusted to 5.0 in static culture. The highest productivity was observed in the M1A05P5 medium that was 5-fold higher than either molasses or Park's medium. Although the molasses medium was proposed as a cost-effective medium, the production price of BC was the lowest in the M1A05P5 medium. Therefore, the newly developed medium and strategy were highly promising candidates for the industrial-scale production of BC.     

Evolutionary adaptation of Kluyveromyces marxianus strain for efficient conversion of whey lactose to bioethanol

The aim of the present work is to develop an osmotolerant yeast strain with high lactose utilization and further use it to ferment lactose rich whey permeate for high ethanol titer and to reduce energy consumption. Ethanol production and growth rate of selected MTCC 1389 strain were quite high in whey containing lactose up to 150 g/L but it remains constant in lactose concentration (200 g/L) as cells encountered osmotic stress. Thus, strain MTCC 1389 was used for an adaptation to lactose concentration 200 g/L for 65 days and used further for fermentation of lactose rich whey. Fermentation with an adapted K. marxianus MTCC 1389 strain in laboratory fermenter resulted in ethanol titer of 79.33 g/L which is nearly 17.5% higher than the parental strain (66.75 g/L). Expression analysis of GPD1, TPS1and TPS2 found upregulated in lactose adapted K. marxianus strain as compared to the parental strain. These results suggest that an adapted K. marxianus strain accumulates glycerol and trehalose in response to lactose stress and improve osmotolerance in K. marxianus cells. Thus, the study illustrates that evolutionary engineering is an efficient strategy to obtain a superior biofuel yeast strain, which efficiently ferments four-fold concentrated cheese whey.     

A laboratory study of producing docosahexaenoic acid from biodiesel-waste glycerol by microalgal fermentation

Crude glycerol is the primary by-product in the biodiesel industry, which is too costly to be purified into to higher quality products used in the health and cosmetics industries. This work investigated the potential of using the crude glycerol to produce docosahexaenoic acid (DHA, 22:6 n-3) through fermentation of the microalga Schizochytrium limacinum. The results showed that crude glycerol supported alga growth and DHA production, with 75–100 g/L concentration being the optimal range. Among other medium and environmental factors influencing DHA production, temperature, trace metal (PI) solution concentration, ammonium acetate, and NH₄Cl had significant effects (P < 0.1). Their optimal values were determined 30 mL/L of PI, 0.04 g/L of NH₄Cl, 1.0 g/L of ammonium acetate, and 19.2 °C. A highest DHA yield of 4.91 g/L with 22.1 g/L cell dry weight was obtained. The results suggested that biodiesel-derived crude glycerol is a promising feedstock for production of DHA from heterotrophic algal culture.     

Optimization of biosurfactant production using waste from biodiesel industry in a new membrane assisted bioreactor

The main byproduct of biodiesel production is glycerol. Here, crude glycerol – byproduct of biodiesel industry – was evaluated as sole carbon source in rhamnolipids production by Pseudomonas aeruginosa. The optimal concentration of crude glycerol and sodium nitrate was assessed using response surface methodology, resulting in about 40–50 mg/L.h of rhamnolipids, which was about four times higher than previously reported in the literature. Fermentation parameters were similar to those observed with commercial glycerol as sole carbon source. The optimized medium was suitable for production using simple (22.9 mg/L.h) and fed-batch (32.4 mg/L.h) fermentation in oxygen-controlled bioreactor without foaming formation. Composition and relative abundance of rhamnolipid congeners showed that crude glycerol had little effect on metabolic pathways involved in their production. CMC values were approximately 130 mg/L and 230–260 mg/L for rhamnolipids from crude and commercial glycerol fermentation, respectively, which were about 2–6 times lower than CMC values of synthetic surfactants.     

Effect of modified MRS medium on production and purification of antimicrobial peptide ST4SA produced by Enterococcus mundtii

Highest antimicrobial activity of peptide ST4SA (51,200 AU/mL) was recorded after 14 h of growth in MRS broth with optimal production at pH 6.0 or 6.5. Growth of strain ST4SA in the presence of tryptone, yeast extract, or a combination of the two, yielded 102,400 AU/mL. An increase in production of peptide ST4SA to 102,400 AU/mL was recorded in the presence of 20.0 g/L fructose, but decreased to 25,600 AU/mL in the presence of lactose (20.0 g/L) or mannose (20.0 g/L) as sole carbon source. Lower activity (25,600 AU/mL) was recorded when 2.0 g/L K₂HPO₄ was replaced by 2.0 g/L KH₂PO₄ in MRS broth. An increase of K₂HPO₄ to 10.0 g/L and 20.0 g/L resulted in higher activity (102,400 AU/mL). Addition of glycerol to MRS broth had a negative effect on peptide ST4SA production. Production of peptide ST4SA required the presence of magnesium sulphate, manganese sulphate and 5.0 g/L sodium acetate. Exclusion of tri-ammonium citrate from the medium resulted in reduction of activity to 3,200 AU/mL. Maximum activity (102,400 AU/mL) was recorded in MRS supplemented with 1.0 ppm Vit. C, DL-6,8-thioctic acid or thiamine, respectively. Growth of Listeria ivanovii susbp. ivanovii ATCC 19119 in the presence of peptide ST4SA (12,800 AU/mL) resulted in 99% cell lysis after 18 h. Improved production of peptide ST4SA was recorded in MRS broth (Biolab) pre-treated with Amberlite XAD-1180. Precipitation with ammonium sulphate, followed by gel filtration chromatography, yielded the highest level of peptide ST4SA. This paper describes the partially deproteination of growth medium to facilitate peptide ST4SA purification.     

Homo-succinic acid production by metabolically engineered Mannheimia succiniciproducens

Succinic acid (SA) is a four carbon dicarboxylic acid of great industrial interest that can be produced by microbial fermentation. Here we report development of a high-yield homo-SA producing Mannheimia succiniciproducens strain by metabolic engineering. The PALFK strain (ldhA^-, pta^-, ackA^-, fruA^-) was developed based on optimization of carbon flux towards SA production while minimizing byproducts formation through the integrated application of in silico genome-scale metabolic flux analysis, omics analyses, and reconstruction of central carbon metabolism. Based on in silico simulation, utilization of sucrose would enhance the SA production and cell growth rates, while consumption of glycerol would reduce the byproduct formation rates. Thus, sucrose and glycerol were selected as dual carbon sources to improve the SA yield and productivity, while deregulation of catabolite-repression was also performed in engineered M. succiniciproducens. Fed-batch fermentations of PALFK with low- and medium-density (OD₆₀₀ of 0.4 and 9.0, respectively) inocula produced 69.2 and 78.4 g/L of homo-SA with yields of 1.56 and 1.64 mol/mol glucose equivalent and overall volumetric SA productivities of 2.50 and 6.02 g/L/h, respectively, using sucrose and glycerol as dual carbon sources. The SA productivity could be further increased to 38.6 g/L/h by employing a membrane cell recycle bioreactor system. The systems metabolic engineering strategies employed here for achieving homo-SA production with the highest overall performance indices reported to date will be generally applicable for developing superior industrial microorganisms and competitive processes for the bio-based production of other chemicals as well.     

Metabolic engineering of fatty acyl-ACP reductase-dependent pathway to improve fatty alcohol production in Escherichia coli

Fatty alcohols are important components of surfactants and cosmetic products. The production of fatty alcohols from sustainable resources using microbial fermentation could reduce dependence on fossil fuels and greenhouse gas emission. However, the industrialization of this process has been hampered by the current low yield and productivity of this synthetic pathway. As a result of metabolic engineering strategies, an Escherichia coli mutant containing Synechococcus elongatus fatty acyl-ACP reductase showed improved yield and productivity. Proteomics analysis and in vitro enzymatic assays showed that endogenous E. coli AdhP is a major contributor to the reduction of fatty aldehydes to fatty alcohols. Both in vitro and in vivo results clearly demonstrated that the activity and expression level of fatty acyl-CoA/ACP reductase is the rate-limiting step in the current protocol. In 2.5-L fed-batch fermentation with glycerol as the only carbon source, the most productive E. coli mutant produced 0.75 g/L fatty alcohols (0.02 g fatty alcohol/g glycerol) with a productivity of up to 0.06 g/L/h. This investigation establishes a promising synthetic pathway for industrial microbial production of fatty alcohols.