2,3-Butanediol production using Klebsiella oxytoca ATCC 8724: Evaluation of biomass derived sugars and fed-batch fermentation process

For this study, 2,3-butanediol (BD) fermentation from pure and biomass-derived sugar were optimized in shake-flask and 5-L bioreactor levels using Klebsiella oxytoca ATCC 8724. The results showed that 70 g/L of single sugar (glucose or xylose) and 90 g/L of mixed-sugar (glucose:xylose = 2:1) were optimum concentrations for efficient 2,3-BD fermentation. At optimum sugar concentrations, 2,3-BD productivities were 1.03, 0.64 and 0.50 gL⁻¹ h⁻¹, and yields were 0.43, 0.36 and 0.35 g/g in glucose, xylose and mixed-sugar medium, respectively. The lack of simultaneous utilization of glucose and xylose led to the lowest productivity in the mixed-sugar medium. Detoxification of biomass hydrolyzates was necessary for efficient 2,3-BD fermentation when sugar concentrations in the medium was 90 g/L or higher, but not with sugar concentrations of 30 g/L or less. A fed-batch fermentation using glucose medium led to an increase 2,3-BD titer to 79.4 g/L and yields 0.47 g/g, while productivity decreased to 0.79 gL⁻¹ h⁻¹. However, the fed-batch process was inefficient using mixed-sugar and biomass hydrolyzates because of poor xylose utilization. These results indicated that appropriate biomass processing technologies must be developed to generate separate glucose and xylose streams to produce high 2,3-BD titer from biomass-derived sugar using a fed-batch process.     

A novel co-culture process with Zymomonas mobilis and Pichia stipitis for efficient ethanol production on glucose/xylose mixtures

An efficient conversion of glucose and xylose is a requisite for a profitable process of bioethanol production from lignocellulose. Considering the approaches available for this conversion, co-culture is a simple process, employing two different organisms for the fermentation of the two sugars. An innovative fermentation scheme was designed, co-culturing immobilized Zymomonas mobilis and free cells of Pichia stipitis in a modified fermentor for the glucose and xylose fermentation, respectively. A sugar mixture of 30 g/l glucose and 20 g/l of xylose was completely converted to ethanol within 19 h. This gave a volumetric ethanol productivity of 1.277 g/l/h and an ethanol yield of 0.49–0.50 g/g, which is more than 96% of the theoretical value. Extension of this fermentation scheme to sugarcane bagasse hydrolysate resulted in a complete sugar utilisation within 26 h; ethanol production peaked at 40 h with a yield of 0.49 g/g. These values are comparable to the best results reported. Cell interaction was observed between Z. mobilis and P. stipitis. Viable cells of Z. mobilis inhibited the cell activity of P. stipitis and the xylose fermentation. Z. mobilis showed evidence of utilising a source other than glucose for growth when co-cultured with P. stipitis.     

Triphasic esterification of butanol and butyric acid in fermentation media

Butanol and butyric acid produced from acetone-butanol-ethanol (ABE) fermentation can be used to produce butyl butyrate, an important fragrance ester. However, low levels of butanol and butyric acid need to be purified from culture media first with energy-intensive distillation processes. In this study, a triphasic (organic/aqueous/fluorous) system is developed to esterify butanol and butyric acid in spent culture media into butyl butyrate directly without purification. The produced butyl butyrate forms a distinct organic phase floating on top and can then be separated easily. In a model system containing 37.1 g/L of butanol and 44.1 g/L of butyric acid, 57% of the butanol is converted to butyl butyrate after 8 h of esterification. With multiple cycles of esterification and product removal, butanol conversion can be further increased to 86%. When spent culture medium containing 7.12 g/L of butanol and 4.81 g/L of butyric acid is used for esterification, 38% of butanol (0.36 mmol) is consumed and 0.33 mmol of butyl butyrate is produced. However, when ABE fermentation and esterification are carried out simultaneously, only 0.042 mmol of butyl butyrate is produced, probably due to the incompatible pH requirements for cell growth (pH 5–7) and esterification (pH 2–3).     

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.     

Development of sequential-co-culture system (Pichia stipitis and Zymomonas mobilis) for bioethanol production from Kans grass biomass

Sequential-co-culture technique was investigated in this study for the production of bioethanol from relatively cheaper lignocellulosic biomass of Kans grass (Saccharum spontaneum). The consortium of Pichia stipitis and Zymomonas mobilis was used to develop a suitable sequential-co-culture system. The Kans grass biomass was hydrolyzed in such a manner that the two sugar fractions, xylose rich and glucose rich were generated (a separate study). The P. stipitis cells and respective fermentation media (xylose rich) were fed to the fermentation vessel, after the set fermentation time Z. mobilis cells and respective media were fed to the same vessel. Different strategies have been followed and experiments were conducted initially at flask level. The selected strategy was then applied at bioreactor level using both synthetic fermentation media and Kans grass hydrolysate media to compare the kinetic parameters. The sequential addition of cultures with their respective media and imposed process conditions, showed better utilization of total sugars added (>95%). Microaerobic condition for P. stipitis and strictly anaerobic condition for Z. mobilis fermentation were found significant. The average ethanol yield (Y_p/s) and overall volumetric productivity (r_po) were found as 0.453 g_p/g_s and 1.580 g/l/h respectively for Kans grass hydrolysate media and 0.474 g_p/g_s and 2.901 g/l/h respectively for synthetic fermentation media.     

Production of butyric acid by a cellulolytic actinobacterium Thermobifida fusca on cellulose

Thermobifida fusca not only produces cellulases, hemicellulases and xylanases, but also excretes butyric acid. In order to achieve a high yield of butyric acid, the effect of different carbon sources: mannose, xylose, lactose, cellobiose, glucose, sucrose and acetates, on butyric acid production was studied. The highest yield of butyric acid was 0.67 g/g C (g-butyric acid/g-carbon input) on cellobiose. The best stir speed and aeration rate for butyric acid production were found to be 400 rpm and 2 vvm in a 5-L fermentor. The maximum titer of 2.1 g/L butyric acid was achieved on 9.66 g/L cellulose. In order to test the production of butyric acid on lignocellulosic biomass, corn stover was used as the substrate, on which there was 2.37 g/L butyric acid produced under the optimized conditions. In addition, butyric acid synthesis pathway was identified involving five genes that catalyzed reactions from acetyl-CoA to butanoyl-CoA in T. fusca.     

Metabolic engineering of Clostridium acetobutylicum for butyric acid production with high butyric acid selectivity

A typical characteristic of the butyric acid-producing Clostridium is coproduction of both butyric and acetic acids. Increasing the butyric acid selectivity important for economical butyric acid production has been rather difficult in clostridia due to their complex metabolic pathways. In this work, Clostridium acetobutylicum was metabolically engineered for highly selective butyric acid production. For this purpose, the second butyrate kinase of C. acetobutylicum encoded by the bukII gene instead of butyrate kinase I encoded by the buk gene was employed. Furthermore, metabolic pathways were engineered to further enhance the NADH-driving force. Batch fermentation of the metabolically engineered C. acetobutylicum strain HCBEKW (pta⁻, buk⁻, ctfB⁻ and adhE1⁻) at pH 6.0 resulted in the production of 32.5 g/L of butyric acid with a butyric-to-acetic acid ratio (BA/AA ratio) of 31.3 g/g from 83.3 g/L of glucose. By further knocking out the hydA gene (encoding hydrogenase) in the HCBEKW strain, the butyric acid titer was not further improved in batch fermentation. However, the BA/AA ratio (28.5 g/g) obtained with the HYCBEKW strain (pta⁻, buk⁻, ctfB⁻, adhE1⁻ and hydA⁻) was 1.6 times higher than that (18.2 g/g) obtained with the HCBEKW strain at pH 5.0, while no improvement was observed at pH 6.0. These results suggested that the buk gene knockout was essential to get a high butyric acid selectivity to acetic acid in C. acetobutylicum.     

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.     

Fermentative production of succinic acid from straw hydrolysate by Actinobacillus succinogenes

In this work, straw hydrolysates were used to produce succinic acid by Actinobacillus succinogenes CGMCC1593 for the first time. Results indicated that both glucose and xylose in the straw hydrolysates were utilized in succinic acid production, and the hydrolysates of corn straw was better than that of rice or wheat straw in anaerobic fermentation of succinic acid. However, cell growth and succinic acid production were inhibited when the initial concentration of sugar, which was from corn straw hydrolysate (CSH), was higher than 60 g l^?1. In batch fermentation, 45.5 g l^?1 succinic acid concentration and 80.7% yield were attained after 48 h incubation with 58 g l^?1 of initial sugar from corn straw hydrolysate in a 5-l stirred bioreactor. While in fed-batch fermentation, concentration of succinic acid achieved 53.2 g l^?1 at a rate of 1.21 g l^?1 h^?1 after 44 h of fermentation. Our work suggested that corn straw could be utilized for the economical production of succinic acid by A. succinogenes.     

Improved 2,3-butanediol production from corncob acid hydrolysate by fed-batch fermentation using Klebsiella oxytoca

Corncob acid hydrolysate, detoxed by sequently boiling, overliming and activated charcoal adsorption, was used for 2,3-butanediol production by Klebsiella oxytoca ACCC 10370. The effects of acetate in hydrolysate and pH on 2,3-butanediol production were investigated. It was found that acetic acid in hydrolysate inhibited the growth of K. oxytoca while benefited the 2,3-butanediol yield. With the increase in acetic acid concentration in medium from 0 to 4 g/l, the lag phase was prolonged and the specific growth rate decreased. The acetic acid inhibition on cell growth can be alleviated by adjusting pH to 6.3 prior to fermentation and a substrate fed-batch strategy with a low initial acetic acid concentration. Under the optimum condition, a maximal 2,3-butanediol concentration of 35.7 g/l was obtained after 60 h of fed-batch fermentation, giving a yield of 0.5 g/g reducing sugar and a productivity of 0.59 g/h l.     

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.     

Enhanced ethanol production by continuous fermentation in a two-tank system with cell recycling

An experimental method for producing ethanol continuously was designed and tested with a cell-recycling two-tank system, which was composed of two fermentors, each of which was individually equipped with a settler for recycling flocculent yeast. This system was effective for the continuous fermentation of ethanol from sucrose at high cell-recycling (r = 0.8–0.9) and dilution (up to 0.48 h^?1) rates. The system has several advantages; the high cell concentration in the fermentors and relief of substrate and product inhibition. Thus, the enhanced productivity using this continuous fermentation with the two-tank cell-recycling system was significantly higher compared with that of the batch fermentation. The results indicate that increased recycling ratios caused an increase in biomass concentration and subsequently, product concentration in the tank. The ethanol productivity increased with the dilution rate, but higher dilution rates could render increasing amounts of sugar unconverted. Continuous fermentation with the sugar feed concentration of 160 g/l at r = 0.9 and dilution rate of 0.2 h^?1 achieved the highest productivity with less than 2% of the unconverted sugar in the product steam. Under the same cell recycling ratios a productivity range of 6.9–7.5 g/l h^?1 could be achieved with feeding concentrations of 80–200 g/l, while batch fermentation at these sugar concentrations led to productivities of 3.85–4.48 g/l h^?1.     

Xylitol production from non-detoxified corncob hemicellulose acid hydrolysate by Candida tropicalis

The interest on use of lignocellulose for producing chemicals is increasing as these feedstocks are low cost, renewable and widespread sources of sugars. Corncob is an attractive raw material for xylitol production due to its high content of xylan. In this study, hemicellulose hydrolysate from corncobs without detoxification was used for xylitol production by Candida tropicalis CCTCC M2012462. Compared with prepared xylose medium, xylitol production with dilute acid hydrolysate medium does not seem to influence specific xylose reductase activity. The decrease in xylitol productivity with dilute acid hydrolysate medium is a result of a lower biomass concentration and lag-phase time. It appears that biomass growth rate is essential for xylitol production. In xylitol fermentation with a low initial inhibitors concentration and substrate feeding strategy, a maximal xylitol concentration of 38.8 g l⁻¹ was obtained after 84 h of fermentation, giving a yield of 0.7 g g⁻¹ xylose and a productivity of 0.46 g l⁻¹ h⁻¹.     

Complete genome sequence,metabolic model construction and phenotypic characterization of Geobacillus LC300, an extremely thermophilic,fast growing,xylose-utilizing bacterium

We have isolated a new extremely thermophilic fast-growing Geobacillus strain that can efficiently utilize xylose, glucose, mannose and galactose for cell growth. When grown aerobically at 72 °C, Geobacillus LC300 has a growth rate of 2.15 h⁻¹ on glucose and 1.52 h⁻¹ on xylose (doubling time less than 30 min). The corresponding specific glucose and xylose utilization rates are 5.55 g/g/h and 5.24 g/g/h, respectively. As such, Geobacillus LC300 grows 3-times faster than E. coli on glucose and xylose, and has a specific xylose utilization rate that is 3-times higher than the best metabolically engineered organism to date. To gain more insight into the metabolism of Geobacillus LC300 its genome was sequenced using PacBio׳s RS II single-molecule real-time (SMRT) sequencing platform and annotated using the RAST server. Based on the genome annotation and the measured biomass composition a core metabolic network model was constructed. To further demonstrate the biotechnological potential of this organism, Geobacillus LC300 was grown to high cell-densities in a fed-batch culture, where cells maintained a high xylose utilization rate under low dissolved oxygen concentrations. All of these characteristics make Geobacillus LC300 an attractive host for future metabolic engineering and biotechnology applications.     

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.     

Enhancement of pilot scale production of l(+)-lactic acid by fermentation coupled with separation using membrane bioreactor

Traditional batch fermentation leads to a higher energy consumption and lower production capability because of longer culture time. In this work, a pilot scale bioreactor composed of a 3000 L fermentor and external ceramic microfiltration equipment was used to perform cell-recycle fermentation. Repeat feeding medium was also used to relieve the substrate inhibition. In such pilot system, the maximum yield and productivity of l(+)-lactic acid production reached 157.22 ± 3.42 g/L and 8.77 ± 0.15 g/L/h which were 4.23% and 315.64% higher than those of batch fermentation, respectively, when equal amount of sugar was consumed. The cost of l(+)-lactic acid production was successfully reduced about two-thirds by the increase of yield and productivity. 12 rounds of cell-recycle fermentations were successfully achieved in the pilot system. The membrane filtration productivity reached to 61.27 ± 2.74 L/m²/h which increased 172.80%, while the cell damaging rate dropped to 3.88 ± 0.18% which decreased 85.77%, compared with those of the ultrafiltration. Furthermore, the ceramic microfiltration membrane showed advantages in tolerance for the temperature, pressure and acid, compared with the organic ultrafiltration membrane. The experimental results indicated that the method could give a reference for low cost production of l(+)-lactic acid in an industrial scale.     

Lipase in biphasic alginate beads as a biocatalyst for esterification of butyric acid and butanol in aqueous media

Esterification of organic acids and alcohols in aqueous media is very inefficient due to thermodynamic constraints. However, fermentation processes used to produce organic acids and alcohols are often conducted in aqueous media. To produce esters in aqueous media, biphasic alginate beads with immobilized lipase are developed for in situ esterification of butanol and butyric acid. The biphasic beads contain a solid matrix of calcium alginate and hexadecane together with 5 mg/mL of lipase as the biocatalyst. Hexadecane in the biphasic beads serves as an organic phase to facilitate the esterification reaction. Under optimized conditions, the beads are able to catalyze the production of 0.16 mmol of butyl butyrate from 0.5 mmol of butyric acid and 1.5 mmol of butanol. In contrast, when monophasic beads (without hexadecane) are used, only trace amount of butyl butyrate is produced. One main application of biphasic beads is in simultaneous fermentation and esterification (SFE) because the organic phase inside the beads is very stable and does not leach out into the culture medium. SFE is successfully conducted with an esterification yield of 6.32% using biphasic beads containing iso-octane even though the solvent is proven toxic to the butanol-producing Clostridium spp.     

Metabolic engineering of Corynebacterium glutamicum for the production of 3-hydroxypropionic acid from glucose and xylose

3-Hydroxypropionic acid (3-HP) is a promising platform chemical which can be used for the production of various value-added chemicals. In this study,Corynebacterium glutamicum was metabolically engineered to efficiently produce 3-HP from glucose and xylose via the glycerol pathway. A functional 3-HP synthesis pathway was engineered through a combination of genes involved in glycerol synthesis (fusion of gpd and gpp from Saccharomyces cerevisiae) and 3-HP production (pduCDEGH from Klebsiella pneumoniae and aldehyde dehydrogenases from various resources). High 3-HP yield was achieved by screening of active aldehyde dehydrogenases and by minimizing byproduct synthesis (gapA^A1GΔldhAΔpta-ackAΔpoxBΔglpK). Substitution of phosphoenolpyruvate-dependent glucose uptake system (PTS) by inositol permeases (iolT1) and glucokinase (glk) further increased 3-HP production to 38.6 g/L, with the yield of 0.48 g/g glucose. To broaden its substrate spectrum, the engineered strain was modified to incorporate the pentose transport gene araE and xylose catabolic gene xylAB, allowing for the simultaneous utilization of glucose and xylose. Combination of these genetic manipulations resulted in an engineered C. glutamicum strain capable of producing 62.6 g/L 3-HP at a yield of 0.51 g/g glucose in fed-batch fermentation. To the best of our knowledge, this is the highest titer and yield of 3-HP from sugar. This is also the first report for the production of 3-HP from xylose, opening the way toward 3-HP production from abundant lignocellulosic feedstocks.     

Mycophenolic acid production in solid-state fermentation using a packed-bed bioreactor

Mycophenolic acid (MPA) was produced from Penicillium brevicompactum by solid-state fermentation (SSF) using pearl barley, and submerged fermentation (SmF) using mannitol. It was found that SSF was superior to SmF in terms of MPA concentration (1219 mg/L vs. 60 mg/L after 144 h fermentation), and the product yields were 6.1 mg/g pearl barley for SSF and 1.2 mg/g mannitol for SmF. The volumetric productivities were 8.5 and 0.42 mg/L h for SSF and SmF, respectively.The optimum solid substrate of SSF for MPA production was pearl barley, producing 5470 mg/kg compared with wheat bran (1601 mg/kg), oat (3717 mg/kg) and rice (2597 mg/kg). The optimum moisture content, incubation time and inoculum concentrations were 70%, 144 h and 6%, respectively. Neither the addition of mannitol or (NH4)₂HPO₄ nor adjustment of media pH within the range of 3–7 significantly enhanced MPA production.MPA production by SSF using a packed-bed bioreactor was performed and an increased maximum production of MPA 6.9 mg/g was achieved at 168 h incubation time. The higher volumetric productivity and concentrations makes SSF an attractive alternative to SmF for MPA production.