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.     

New integrated bioprocess for the continuous production,extraction and purification of lipopeptides produced by Bacillus subtilis in membrane bioreactor

Recently, a bubbleless membrane bioreactor (BMBR) has been successfully developed for biosurfactant production by Bacillus subtilis [1]. In this study, for the first time, continuous culture were carried out for the production of surfactin in a BMBR, both with or without a coupled microfiltration membrane. Results from continuous culture showed that a significant part of biomass was immobilized onto the air/liquid membrane contactor. Immobilized biomass activity onto the air/liquid membrane contactor was monitored using a respirometric analysis. Kinetics of growth, surfactin and primary metabolites production were investigated. Planktonic biomass, immobilized biomass and surfactin production and productivity obtained in batch culture (3 L) of 1.5 days of culture were 4.5 g DW, 1.3 g DW, 1.8 g and 17.4 mg L^?1 h^?1, respectively. In continuous culture without total cell recycling (TCR), the planktonic biomass was leached, but immobilized biomass reached a steady state at an estimated 6.6 g DW. 11.5 g of surfactin was produced after 3 days of culture, this gave an average surfactin productivity of 54.7 mg L^?1 h^?1 for the continuous culture, which presented a surfactin productivity of 30 mg L^?1 h^?1 at the steady state. TCR was then investigated for the continuous production, extraction and purification of surfactin using a coupled ultrafiltration step. In continuous culture with TCR at a dilution rate of 0.1 h^?1, planktonic biomass, immobilized biomass, surfactin production and productivity reached 7.5 g DW, 5.5 g DW, 7.1 g and 41.6 mg L^?1 h^?1 respectively, after 2 days of culture. After this time, biomass and surfactin productions stopped. Increasing dilution rate to 0.2 h^?1 led to the resumption of biomass and surfactin production and these values reached 11.1 g DW, 10.5 g DW, 7.9 g and 110.1 mg L^?1 h^?1, respectively, after 3 days of culture. This study has therefore shown that with this new integrated bioprocess, it was possible to continuously extract and purify several grams of biosurfactant, with purity up to 95%.     

Continuous succinic acid fermentation by Actinobacillus succinogenes

Fermentations were performed in an external recycle bioreactor using CO₂ and d-glucose at feed concentrations of 20 and 40 g L⁻¹. Severe biofilm formation prevented kinetic analysis of suspended cell (‘chemostat’) fermentation, while perlite packing enhanced the volumetric productivity by increasing the amount of immobilised cells. The highest productivity of 6.35 g L⁻¹ h⁻¹ was achieved at a dilution rate of 0.56 h⁻¹. A constant succinic acid yield of 0.69 ± 0.02 g/(g of glucose consumed) was obtained and found to be independent of the dilution rate, transient state and extent of biofilm build-up – approximately 56% of the carbon that formed phosphoenolpyruvate ended up as succinate. Byproduct analysis indicated that pyruvate oxidation proceeded solely via the formate-lyase pathway. Cell growth and corresponding biofilm formation were rapid at dilution rates higher than 0.35 h⁻¹ when the product concentrations were low (succinic acid < 10 g L⁻¹), while minimal growth was observed at succinic acid concentrations above this threshold.     

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.     

Ability of a perfluoropolymer membrane to tolerate by-products of ethanol fermentation broth from dilute acid-pretreated rice straw

A perfluoropolymer (PFP) membrane has been prepared for use in vapor permeation to separate aqueous ethanol mixtures produced from rice straw with xylose-assimilating recombinant Saccharomyces cerevisiae. PFP membranes commonly have been used for dehydration process and possess good selectivity and high permeances. The effects of by-products during dilute acid pretreatment, addition of yeast extract, and ethanol fermentation on PFP membrane performance were investigated. While feeding mixtures of ethanol (90 wt%) in water, to which individual by-products (0.1–2 g/L) were added, the PFP membrane demonstrated no clear change in permeation rate (439–507 g m⁻² h⁻¹) or separation factor (14.9–23.5) from 2 to 4 h of the process. The PFP membrane also showed no clear change in permeation rate (751–859 g m⁻² h⁻¹) or separation factor (12.5–13.8) while feeding the mixture (final ethanol conc.: 61 wt%) of ethanol and distillation of the fermentation broth using a suspended fraction of dilute acid-pretreated rice straw for 20 h. These results suggest that the PFP membrane can tolerate actual distillation liquids from ethanol fermentation broth obtained from lignocellulosic biomass pretreated with dilute acid.     

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.     

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.     

Improved β-carotene production of Rhodotorula glutinis in fermented radish brine by continuous cultivation

Fermentation kinetics of growth and β-carotene production by Rhodotorula glutinis DM28 in batch and continuous cultures using fermented radish brine, a waste generated from fermented vegetable industry, as a cultivation medium were investigated. The suitable brine concentration for β-carotene production by R. glutinis DM28 was 30 g l^?1. Its growth and β-carotene production obtained by batch culture in shake flasks were 2.2 g l^?1 and 87 μg l^?1, respectively, while, in a bioreactor were 2.6 g l^?1 and 186 μg l^?1, respectively. Furthermore, its maximum growth rate and β-carotene productivity in continuous culture obtained at the dilution rate of 0.24 h^?1 were 0.3 g l^?1 h^?1 and 19 μg l^?1 h^?1, respectively, which were significantly higher than those in the batch. Therefore, improved growth rate and β-carotene productivity of R. glutinis in fermented radish brine could be accomplished by continuous cultivation.     

A stable immobilized d-psicose 3-epimerase for the production of d-psicose in the presence of borate

Maximal activity of the immobilized d-psicose 3-epimerase from Agrobacterium tumefaciens on Duolite A568 beads was achieved at pH 9.0 and 55 °C with borate, and at pH 8.5 and 50 °C without borate. The half-lives of the immobilized enzyme at 50 °C with and without borate were increased 4.2- and 128-fold compared to that of the free enzyme without borate, respectively. The immobilized enzyme with borate produced 441 g l^?1 psicose from 700 g l^?1 fructose at pH 9.0 and 55 °C, whereas 193 g l^?1 psicose was produced without borate at pH 8.5 and 50 °C after 120 min in a batch reaction. The immobilized enzyme in a packed-bed bioreactor without borate was produced continuously 325 g l^?1 psicose from 500 g l^?1 fructose at a dilution rate of 1.62 h^?1 over a 236 h period with productivity of 527 g l^?1 h^?1 while that without borate produced 146 g l^?1 psicose at 4.15 h^?1 over a 384-h period with productivity of 606 g l^?1 h^?1. The operational half-lives of the enzyme with and without borate in the bioreactor were 601 and 645 h, respectively. In the present study, psicose was produced stably with high productivity using the immobilized d-psicose 3-epimerase in the presence of borate.     

Effects of feed sugar concentration on continuous ethanol fermentation of cheese whey powder solution (CWP)

Cheese whey powder (CWP) solution with different CWP or sugar concentrations was fermented to ethanol in a continuous fermenter using pure culture of Kluyveromyces marxianus (DSMZ 7239). Sugar concentration of the feed CWP solution varied between 55 and 200 g l⁻¹ while the hydraulic residence time (HRT) was kept constant at 54 h. Ethanol formation, sugar utilization and biomass formation were investigated as functions of the feed sugar concentration. Percent sugar utilization and biomass concentrations decreased and the effluent sugar concentration increased with increasing feed sugar concentrations especially for the feed sugar contents above 100 g l⁻¹. Ethanol concentration and productivity (DP) increased with increasing feed sugar up to 100 g l⁻¹ and then decreased with further increases in the feed sugar content. The highest ethanol concentration (3.7%, v v⁻¹) and productivity (0.54 gE l⁻¹ h⁻¹) were obtained with the feed sugar content of 100 g l⁻¹ or 125 g l⁻¹. The ethanol yield coefficient (Y_P/S) was also maximum (0.49 gE gS⁻¹) when the feed sugar was between 100 and 125 g l⁻¹. The growth yield coefficient (Y_X/S) decreased steadily from 0.123 to 0.063 gX gS⁻¹ when the feed sugar increased from 55 to 200 g l⁻¹ due to adverse effects of high sugar contents on yeast growth. The optimal feed sugar concentration maximizing the ethanol productivity and sugar utilization was between 100 and 125 g l⁻¹ under the specified experimental conditions.     

Effect of inoculum and pepsin–pancreatin hydrolysis on fibre fermentation measured by the gas production technique in pigs

Two experiments were undertaken to adapt the in vitro gas production technique in syringes, used for ruminants, to fibre fermentation studies in the large intestine of pigs.In a first experiment, two inocula (faeces and large intestine content) were compared at four dilution levels in a buffer solution (0.025, 0.05, 0.1 and 0.2 g ml⁻¹) with two substrates: wheat bran and sugar–beet pulp. The accumulated gas produced over 72 h was modelled and the kinetics parameters compared. The time to half asymptote was lower for the intestinal inoculum (5.5 versus 8.0 h, P<0.02), but the 2 inocula yielded similar fractional rates of degradation (0.16 h⁻¹) and gave equal final gas production (252 ml g⁻¹ substrate). No interaction (P>0.05) was observed between inocula and substrates. The dilution of the samples in the buffer solution increased (P<0.001) the lag time (from 0.9 to 2.1 h for dilution rates ranging from 0.2 to 0.025 g ml⁻¹, respectively) and decreased (P<0.001) the rates of substrate degradation (from 0.18 to 0.13 h⁻¹).A second experiment aimed to study the effect of an in vitro pepsin–pancreatin hydrolysis of the sample prior to the gas test. Six substrates were tested: maize, wheat bran, sugar–beet pulp, lupins, peas and soybean meal. The enzymatic hydrolysis affected (P<0.001) the kinetics parameters and the ranking order of the fermented substrates. The lag times also increased for all ingredients. The rate of degradation decreased when peas, lupins, maize and wheat bran were hydrolysed (P<0.001) but it increased with soybean meal (P=0.014) and sugar–beet pulp (P<0.001). Final gas production increased with peas and soybean meal (P<0.001), remained unchanged for lupins and decreased for the other substrates (P<0.001).In conclusion, the method using faeces as a source of microbial inoculum is reliable to characterise the fermentation kinetics of ingredients in the large intestine of pigs. However, it is important to hydrolyse the substrates with pepsin and pancreatin before the gas tests.     

Poly(3-hydroxybutyrate) production by Cupriavidus necator using waste glycerol

Polyhydroxyalkanoates (PHAs) have been recognized as good substitutes for the non-biodegradable petrochemically produced polymers. However, their high (real or estimated) current production cost limits their industrial applications. This work exploits two strategies to enhance PHAs substitution potential: the increase in PHA volumetric productivity in high density cultures and the use of waste glycerol (GRP), a by-product from the biodiesel industry, as primary carbon source for cell growth and polymer synthesis. Cupriavidus necator DSM 545 was used to accumulate poly(3-hydroxybutyrate) (P(3HB)) from GRP and from commercial glycerol (PG) as control substrate. On PG, productivities between 0.6 g_PHB L^?1 h^?1 and 1.5 g_PHB L^?1 h^?1 were attained. The maximum cell DW was 82.5 g_DW L^?1, the P(3HB) content being 62%. When GRP was used, 68.8 g_DW L^?1 with a P(3HB) accumulation of 38% resulting in a final productivity of 0.84 g_PHB L^?1 h^?1 was obtained. By decreasing the biomass concentration at which accumulation was triggered, a productivity of 1.1 g_PHB L^?1 h^?1 (50% P(3HB), w/w) was attained using GRP. P(3HB) molecular weights (M_w) ranged from 7.9 × 10⁵ to 9.6 × 10⁵ Da.     

Continuous 2-Keto-l-gulonic acid fermentation by mixed culture of Ketogulonicigenium vulgare DSM 4025 and Bacillus megaterium or Xanthomonas maltophilia

The fermentation process of 2-keto-L-gulonic acid (2KGA) from L-sorbose was developed using a two-stage continuous fermentation system. The mixed culture of Ketogulonicigenium vulgare DSM 4025 and Bacillus megaterium DSM 4026 produced 90 g/L of 2KGA from 120 g/L of L-sorbose at the dilution rate of 0.01 h⁻¹ in a single-stage continuous fermentation process. But after the production period was beyond 150 h, the significant decrease of 2KGA productivity was observed. When the non-spore forming bacteria Xanthomonas maltophilia IFO 12692 was used instead of B. megaterium DSM 4026 as a partner strain for K. vulgare DSM 4025, the 2KGA productivity was significantly improved in a two-stage continuous culture mode, in which two fermentors of the same size and volume were connected in series. In this mode, with two sets of 3-L jar fermentors, the steady state could be continued to over 1,331.5 h at least, when the dilution rates were 0.0382 h⁻¹ and 0.0380 hour⁻¹, respectively, for the first and second fermentors. The overall productivity was calculated to be 2.15 g/L/h at 113.1 g/L and a molar conversion yield of 90.1%. In the up-scaling fermentation to 30-L jar fermentors, 118.5 g/L of 2KGA was produced when dilution rates in both stages were 0.0430 hour⁻¹, and the overall productivity was calculated to be 2.55 g/L/h.     

Simultaneous saccharification and fermentation of cellulose by Fusarium oxysporum F3—growth characteristics and metabolite profiling

The simultaneous saccharification and fermentation (SSF) of cellulose by Fusarium oxysporum was investigated in the present study. It was found that F. oxysporum grow with a maximum specific growth rate of 0.023 h⁻¹ on cellulose at aerobic conditions and that it can produce ethanol with a volumetric productivity of 0.044 g/L/h and a yield of 0.35 g/g cellulose under anaerobic conditions. The cellulase system in F. oxysporum is well balanced as no cellobiose accumulated. The profile of the phosphorylated intermediates from Pentose Phosphate Pathway (PPP), Embden–Meyerhof–Parnas Pathway (EMP) and the key intermediates of the glycolytic pathway as well as extracellular organic and amino acids were determined during the fermentation in order to investigate the potential metabolic bottlenecks of the process. The high levels of intracellular glucose-1,6-DP a metabolic downstream of phoshoglucomutase also indicates limiting activities of this enzyme and difficulty of glucose to be channelled into biosynthetic and glycolytic pathways. The presence of high levels of γ-aminobutyrate (GABA) under anaerobic conditions suggests a functional GABA bypass and possible block in the Krebs cycle.     

Catalytic removal of sulfide by an immobilized sulfide-oxidase bioreactor

A bioreactor packed with chitosan immobilized sulfide-oxidase from Streptomyces species LD048 was developed to treat a liquid stream of sulfide. The inoculation system was composed of glass with a 0.7 L working volume and enzyme activity of 2 mmol S g^?1 carrier. The sulfide removal efficiency was almost 100% when the volumetric loading was increased up to 3.9 mmol S L^?1 h^?1 at a space velocity of 18 h^?1. The maximal elimination capacity was 22.1 mmol S L^?1 h^?1 with a space velocity of 72 h^?1. When the aeration was increased from 0.05 to 0.1 L min^?1, the average removal efficiency improved from 81% to 94%. A removal efficiency of 90% was obtained after 15 days of operation with a load rate of 8.9 mmol S L^?1 h^?1 and a space velocity of 14.28 h^?1. An operational equation based on the ideal plug flow bioreactor and the Michaelis–Menten model predicted the performance of this bioreactor.     

Continuous production of selenomethionine-enriched Chlorella sorokiniana biomass in a photobioreactor

This article describes the enrichment of the fresh-water green microalga Chlorella sorokiniana in selenomethionine (SeMet). The microalga was cultivated in a 2.2 L glass-vessel photobioreactor, in a culture medium supplemented with selenate (SeO₄^2?) concentrations ranging from 5 to 50 mg L^?1. Although selenate exposure lowered culture viability, C. sorokiniana grew well at all tested selenate concentrations, however cultures supplemented with 50 mg L^?1 selenate did not remain stable at steady state. A suitable selenate concentration in fresh culture medium for continuous operation was determined, which allowed stable long-term cultivation at steady state and maximal SeMet productivity. In order to do that, the effect of dilution rate on biomass productivity, viability and SeMet content of C. sorokiniana at several selenate concentrations were determined in the photobioreactor. A maximal SeMet productivity of 21 μg L^?1 day^?1 was obtained with 40 mg L^?1 selenate in the culture medium. Then a continuous cultivation process at several dilution rates was performed at 40 mg L^?1 selenate obtaining a maximum of 246 μg L^?1 day^?1 SeMet at a low dilution rate of 0.49 day^?1, calculated on total daily effluent volume. This paper describes for the first time an efficient long-term continuous cultivation of C. sorokiniana for the production of biomass enriched in the high value amino acid SeMet, at laboratory scale.     

Quasi-continuous fermentation in a reverse-flow diafiltration bioreactor

Heterogeneities occur in various bioreactor designs including cell retention devices. Whereas in external devices changing environmental conditions cannot be prevented, cells are retained in their optimal environment in internal devices. Conventional reverse-flow diafiltration utilizes an internal membrane device, but pulsed feeding causes spatial heterogeneities. In this study, the influence of conventional reverse-flow diafiltration on the yeast Hansenula polymorpha is investigated. Alternating 180 s of feeding with 360 s of non-feeding at a dilution rate of 0.2 h⁻¹ results in an oscillating DOT signal with an amplitude of 60%. Thereby, induced short-term oxygen limitations result in the formation of ethanol and a reduced product concentration of 25%. This effect is enforced at increased dilution rate. To overcome this cyclic problem, sequential operation of three membranes is introduced. Thus, quasi-continuous feeding is achieved reducing the oscillation of the DOT signal to an amplitude of 20% and 40% for a dilution rate of 0.2 h⁻¹ and 0.5 h⁻¹, respectively. Fermentation conditions characterized by complete absence of oxygen limitation and without formation of overflow metabolites could be obtained for dilution rates from 0.1 h⁻¹ to 0.5 h⁻¹. Thus, sequential operation of three membranes minimizes oscillations in the DOT signal providing a nearly homogenous culture over time.     

Challenges in enzymatic hydrolysis and fermentation of pretreated Arundo donax revealed by a comparison between SHF and SSF

The perennial herbaceous crop Arundo donax is a potential feedstock for second-generation bioethanol production. In the present work, two different process options were investigated for the conversion of two differently steam-pretreated batches of A. donax. The pretreated raw material was converted to ethanol with a xylose-consuming Saccharomyces cerevisiae strain, VTT C-10880, by applying either separate hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF). The highest overall ethanol yield and final ethanol concentration were achieved using SHF (0.27 g g^?1 and 20.6 g L^?1 compared to 0.24 g g^?1 and 19.0 g L^?1 when SSF was used). The performance of both SHF and SSF was improved by complementing the cellulolytic enzymes with hemicellulases. The higher amount of acetic acid in one of the batches was shown to strongly affect xylose consumption in the fermentation. Only half of the xylose was consumed when batch 1 (high acetic acid) was fermented, compared to that 94% of the xylose was consumed in fermentation of batch 2 (lower acetic acid). Furthermore, the high amount of xylooligomers present in the pretreated materials considerably inhibited the enzymatic hydrolysis. Both the formation of xylooligomers and acetic acid thus need to be considered in the pretreatment process in order to achieve efficient conversion of A. donax to ethanol.