Fibrous Bed Bioreactor Research Articles

Enhanced butyric acid tolerance and production by Class I heat shock protein-overproducing Clostridium tyrobutyricum ATCC 25755.

The response of Clostridium tyrobutyricum to butyric acid stress involves various stress-related genes, and therefore overexpression of stress-related genes can improve butyric acid tolerance and yield. Class I heat shock proteins (HSPs) play an important role in the process of protecting bacteria from sudden changes of extracellular stress by assisting protein folding correctly. The results of quantitative real-time PCR indicated that the Class I HSGs grpE, dnaK, dnaJ, groEL, groES, and htpG were significantly upregulated under butyric acid stress, especially the dnaK and groE operons. Overexpression of groESL and htpG could significantly improve the tolerance of C. tyrobutyricum to butyric acid, while overexpression of dnaK and dnaJ showed negative effects on butyric acid tolerance. Acid production was also significantly promoted by increased GroESL expression levels; the final butyric acid and acetic acid concentrations were 28.2 and 38% higher for C. tyrobutyricum ATCC 25755/groESL than for the wild-type strain. In addition, when fed-batch fermentation was carried out using cell immobilization in a fibrous-bed bioreactor, the butyric acid yield produced by C. tyrobutyricum ATCC 25755/groESL reached 52.2g/L, much higher than that for the control. The improved butyric acid yield is probably attributable to the high GroES and GroEL levels, which can stabilize the biosynthetic machinery of C. tyrobutyricum under extracellular butyric acid stress.

Moderate alkali-thermophilic ethanologenesis by locally isolated Bacillus licheniformis from Pakistan employing sugarcane bagasse: a comparative aspect of aseptic and non-aseptic fermentations

BackgroundBiofuels obtained from first-generation (1G) sugars-starch streams have been proven unsustainable as their constant consumption is not only significantly costly for commercial scale production systems, but it could potentially lead to problems associated with extortionate food items for human usage. In this regard, biofuels’ production in alkali-thermophilic environs from second-generation (2G) bio-waste would not only be markedly feasible, but these extreme conditions might be able to sustain aseptic fermentations without spending much for sterilization.ResultsPresent investigation deals with the valuation of ethanologenic potential of locally isolated moderate alkali-thermophilic fermentative bacterium, Bacillus licheniformis KU886221 employing sugarcane cane bagasse (SCB) as substrate. A standard 2-factor central composite response surface design was used to estimate the optimized cellulolytic and hemicellulolytic enzymatic hydrolysis of SCB into maximum fermentable sugars. After elucidation of optimized levels of fermentation factors affecting ethanol fermentation using Taguchi OA L27 (3^13) experimental design, free cell batch culture was carried out in bench-scale stirred-tank bioreactor for ethanol fermentation. Succeeding fermentation modifications included subsequent substrate addition, immobilized cells fibrous-bed bioreactor (FBB) incorporation to the basic setup, and performance of in situ gas stripping for attaining improved ethanol yield. Highest ethanol yield of 1.1406 mol ethanol/mol of equivalent sugars consumed was obtained when gas stripping was performed during fed-batch fermentation involving FBB under aseptic conditions. Despite the fact that under non-aseptic conditions, 30.5% lesser ethanol was formed, still, reduced yield might be considered influential as it saved the cost of sterilization for ethanol production.ConclusionEffectual utilization of low-priced abundantly available lignocellulosic waste sugarcane bagasse under non-aseptic moderate alkali-thermophilic fermentation conditions as directed in this study has appeared very promising for large-scale cost-effective bioethanol generation processes.

N-Butanol production from sucrose and sugarcane juice by engineered Clostridium tyrobutyricum overexpressing sucrose catabolism genes and adhE2

The production of n-butanol from sugarcane juice by metabolically engineered Clostridium tyrobutyricum Ct(Δack)-pscrBAK overexpressing scr operon genes (scrB, scrA, and scrK) for sucrose catabolism and an aldehyde/alcohol dehydrogenase gene (adhE2) for butanol biosynthesis was studied with corn steep liquor (CSL) as a low-cost nitrogen source. In free cell fermentation, butanol production of ∼16g/L at a yield of 0.31±0.02g/g and productivity of 0.33±0.02g/L·h was obtained from sucrose and yield of 0.24±0.02g/g and productivity of 0.30±0.01g/L·h from sugarcane juice containing sucrose, glucose and fructose. The fermentation was also studied in a fibrous bed bioreactor (FBB) operated in a repeated batch mode for 10 consecutive cycles in 10days, achieving an average butanol yield of 0.21±0.02g/g and productivity of 0.53±0.05g/L·h from sugarcane juice, demonstrating its long-term stability without applying the antibiotic selection pressure.

High efficiency succinic acid production from glycerol via in situ fibrous bed bioreactor with an engineered Yarrowia lipolytica

In this study, in situ fibrous bed bioreactor (isFBB) was developed at the first time for efficient succinic acid (SA) production by Yarrowia lipolytica. After optimization, SA titer, productivity and yield of 51.9g/L, 1.46g/L/h and 0.42g/g were obtained respectively via isFBB fermentation under conditions of 750cm2 cotton towel, 120g/L initial glycerol and 3L/min aeration rate. By fed batch strategy, SA titer raised up to 198.2g/L was achieved, which was the highest value ever reported. In operation stability study, SA productivity showed no obvious decrease after 12 repeated batches of 460h fermentation, and cell viability even recovered within two repeated batches after intentional interruption. This study successfully attained a highly efficient and stable isFBB for enhanced SA production by Y. lipolytica.

Enhanced butanol production by solvent tolerance Clostridium acetobutylicum SE25 from cassava flour in a fibrous bed bioreactor

To enhance the butanol productivity and reduce the material cost, acetone, butanol, and ethanol fermentation by Clostridium acetobutylicum SE25 was investigated using batch, repeated-batch and continuous cultures in a fibrous bed bioreactor, where cassava flour was used as the substrate. With periodical nutrient supplementation, stable butanol production was maintained for about 360h in a 6-cycle repeated-batch fermentation with an average butanol productivity of 0.28g/L/h and butanol yield of 0.32g/g-starch. In addition, the highest butanol productivity of 0.63g/L/h and butanol yield of 0.36g/g-starch were achieved when the dilution rate were investigated in continuous production of acetone, butanol, and ethanol using a fibrous bed bioreactor, which were 231.6% and 28.6% higher than those of the free-cell fermentation. On the other hand, this study also successfully comfirmed that the biofilm can provide an effective protection for the microbial cells which are growing in stressful environment.

Improved production of propionic acid driven by hydrolyzed liquid containing high concentration of l-lactic acid from co-fermentation of food waste and sludge

This study investigated the feasibility of improved production propionic acid-enriched volatile fatty acid (VFA) from high concentration (Cs) of food waste and waste activated sludge (WAS) via lactic acid pathway by using of Propionibacterium acidipropionici. It was observed that production of l-lactate overwhelmed to d-lactate at first stage, which improved from 3.21 to 35.45gCOD/L with increase of substrate Cs. However, kinetic model analysis indicated that P. acidipropionici growth rate μmax was decreased with increase of l-lactate concentration, which explained second stage free cell fermentation of propionic acid was inhibited when fed by first stage liquid from R-40, R-55 and R-70. Then, the fibrous bed bioreactor was employed to eliminate the feed inhibition. The maximal percentage of propionic acid (68.3%) and production (16.31gCOD/L) was obtained by feeding liquid of R-55, which was improved by 3.33 folds compared to the free cell fermentation.

Butyric acid production from oilseed rape straw by Clostridium tyrobutyricum immobilized in a fibrous bed bioreactor

Abstract Butyric acid is an important specialty chemical with wide industrial applications, while economical production via feasible large-scale fermentation requires low-cost substrate and an efficient process. In this study, hydrolyzed oilseed rape (OSR) straw was used as an alternative carbon source for immobilized-cell fermentation of Clostridium tyrobutyricum to produce butyric acid. Compared to the free-cell fermentation, higher butyrate yield (0.43 g/g vs. 0.37 g/g), productivity (2.46 g/L/h vs. 0.93 g/L/h) and concentration (13.6 g/L vs. 11.6 g/L) were obtained with immobilized cells of C. tyrobutyricum in repeated-batch fermentation. Furthermore, fed-batch fermentation using pretreated OSR straw hydrolysate resulted in a high butyric acid concentration of 50.2 g/L, with yield of 0.38 g/g. Finally, the repeated batch fermentation was carried out, which achieved high butyrate yield and volumetric productivity during long-term operation. With desirable characteristics such as economic viability and competitiveness, as well as being environmentally friendly, the fermentation developed in this work should provide an efficient process for industrial production of butyric acid.

Production of β-glucosidase from wheat bran and glycerol by Aspergillus niger in stirred tank and rotating fibrous bed bioreactors

Aspergillus niger NRRL 3112 can produce considerable amounts of β-1,4-glucosidase (BGL) when grown on wheat bran and glycerol as co-substrates. BGL production was first investigated in a stirred-tank bioreactor (STR) at 450rpm and 2 vvm. About 5.4U/mL BGL was obtained using spore suspension as inoculum, whereas a higher production of 9.3U/mL was obtained using precultured cell pellets. The production of BGL in batch, fed-batch and repeated batch modes in a rotating fibrous bed bioreactor (RFBB) was then studied and compared to the STR. The highest BGL productivity of 1.78U/mL/day was obtained in the RFBB operated at a repeated batch mode, which was slightly higher than that (1.65U/mL/day) obtained in the STR with preformed cell pellets and about 1.75-fold of that (1.02U/mL/day) for the free-cell fermentation in STR inoculated with spores. This work demonstrated that the RFBB could provide an efficient process for β-glucosidase production from low-cost wheat bran and glycerol.

Production of 1,3-propanediol by Clostridium beijerinckii DSM 791 from crude glycerol and corn steep liquor: Process optimization and metabolic engineering

1,3-Propanediol (1,3-PDO) production from crude glycerol, a byproduct from biodiesel manufacturing, by Clostridium beijerinckii DSM 791 was studied with corn steep liquor as an inexpensive nitrogen source replacing yeast extract in the fermentation medium. A stable, long-term 1,3-PDO production from glycerol was demonstrated with cells immobilized in a fibrous bed bioreactor operated in a repeated batch mode, which partially circumvented the 1,3-PDO inhibition problem. The strain was then engineered to overexpress Escherichia coli gldA encoding glycerol dehydrogenase (GDH) and dhaKLM encoding dihydroxyacetone kinase (DHAK), which increased 1,3-PDO productivity by 26.8–37.5% compared to the wild type, because of greatly increased specific growth rate (0.25–0.40h−1 vs. 0.13–0.20h−1 for the wild type). The engineered strain gave a high 1,3-PDO titer (26.1g/L), yield (0.55g/g) and productivity (0.99g/L·h) in fed-batch fermentation. Overexpressing GDH and DHAK was thus effective in increasing 1,3-PDO production from glycerol.

Expansion of human mesenchymal stem cells in fibrous bed bioreactor

Expansion of human mesenchymal stem cells (hMSCs) in bioreactor while preserving their innate properties is important in translation of hMSC-based therapy to clinical applications. The present study investigates the feasibility of hMSC expansion in a 2.5L CelliGen® 310 Bioreactor packed with Fibra-Cel® disks. After 9 days of expansion, a 9.2-fold increase in cell number with the population doubling (PD) time of 2.8 days (67.2h) was achieved and that the specific glucose consumption and lactate production were measured to be 12.48pmol/cell/day and 20.95pmol/cell/day, respectively. hMSCs harvested from the bioreactor maintained their properties based on the analysis of phenotypic surface markers, colony forming unit-fibroblasts (CFU-F) number, and multilineage differentiation ability. The results demonstrate the feasibility and the potential of the fibrous bed bioreactor for large scale hMSC expansion.

High production of butyric acid by Clostridium tyrobutyricum mutant

The objective of this study was to improve the production of butyric acid by process optimization using the metabolically engineered mutant of Clostridium tyrobutyricum (PAK-Em). First, the free-cell fermentation at pH 6.0 produced butyric acid with concentration of 38.44 g/L and yield of 0.42 g/g. Second, the immobilizedcell fermentations using fibrous-bed bioreactor (FBB) were run at pHs of 5.0, 5.5, 6.0, 6.5 and 7.0 to optimize fermentation process and improve the butyric acid production. It was found that the highest titer of butyric acid, 63.02 g/L, was achieved at pH 6.5. Finally, the metabolic flux balance analysis was performed to investigate the carbon rebalance in C. tyrobutyricum. The results show both gene manipulation and fermentation pH change redistribute carbon between biomass, acetic acid and butyric acid. This study demonstrated that high butyric acid production could be obtained by integrating metabolic engineering and fermentation process optimization. Open image in new window

Butyric acid production from recycled waste paper by immobilized Clostridium tyrobutyricum in a fibrous‐bed bioreactor

AbstractBACKGROUNDRecycled waste paper (RWP) has been considered as a low‐cost substrate for cost‐effective production of various biofuels and chemicals. The aim of this work was to utilize the fermentable sugar obtained from RWP, and thus to develop an alternative source of butyric acid to replace the petroleum‐based product.RESULTSRWP hydrolysate was successfully utilized for enhanced production of butyric acid in a fibrous‐bed bioreactor (FBB) with a high titer (53.42 g L−1) and high selectivity (43.43 g g−1 butyrate/acetate ratio) in fed‐batch fermentation. A repeated‐batch fermentation process was carried out in which highly consistent butyrate yield (0.41 g g−1 utilized sugar), titer (20.96 g L−1), and productivity (1.62 g L−1 h−1) were achieved, demonstrating a stable and reliable long‐term performance. Glucose and xylose, two major fermentable sugars in RWP hydrolysate, could be simultaneously consumed for butyrate production, providing high substrate utilization efficiency. Moreover, the presence of a metabolic pathway shift from acetate‐forming pathway towards butyrate synthesis was confirmed.CONCLUSIONHigh concentration fermentable sugar solutions were obtained from recycled waste paper, and could be utilized successfully for butyric acid production. The results also showed that the process developed was cost‐effective and benign for environmental protection. © 2015 Society of Chemical Industry

Adaptation and transcriptome analysis of Aureobasidium pullulans in corncob hydrolysate for increased inhibitor tolerance to malic acid production.

Malic acid is a dicarboxylic acid widely used in the food industry, and is also a potential C4 platform chemical. Corncob is a low-cost renewable feedstock from agricultural industry. However, side-reaction products (furfural, 5-hydroxymethylfurfural (HMF), formic acid, and acetic acid) that severely hinder fermentation are formed during corncob pretreatment. The process for producing malic acid from a hydrolysate of corncob was investigated with a polymalic acid (PMA)-producing Aureobasidium pullulans strain. Under the optimal hydrolysate sugar concentration 110 g/L, A. pullulans was further adapted in an aerobic fibrous bed bioreactor (AFBB) by gradually increasing the sugar concentration of hydrolysate. After nine batches of fermentation, the production and productivity of malic acid reached 38.6 g/L and 0.4 g/L h, respectively, which was higher than that in the first batch (27.6 g/L and 0.29 g/L h, respectively). The adapted strain could grow under the stress of 0.5 g/L furfural, 3 g/L HMF, 2g/L acetic acid, and 0.5 g/L formic acid, whereas the wild type did not. Transcriptome analysis revealed that the differentially expressed genes were related to carbohydrate transport and metabolism, lipid transport and metabolism, signal transduction mechanism, redox metabolism, and energy production and conversion under 0.5 g/L furfural and 3 g/L HMF stress conditions. In total, 42 genes in the adapted strain were upregulated by 15-fold or more, and qRT-PCR also confirmed that the expression levels of key genes (i.e. SIR, GSS, CYS, and GSR) involved in sulfur assimilation pathway were upregulated by over 10-fold in adapted strain for cellular protection against oxidative stress.

D-lactic acid production by Sporolactobacillus inulinus Y2-8 immobilized in fibrous bed bioreactor using corn flour hydrolyzate.

In this study, a fibrous bed bioreactor (FBB) was used for D-lactic acid (D-LA) production by Sporolactobacillus inulinus Y2-8. Corn flour hydrolyzed with α-amylase and saccharifying enzyme was used as a cost-efficient and nutrient-rich substrate for D-LA production. A maximal starch conversion rate of 93.78% was obtained. The optimum pH for D-LA production was determined to be 6.5. Ammonia water was determined to be an ideal neutralizing agent, which improved the D-LA production and purification processes. Batch fermentation and fedbatch fermentation, with both free cells and immobilized cells, were compared to highlight the advantages of FBB fermentation. In batch mode, the D-LA production rate of FBB fermentation was 1.62 g/l/h, which was 37.29% higher than that of free-cell fermentation, and the D-LA optical purities of the two fermentation methods were above 99.00%. In fed-batch mode, the maximum D-LA concentration attained by FBB fermentation was 218.8 g/l, which was 37.67% higher than that of free-cell fermentation. Repeated-batch fermentation was performed to determine the long-term performance of the FBB system, and the data indicated that the average D-LA production rate was 1.62 g/l/h and the average yield was 0.98 g/g. Thus, hydrolyzed corn flour fermented by S. inulinus Y2-8 in a FBB may be used for improving D-LA fermentation by using ammonia water as the neutralizing agent.

Long-Term Production of Fuel Ethanol by Immobilized Yeast in Repeated-Batch Simultaneous Saccharification and Fermentation of Cassava

In this study, a fibrous matrix with chemical modification was used as a support material for the immobilization of Saccharomyces cerevisiae 1308. Cassava hydrolysate without filteration was used for ethanol fermentation. An average utilization rate of starch of 83.5% was obtained by the immobilized cells during the first seven repeated simultaneous saccharification and fermentation. The utilization rate of starch of the immobilized cells was 2.1% higher than that of free cells with an inoculation quantity of 15% (v/v) under the same fermentation conditions. In addition, the effects of enzyme feeding strategies on immobilized yeast fermentation were investigated. The highest utilization rate of starch of 85.9% was obtained at 35 °C with the optimal enzyme feeding strategy. Fermentation performed successfully for about 6 months with 87 batches suggests that ethanol production from cassava using immobilized yeast in a fibrous-bed bioreactor is feasible and may meet the demands of industrial production.

Engineering Clostridium acetobutylicum with a histidine kinase knockout for enhanced n-butanol tolerance and production.

Clostridium acetobutylicum JB200, a mutant strain of C. acetobutylicum ATCC 55025 obtained through strain evolution in a fibrous bed bioreactor, had high butanol tolerance and produced up to ~21 g/L butanol from glucose in batch fermentation, an improvement of ~67 % over the parental strain (~12.6 g/L). Comparative genomic analysis revealed a single-base deletion in the cac3319 gene leading to C-terminal truncation in its encoding histidine kinase (HK) in JB200. To study the effects of cac3319 mutation on cell growth and fermentation, the cac3319 gene in ATCC 55025 was disrupted using the ClosTron group II intron-based gene inactivation system. Compared to ATCC 55025, the cac3319 HK knockout mutant, HKKO, produced 44.4 % more butanol (18.2 ± 1.3 vs. 12.6 ± 0.2 g/L) with a 90 % higher productivity (0.38 ± 0.03 vs. 0.20 ± 0.02 g/L h) due to increased butanol tolerance, confirming, for the first time, that cac3319 plays an important role in regulating solvent production and tolerance in C. acetobutylicum. This work also provides a novel metabolic engineering strategy for generating high-butanol-tolerant and high-butanol-producing strains for industrial applications.

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 80g/L, 0.051/h, and 6.0–6.5, respectively. A SA concentration of 55.3±0.8g/L, productivity of 2.77±0.04g/L/h, and yield of 0.8±0.02g/g were obtained, and the continuous fermentation exhibited long-term stability for as long as 18 days (440h) 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 60g/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.

Enhancement of n-butanol production by in situ butanol removal using permeating-heating-gas stripping in acetone-butanol-ethanol fermentation.

Butanol recovery from acetone-butanol-ethanol (ABE) fed-batch fermentation using permeating-heating-gas was determined in this study. Fermentation was performed with Clostridium acetobutylicum B3 in a fibrous bed bioreactor and permeating-heating-gas stripping was used to eliminate substrate and product inhibition, which normally restrict ABE production and sugar utilization to below 20 g/L and 60 g/L, respectively. In batch fermentation (without permeating-heating-gas stripping), C. acetobutylicum B3 utilized 60 g/L glucose and produced 19.9 g/L ABE and 12 g/L butanol, while in the integrated process 290 g/L glucose was utilized and 106.27 g/L ABE and 66.09 g/L butanol were produced. The intermittent gas stripping process generated a highly concentrated condensate containing approximately 15% (w/v) butanol, 4% (w/v) acetone, a small amount of ethanol (<1%), and almost no acids, resulting in a highly concentrated butanol solution [∼ 70% (w/v)] after phase separation. Butanol removal by permeating-heating-gas stripping has potential for commercial ABE production.

Stable high-titer n-butanol production from sucrose and sugarcane juice by Clostridium acetobutylicum JB200 in repeated batch fermentations.

The production of n-butanol, a widely used industrial chemical and promising transportation fuel, from abundant, low-cost substrates, such as sugarcane juice, in acetone-butanol-ethanol (ABE) fermentation was studied with Clostridium acetobutylicum JB200, a mutant with high butanol tolerance and capable of producing high-titer (>20 g/L) n-butanol from glucose. Although JB200 is a favorable host for industrial bio-butanol production, its fermentation performance with sucrose and sugarcane juice as substrates has not been well studied. In this study, the long-term n-butanol production from sucrose by JB200 was evaluated with cells immobilized in a fibrous-bed bioreactor (FBB), showing stable performance with high titer (16-20 g/L), yield (∼ 0.21 g/g sucrose) and productivity (∼ 0.32 g/Lh) for 16 consecutive batches over 800 h. Sugarcane thick juice as low-cost substrate was then tested in 3 consecutive batches, which gave similar n-butanol production, demonstrating that JB200 is a robust and promising strain for industrial ABE fermentation.

Long-term production of butyric acid through immobilization ofClostridium tyrobutyricumin a moving fibrous-bed bioreactor (MFBB)

BACKGROUND Butyric acid is an important chemical with wide industrial applications, but large-scale bio-production still requires an efficient bioprocess. For this purpose, one novel moving fibrous-bed bioreactor (MFBB) was developed in this work for economical production of butyric acid. RESULTS The cells of Clostridium tyrobutyricum were immobilized on a fibrous matrix packed in porous levitated sphere carriers. Each carrier was packed with a fiber quantity 2 cm × 6 cm (0.42 g) and was rotated at 100 rpm in a stirred-tank fermentor having pH and temperature controls. Batch and repeated-batch fermentations were carried out to produce butyric acid with a high yield (0.48 ± 0.015 g g-1). Subsequent fed-batch fermentation using the MFBB resulted in a high concentration of butyric acid (65.4 ± 1.4 g L-1). This novel MFBB could be employed to produce high concentration butyric acid (64.5 ± 1.2 g L-1) by a simple repeated-batch addition of a high concentration of glucose (>100 g L-1). CONCLUSIONS The MFBB system developed could practically and economically produce butyric acid from glucose for long-term operation. © 2013 Society of Chemical Industry