Publications by authors named "Shang-Tian Yang"

144 Publications

Comparative transcriptome analysis of Clostridium tyrobutyricum expressing a heterologous uptake hydrogenase.

Sci Total Environ 2020 Dec 16;749:142022. Epub 2020 Sep 16.

State Key Laboratory of Urban Water Resource and Environment, School of Environment, Harbin Institute of Technology, Harbin 150090, China. Electronic address:

Clostridium tyrobutyricum is a promising microbial cell factory to produce biofuels. In this study, an uptake hydrogenase (hyd2293) from Ethanoligenens harbinense was overexpressed in C. tyrobutyricum and significantly affected the redox reactions and metabolic profiles. Compared to the parental strain (Ct-WT), the mutant strain Ct-Hyd2293 produced ~34% less butyrate, ~148% more acetate, and ~11% less hydrogen, accompanied by the emerging genesis of butanol. Comparative transcriptome analysis revealed that 666 genes were significantly differentially expressed after the overexpression of hyd2293, including 82 up-regulated genes and 584 down-regulated genes. The up-regulated genes were mainly involved in carbohydrate and energy metabolisms while the down-regulated genes were distributed in nearly all pathways. Genes involved in glucose transportation, glycolysis, different fermentation pathways and hydrogen metabolism were studied and the gene expression changes showed the mechanism of the metabolic flux redistribution in Ct-Hyd2293. The overexpression of uptake hydrogenase redirected electrons from hydrogen and butyrate to butanol. The key enzymes participating in the energy conservation and sporulation were also identified and their transcription levels were generally reduced. This study demonstrated the transcriptomic responses of C. tyrobutyricum to the expression of a heterologous uptake hydrogenase, which provided a better understanding of the metabolic characteristics of C. tyrobutyricum and demonstrated the potential role of redox manipulation in metabolic engineering for biofuel productions.
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http://dx.doi.org/10.1016/j.scitotenv.2020.142022DOI Listing
December 2020

Regulator RcsB Controls Prodigiosin Synthesis and Various Cellular Processes in Serratia marcescens JNB5-1.

Appl Environ Microbiol 2021 01 4;87(2). Epub 2021 Jan 4.

Key Laboratory of Industrial Biotechnology of the Ministry of Education, Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, China

Prodigiosin (PG), a red linear tripyrrole pigment normally secreted by , has received attention for its reported immunosuppressive, antimicrobial, and anticancer properties. Although several genes have been shown to be important for prodigiosin synthesis, information on the regulatory mechanisms behind this cellular process remains limited. In this work, we identified that the transcriptional regulator RcsB encoding gene () negatively controlled prodigiosin biosynthesis in Disruption of conferred a remarkably increased production of prodigiosin. This phenotype corresponded to negative control of transcription of the prodigiosin-associated operon by RcsB, probably by binding to the promoter region of the prodigiosin synthesis positive regulator FlhDC. Moreover, using transcriptomics and further experiments, we revealed that RcsB also controlled some other important cellular processes, including swimming and swarming motilities, capsular polysaccharide production, biofilm formation, and acid resistance (AR), in Collectively, this work proposes that RcsB is a prodigiosin synthesis repressor in and provides insight into the regulatory mechanism of RcsB in cell motility, capsular polysaccharide production, and acid resistance in RcsB is a two-component response regulator in the Rcs phosphorelay system, and it plays versatile regulatory functions in However, information on the function of the RcsB protein in bacteria, especially in , remains limited. In this work, we illustrated experimentally that the RcsB protein was involved in diverse cellular processes in , including prodigiosin synthesis, cell motility, capsular polysaccharide production, biofilm formation, and acid resistance. Additionally, the regulatory mechanism of the RcsB protein in these cellular processes was investigated. In conclusion, this work indicated that RcsB could be a regulator for prodigiosin synthesis and provides insight into the function of the RcsB protein in .
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http://dx.doi.org/10.1128/AEM.02052-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7783331PMC
January 2021

Sustainable production and biomedical application of polymalic acid from renewable biomass and food processing wastes.

Crit Rev Biotechnol 2021 Mar 5;41(2):216-228. Epub 2020 Nov 5.

William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, USA.

Polymalic acid (PMA), a homopolymer of L-malic acid (MA) generated from a yeast-like fungus , has unique properties and many applications in food, biomedical, and environmental fields. Acid hydrolysis of PMA, releasing the monomer MA, has become a novel process for the production of bio-based MA, which currently is produced by chemical synthesis using petroleum-derived feedstocks. Recently, current researches attempted to develop economically competitive process for PMA and MA production from renewable biomass feedstocks. Compared to lignocellulosic biomass, PMA and MA production from low-value food processing wastes or by-products, generated from corn, sugarcane, or soybean refinery industries, showed more economical and sustainable for developing a MA derivatives platform from biomass biorefinery to chemical conversion. In the review, we compared the process feasibility for PMA fermentation with lignocellulosic biomass and food process wastes. Some useful strategies for metabolic engineering are summarized. Its changeable applicability and future prospects in food and biomedical fields are also discussed.
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http://dx.doi.org/10.1080/07388551.2020.1844632DOI Listing
March 2021

Effects of benzyl viologen on increasing NADH availability, acetate assimilation, and butyric acid production by Clostridium tyrobutyricum.

Biotechnol Bioeng 2021 Feb 4;118(2):770-783. Epub 2020 Nov 4.

William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, Ohio, USA.

Clostridium tyrobutyricum produces butyric and acetic acids from glucose. The butyric acid yield and selectivity in the fermentation depend on NADH available for acetate reassimilation to butyric acid. In this study, benzyl viologen (BV), an artificial electron carrier that inhibits hydrogen production, was used to increase NADH availability and butyric acid production while eliminating acetic acid accumulation by facilitating its reassimilation. To better understand the mechanism of and find the optimum condition for BV effect on enhancing acetate assimilation and butyric acid production, BV at various concentrations and addition times during the fermentation were studied. Compared with the control without BV, the addition of 1 μM BV increased butyric acid production from glucose by ∼50% in yield and ∼29% in productivity while acetate production was completely inhibited. Furthermore, BV also increased the coutilization of glucose and exogenous acetate for butyric acid production. At a concentration ratio of acetate (g/L) to BV (mM) of 4, both acetate assimilation and butyrate biosynthesis increased with increasing the concentrations of BV (0-6.25 μM) and exogenous acetate (0-25 g/L). In a fed-batch fermentation with glucose and ∼15 g/L acetate and 3.75 μM BV, butyrate production reached 55.9 g/L with productivity 0.93 g/L/h, yield 0.48 g/g, and 97.4% purity, which would facilitate product purification and reduce production cost. Manipulating metabolic flux and redox balance via BV and acetate addition provided a simple to implement metabolic process engineering approach for butyric acid production from sugars and biomass hydrolysates.
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http://dx.doi.org/10.1002/bit.27602DOI Listing
February 2021

Recent advances in n-butanol and butyrate production using engineered Clostridium tyrobutyricum.

World J Microbiol Biotechnol 2020 Aug 14;36(9):138. Epub 2020 Aug 14.

Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, 43210, USA.

Acidogenic clostridia naturally producing acetic and butyric acids has attracted high interest as a novel host for butyrate and n-butanol production. Among them, Clostridium tyrobutyricum is a hyper butyrate-producing bacterium, which re-assimilates acetate for butyrate biosynthesis by butyryl-CoA/acetate CoA transferase (CoAT), rather than the phosphotransbutyrylase-butyrate kinase (PTB-BK) pathway widely found in clostridia and other microbial species. To date, C. tyrobutyricum has been engineered to overexpress a heterologous alcohol/aldehyde dehydrogenase, which converts butyryl-CoA to n-butanol. Compared to conventional solventogenic clostridia, which produce acetone, ethanol, and butanol in a biphasic fermentation process, the engineered C. tyrobutyricum with a high metabolic flux toward butyryl-CoA produced n-butanol at a high yield of > 0.30 g/g and titer of > 20 g/L in glucose fermentation. With no acetone production and a high C4/C2 ratio, butanol was the only major fermentation product by the recombinant C. tyrobutyricum, allowing simplified downstream processing for product purification. In this review, novel metabolic engineering strategies to improve n-butanol and butyrate production by C. tyrobutyricum from various substrates, including glucose, xylose, galactose, sucrose, and cellulosic hydrolysates containing the mixture of glucose and xylose, are discussed. Compared to other recombinant hosts such as Clostridium acetobutylicum and Escherichia coli, the engineered C. tyrobutyricum strains with higher butyrate and butanol titers, yields and productivities are the most promising hosts for potential industrial applications.
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http://dx.doi.org/10.1007/s11274-020-02914-2DOI Listing
August 2020

Acetone, butanol, and ethanol production from puerariae slag hydrolysate through ultrasound-assisted dilute acid by Clostridium beijerinckii YBS3.

Bioresour Technol 2020 Nov 24;316:123899. Epub 2020 Jul 24.

College of Bioscience and Bioengineering, Jiangxi Agricultural University, Jiangxi Engineering Laboratory for the Development and Utilization of Agricultural Microbial Resources, Jiangxi Key Laboratory for Conservation and Utilization of Fungal Resources, Nanchang, Jiangxi 330045, China. Electronic address:

In this study, puerariae slag (PS) was evaluated as a renewable raw material for acetone-butanol-ethanol (ABE) fermentation. To accelerate the hydrolysis of PS, the method of ultrasound-assisted dilute acid hydrolysis (UAAH) was used. With this effort, 0.69 g reducing sugar was obtained from 1 g raw material under the optimal pretreatment condition. Subsequently, the butanol and total solvent production of 8.79 ± 0.16 g/L and 12.32 ± 0.26 g/L were obtained from the non-detoxified diluted hydrolysate, and the yield and productivity of butanol were 0.19 g/g and 0.12 g/L/h, respectively. Additionally, the changes in the structure of PS after different pretreatment methods were observed using SEM and FT-IR. UAAH resulted in more severe and distinct damage to the dense structure of PS. This study suggests that the UAAH is an attainable but effective pretreatment method, thereby is a promising technique for lignocellulose hydrolysis and improve butanol production.
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http://dx.doi.org/10.1016/j.biortech.2020.123899DOI Listing
November 2020

Improved Prodigiosin Production by Relieving CpxR Temperature-Sensitive Inhibition.

Front Bioeng Biotechnol 2020 3;8:344. Epub 2020 Jun 3.

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.

Prodigiosin (PG) is a typical secondary metabolite mainly produced by . CpxR protein is an OmpR family transcriptional regulator in Gram-negative bacteria. Firstly, it was found that insertion mutation of in JNB 5-1 by a transposon Tn5G increased the production of PG. Results from the electrophoretic mobility shift assay (EMSA) indicated that CpxR could bind to the promoter of the gene cluster and repress the transcription levels of genes involved in PG biosynthesis in JNB 5-1. In the Δ mutant strain, the transcription levels of the gene cluster and the genes involved in the pathways of PG precursors, such as proline, pyruvate, serine, methionine, and S-adenosyl methionine, were significantly increased, hence promoting the production of PG. Subsequently, a fusion segment composed of the genes , and , responsible for proline, serine, and methionine, was inserted into the gene in JNB 5-1. On fermentation by the resultant engineered , the highest PG titer reached 5.83 g/L and increased by 41.9%, relative to the parental strain. In this study, we revealed the role of CpxR in PG biosynthesis and provided an alternative strategy for the engineering of to enhance PG production.
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http://dx.doi.org/10.3389/fbioe.2020.00344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7283389PMC
June 2020

A novel β-galactosidase from Klebsiella oxytoca ZJUH1705 for efficient production of galacto-oligosaccharides from lactose.

Appl Microbiol Biotechnol 2020 Jul 21;104(14):6161-6172. Epub 2020 May 21.

William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 151 West Woodruff Avenue, Columbus, OH, 43210, USA.

Galacto-oligosaccharides (GOS), which can be produced by enzymatic transgalactosylation of lactose with β-galactosidases, have attracted much attention in recent years because of their prebiotic functions and wide uses in infant formula, infant foods, livestock feed, and pet food industries. In this study, a novel β-galactosidase-producing Klebsiella oxytoca ZJUH1705, identified by its 16S rRNA sequence (GenBank accession no. MH981243), was isolated. Two β-galactosidase genes, bga 1 encoding a 2058-bp fragment (GenBank accession no. MH986613) and bga 2 encoding a 3108-bp fragment (GenBank accession no. MN182756), were cloned from K. oxytoca ZJUH1705 and expressed in E. coli. The purified β-gal 1 and β-gal 2 had the specific activity of 217.56 U mg and 57.9 U mg, respectively, at the optimal pH of 7.0. The reaction kinetic parameters K, V, and K with oNPG as the substrate at 40 °C were 5.62 mM, 167.1 μmol mg min, and 218.1 s, respectively, for β-gal 1 and 3.91 mM, 14.6 μmol mg min, and 28.9 s, respectively, for β-gal 2. Although β-gal 1 had a higher enzyme activity for lactose hydrolysis, only β-gal 2 had a high transgalactosylation capacity. Using β-gal 2 with the addition ratio of ~ 2.5 U g lactose, a high GOS yield of 45.5 ± 2.3% (w/w) was obtained from lactose (40% w/w or 480 g L) in a phosphate buffer (100 mM, pH 7.0) at 40 °C in 48 h. Thus, the β-gal 2 from K. oxytoca ZJUH1705 would be a promising biocatalyst for GOS production from lactose.Key Points• A novel bacterial β-galactosidase producer was isolated and identified.• β-Galactosidases were cloned and expressed in heterologous strain and characterized.• Both enzymes have hydrolytic activity but only one have transglycosilation activity.• The developed process with β-gal 2 could provide an alternative for GOS production.
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http://dx.doi.org/10.1007/s00253-020-10679-9DOI Listing
July 2020

Development of an in vivo fluorescence based gene expression reporter system for Clostridium tyrobutyricum.

J Biotechnol 2019 Nov 28;305:18-22. Epub 2019 Aug 28.

William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 151 Woodruff Ave., Columbus, OH 43210, USA. Electronic address:

C. tyrobutyricum, an acidogenic Clostridium, has aroused increasing interest due to its potential to produce biofuel efficiently. However, construction of recombinant C. tyrobutyricum for enhanced biofuel production has been impeded by the limited genetic engineering tools. In this study, a flavin mononucleotide (FMN)-dependent fluorescent protein Bs2-based gene expression reporter system was developed to monitor transformation and explore in vivo strength and regulation of various promoters in C. tyrobutyricum and C. acetobutylicum. Unlike green fluorescent protein (GFP) and its variants, Bs2 can emit green light without oxygen, which makes it extremely suitable for promoter screening and transformation confirmation in organisms grown anaerobically. The expression levels of bs2 under thiolase promoters from C. tyrobutyricum and C. acetobutylicum were measured and compared based on fluorescence intensities. The capacities of the two promoters in driving secondary alcohol dehydrogenase (adh) gene for isopropanol production in C. tyrobutyricum were distinguished, confirming that this reporter system is a convenient, effective and reliable tool for promoter strength assay and real time monitoring in C. tyrobutyricum, while demonstrating the feasibility of producing isopropanol in C. tyrobutyricum for the first time.
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http://dx.doi.org/10.1016/j.jbiotec.2019.08.019DOI Listing
November 2019

Production of n-butanol from cassava bagasse hydrolysate by engineered Clostridium tyrobutyricum overexpressing adhE2: Kinetics and cost analysis.

Bioresour Technol 2019 Nov 8;292:121969. Epub 2019 Aug 8.

William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 151 West Woodruff Avenue, Columbus, OH 43210, USA. Electronic address:

The production of biofuels such as butanol is usually limited by the availability of inexpensive raw materials and high substrate cost. Using food crops as feedstock in the biorefinery industry has been criticized for its competition with food supply, causing food shortage and increased food prices. In this study, cassava bagasse as an abundant, renewable, and inexpensive byproduct from the cassava starch industry was used for n-butanol production. Cassava bagasse hydrolysate containing mainly glucose was obtained after treatments with dilute acid and enzymes (glucoamylases and cellulases) and then supplemented with corn steep liquor for use as substrate in repeated-batch fermentation with engineered Clostridium tyrobutyricum CtΔack-adhE2 in a fibrous-bed bioreactor. Stable butanol production with high titer (>15.0 g/L), yield (>0.30 g/g), and productivity (~0.3 g/L∙h) was achieved, demonstrating the feasibility of an economically competitive process for n-butanol production from cassava bagasse for industrial application.
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http://dx.doi.org/10.1016/j.biortech.2019.121969DOI Listing
November 2019

n-Butanol production from lignocellulosic biomass hydrolysates without detoxification by Clostridium tyrobutyricum Δack-adhE2 in a fibrous-bed bioreactor.

Bioresour Technol 2019 Oct 3;289:121749. Epub 2019 Jul 3.

William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 151 West Woodruff Avenue, Columbus, OH 43210, USA. Electronic address:

Acetone-butanol-ethanol fermentation suffers from high substrate cost and low butanol titer and yield. In this study, engineered Clostridium tyrobutyricum CtΔack-adhE2 immobilized in a fibrous-bed bioreactor was used for butanol production from glucose and xylose present in the hydrolysates of low-cost lignocellulosic biomass including corn fiber, cotton stalk, soybean hull, and sugarcane bagasse. The biomass hydrolysates obtained after acid pretreatment and enzymatic hydrolysis were supplemented with corn steep liquor and used in repeated-batch fermentations. Butanol production with high titer (∼15 g/L), yield (∼0.3 g/g), and productivity (∼0.3 g/L∙h) was obtained from cotton stalk, soybean hull, and sugarcane bagasse hydrolysates, while corn fiber hydrolysate with higher inhibitor contents gave somewhat inferior results. The fermentation process was stable for long-term operation without any noticeable degeneration, demonstrating its potential for industrial application. A techno-economic analysis showed that n-butanol could be produced from lignocellulosic biomass using this novel fermentation process at ∼$2.5/gal for biofuel application.
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http://dx.doi.org/10.1016/j.biortech.2019.121749DOI Listing
October 2019

Engineering Clostridium for improved solvent production: recent progress and perspective.

Appl Microbiol Biotechnol 2019 Jul 29;103(14):5549-5566. Epub 2019 May 29.

Department of Chemical and Biomolecular Engineering, The Ohio State University, 151 West Woodruff Avenue, Columbus, OH, 43210, USA.

Clostridia are Gram-positive, spore-forming, obligate anaerobic bacteria that can produce solvents such as acetone, ethanol, and butanol, which can be used as biofuels or building block chemicals. Many successful attempts have been made to improve solvent yield and titer from sugars through metabolic engineering of solventogenic and acidogenic clostridia. More recently, cellulolytic and acetogenic clostridia have also attracted high interests for their ability to utilize low-cost renewable substrates such as cellulose and syngas. Process engineering such as in situ butanol recovery and consolidated bioprocessing (CBP) has been developed for improved solvent titer and productivity. This review focuses on metabolic and process engineering strategies for solvent production from sugars, lignocellulosic biomass, and syngas by various clostridia, including conventional solventogenic Clostridium acetobutylicum, engineered acidogens such as C. tyrobutyricum and C. cellulovorans, and carboxydotrophic acetogens such as C. carboxidivorans and C. ljungdahlii.
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http://dx.doi.org/10.1007/s00253-019-09916-7DOI Listing
July 2019

Development of a shuttle plasmid without host restriction sites for efficient transformation and heterologous gene expression in Clostridium cellulovorans.

Appl Microbiol Biotechnol 2019 Jul 21;103(13):5391-5400. Epub 2019 May 21.

William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 151 W. Woodruff Avenue, Columbus, OH, 43210, USA.

Clostridium cellulovorans capable of producing large amounts of acetate and butyrate from cellulose is a promising candidate for biofuels and biochemicals production from lignocellulosic biomass. However, the restriction modification (RM) systems of C. cellulovorans hindered the application of existing shuttle plasmids for metabolic engineering of this organism. To overcome the hurdle of plasmid digestion by host, a new shuttle plasmid (pYL001) was developed to remove all restriction sites of two major RM systems of C. cellulovorans, Cce743I and Cce743II. The pYL001 plasmid remained intact after challenge by C. cellulovorans cell extract. Post-electroporation treatments and culturing conditions were also modified to improve cell growth and colony formation on agar plates. With the improvements, the pYL001 plasmid, without in vivo methylation, was readily transformed into C. cellulovorans with colonies of recombinant cells formed on agar plates within 24 h. Three pYL001-derived recombinant plasmids free of Cce743I/Cce743II restriction sites, after synonymous mutation of the heterologous genes, were constructed and transformed into C. cellulovorans. Functional expression of these genes was confirmed with butanol and ethanol production from glucose in batch fermentations by the transformants. The pYL001 plasmid and improved transformation method can facilitate further metabolic engineering of C. cellulovorans for cellulosic butanol production.
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http://dx.doi.org/10.1007/s00253-019-09882-0DOI Listing
July 2019

Metabolic engineering of Clostridium carboxidivorans for enhanced ethanol and butanol production from syngas and glucose.

Bioresour Technol 2019 Jul 30;284:415-423. Epub 2019 Mar 30.

William G. Lowrie Department of Chemical & Biomolecular Engineering, The Ohio State University, 151 West Woodruff Ave, Columbus, OH 43210, USA. Electronic address:

Clostridium carboxidivorans can convert CO, CO and H to ethanol and n-butanol; however, its industrial application is limited by the lack of tools for metabolic pathway engineering. In this study, C. carboxidivorans was successfully engineered to overexpress aor, adhE2, and fnr together with adhE2 or aor. In glucose fermentation, all engineered strains showed higher alcohol yields compared to the wild-type. Strains overexpressing aor showed CO re-assimilation during heterotrophic growth. In syngas fermentation, compared to the wild-type, the strain overexpressing adhE2 produced ∼50% more ethanol and the strain overexpressing adhE2 and fnr produced ∼18% more butanol and ∼22% more ethanol. Interestingly, both strains showed obvious acid re-assimilation, with <0.15 g/L total acid remaining at the end of fermentation. Overexpressing fnr with adhE2 enhanced butanol production compared to only adhE2. This is the first report of overexpressing homologous and heterologous genes in C. carboxidivorans for enhancing alcohols production from syngas and glucose.
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http://dx.doi.org/10.1016/j.biortech.2019.03.145DOI Listing
July 2019

n-Butanol and ethanol production from cellulose by Clostridium cellulovorans overexpressing heterologous aldehyde/alcohol dehydrogenases.

Bioresour Technol 2019 Aug 3;285:121316. Epub 2019 Apr 3.

William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 151 W. Woodruff Avenue, Columbus, OH 43210, USA. Electronic address:

With high cellulolytic and acetic/butyric acids production abilities, Clostridium cellulovorans is promising for use to produce cellulosic n-butanol. Here, we introduced three different aldehyde/alcohol dehydrogenases encoded by bdhB, adhE1, and adhE2 from Clostridium acetobutylicum into C. cellulovorans and studied their effects on ethanol and n-butanol production. Compared to AdhE2, AdhE1 was more specific for n-butanol biosynthesis over ethanol. Co-expressing adhE1 with bdhB produced a comparable amount of butanol but significantly less ethanol, leading to a high butanol/ethanol ratio of 7.0 and 5.6 (g/g) in glucose and cellulose fermentation, respectively. Co-expressing adhE1 or adhE2 with bdhB did not increase butanol production because the activity of BdhB was limited by the NADPH availability in C. cellulovorans. Overall, the strain overexpressing adhE2 alone produced the most n-butanol (4.0 g/L, yield: 0.22 ± 0.01 g/g). Based on the insights from this study, further metabolic engineering of C. cellulovorans for cellulosic n-butanol production is suggested.
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http://dx.doi.org/10.1016/j.biortech.2019.121316DOI Listing
August 2019

An engineered mouse embryonic stem cell model with survivin as a molecular marker and EGFP as the reporter for high throughput screening of embryotoxic chemicals in vitro.

Biotechnol Bioeng 2019 07 12;116(7):1656-1668. Epub 2019 Apr 12.

William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 151 West Woodruff Avenue, Columbus, Ohio.

Embryonic stem cell test (EST) is the only generally accepted in vitro method for assessing embryotoxicity without animal sacrifice. However, the implementation and application of EST for regulatory embryotoxicity screening are impeded by its technical complexity, long testing period, and limited endpoint data. In this study, a high throughput embryotoxicity screening based on mouse embryonic stem cells (mESCs) expressing enhanced green fluorescent protein (EGFP) driven by a human survivin promoter and a human cytomegalovirus promoter, respectively, was developed. These EGFP expressing mESCs were cultured in three-dimensional (3D) fibrous scaffolds in microbioreactors on a multiwell plate with EGFP fluorescence signals as cell responses to chemicals monitored noninvasively in a high throughput manner. Nine chemicals with known developmental toxicity were used to validate the survivin-based embryotoxicity assay, which showed that strongly embryotoxic compounds such as 5-fluorouracil, retinoic acid, and methotrexate downregulated survivin expression by more than 50% in 3 days, while weakly embryotoxic compounds such as boric acid, methoxyacetic acid, and tetracyclin showed modest downregulation effect and nonembryotoxic saccharin, penicillin G, and acrylamide had negligible downregulation effect on survivin expression, confirming that survivin can be used as a molecular endpoint for high throughput screening of embryotoxicants. The potential developmental toxicity of three Chinese herbal medicines were also evaluated using this assay, demonstrating its application in in vitro developmental toxicity test for drug safety assessment.
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http://dx.doi.org/10.1002/bit.26977DOI Listing
July 2019

Biosynthesis of polymalic acid in fermentation: advances and prospects for industrial application.

Crit Rev Biotechnol 2019 May 10;39(3):408-421. Epub 2019 Feb 10.

b William G. Lowrie Department of Chemical and Biomolecular Engineering , The Ohio State University , Columbus , OH , USA.

Some microorganisms naturally produce β-poly(l-malic acid) (PMA), which has excellent water solubility, biodegradability, and biocompatibility properties. PMA has broad prospective applications as novel biopolymeric materials and carriers in the drug, food, and biomedical fields. Malic acid, a four-carbon dicarboxylic acid, is widely used in foods and pharmaceuticals, as a platform chemical. Currently, malic acid produced through chemical synthesis and is available as a racemic mixture of l- and d-forms. The d-form malic acid exhibits safety concerns for human consumption. There is extensive interest to develop economical bioprocesses for l-malic acid and PMA production from renewable biomass feedstocks. In this review, we focus on PMA biosynthesis by Aureobasidium pullulans, a black yeast with a large genome containing genes encoding many hydrolases capable of degrading various plant materials. The metabolic and regulatory pathways for PMA biosynthesis, metabolic engineering strategies for strain development, process factors affecting fermentation kinetics and PMA production, and downstream processing for PMA recovery and purification are discussed. Prospects of microbial PMA and malic acid production are also considered.
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http://dx.doi.org/10.1080/07388551.2019.1571008DOI Listing
May 2019

A fluorescent 3D cell culture assay for high throughput screening of cancer drugs down-regulating survivin.

J Biotechnol 2019 Jan 22;289:80-87. Epub 2018 Nov 22.

William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 151 West Woodruff Ave., Columbus, Ohio, 43210, USA. Electronic address:

Survivin, a member of inhibitor of apoptosis family, is currently undergoing intensive investigations as a promising cancer marker due to its overexpression in multiple tumor tissues and close relationship with chemotherapy resistance. In this study, a novel 3D survivin promoter assay was developed, using enhanced green fluorescent protein (EGFP) as the reporter to assess survivin promoter activity for cancer drug screening. Breast cancer MCF-7 cells were engineered to express EGFP controlled by a human survivin promoter and a CMV promoter, respectively. These cells were cultured in three-dimensional (3D) polymer-based scaffolds on a 40-microbioreactor platform (40-MBR) with real-time monitoring of EGFP signals. The EGFP production driven by the survivin promoter was strongly correlated with survivin transcriptional level in MCF-7 cells treated with YM155, a small-molecule survivin promoter suppressant. Moreover, the potential inhibition effects of doxorubicin and cisplatin on survivin and their cytotoxicity were also evaluated in this system. This study demonstrated the potential application of the novel 3D survivin promoter-EGFP reporter assay for high-throughput screening of chemicals down-regulating survivin as a molecular target for cancer therapy.
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http://dx.doi.org/10.1016/j.jbiotec.2018.11.018DOI Listing
January 2019

Deciphering mixotrophic Clostridium formicoaceticum metabolism and energy conservation: Genomic analysis and experimental studies.

Genomics 2019 12 20;111(6):1687-1694. Epub 2018 Nov 20.

William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 151 W. Woodruff Avenue, Columbus, OH 43210, USA. Electronic address:

Clostridium formicoaceticum, a Gram-negative mixotrophic homoacetogen, produces acetic acid as the sole metabolic product from various carbon sources, including fructose, glycerol, formate, and CO. Its genome of 4.59-Mbp contains a highly conserved Wood-Ljungdahl pathway gene cluster with the same layout as that in other mixotrophic acetogens, including Clostridium aceticum, Clostridium carboxidivorans, and Clostridium ljungdahlii. For energy conservation, C. formicoaceticum does not have all the genes required for the synthesis of cytochrome or quinone used for generating proton gradient in H-dependent acetogens such as Moorella thermoacetica; instead, it has the Rnf system and a Na-translocating ATPase similar to the one in Acetobacterium woodii. Its growth in both heterotrophic and autotrophic media were dependent on the sodium concentration. C. formicoaceticum has genes encoding acetaldehyde dehydrogenases, alcohol dehydrogenases, and aldehyde oxidoreductases, which could convert acetyl-CoA and acetate to ethanol and butyrate to butanol under excessive reducing equivalent conditions.
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http://dx.doi.org/10.1016/j.ygeno.2018.11.020DOI Listing
December 2019

Butyric acid: Applications and recent advances in its bioproduction.

Biotechnol Adv 2018 12 26;36(8):2101-2117. Epub 2018 Sep 26.

Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH 43210, USA. Electronic address:

Butyric acid is an important C4 organic acid with broad applications. It is currently produced by chemosynthesis from petroleum-based feedstocks. However, the fermentative production of butyric acid from renewable feedstocks has received growing attention because of consumer demand for green products and natural ingredients in foods, pharmaceuticals, animal feed supplements, and cosmetics. In this review, strategies for improving microbial butyric acid production, including strain engineering and novel fermentation process development are discussed and compared regarding product yield, titer, purity and productivity. Future perspectives on strain and process improvements for butyric acid production are also discussed.
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http://dx.doi.org/10.1016/j.biotechadv.2018.09.005DOI Listing
December 2018

Response Surface Methodology for Optimization of Genistein Content in Soy Flour and its Effect on the Antioxidant Activity.

Iran J Pharm Res 2018 ;17(3):1026-1035

William G. Lowrie Department of Chemical and Biomolecular Engineering, Ohio State University, Columbus, Ohio, USA.

Biotransformation of isoflavones glycosides into the aglycone form is essential to attain the maximum bioavailability. The factors affecting deglycosylation of genistin in soy flour using commercial -glucosidase enzyme were evaluated. The presence of genistin in soy flour was confirmed by isolation through chromatographic fractionation and identification by spectral method. Two-levels Plackett-Burman design was applied and effective variables for genistein production were determined. Agitation rate, enzyme concentration, and reaction time, owing to their significant positive effect, and pH, owing to its significant negative effect, were further evaluated using Box-Behnken model. Accordingly the optimal combination of the major reaction affecting factors was "enzyme concentration, 1 IU; agitation speed, 250 rpm; reaction time, 5 h and pH 4. The concentration of genistein can be increased by 9.91 folds (from 0.8 mg/g in the non biotransformed soy flour to 7.93 mg/g in the biotransformed one) using the determined optimal combination of major reaction affecting factors. The antioxidant activity of the non biotransformed and biotransformed soy flour extracts was determined by DPPH method. It was found that biotransformation increase the antioxidant activity by two folds. The concentration causing a 50% reduction of DPPH absorbance (EC) were 10 and 5 mg/mL for the non biotransformed and biotransformed soy flour extracts, respectively.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6094413PMC
January 2018

Production of butyric acid from acid hydrolysate of corn husk in fermentation by : kinetics and process economic analysis.

Biotechnol Biofuels 2018 15;11:164. Epub 2018 Jun 15.

1College of Animal Science, Zhejiang University, No. 866 Yuhangtang Road, Hangzhou, 310058 People's Republic of China.

Background: Butyric acid is an important chemical currently produced from petrochemical feedstocks. Its production from renewable, low-cost biomass in fermentation has attracted large attention in recent years. In this study, the feasibility of corn husk, an abundant agricultural residue, for butyric acid production by using immobilized in a fibrous bed bioreactor (FBB) was evaluated.

Results: Hydrolysis of corn husk (10% solid loading) with 0.4 M HSO at 110 °C for 6 h resulted in a hydrolysate containing ~ 50 g/L total reducing sugars (glucose:xylose = 1.3:1.0). The hydrolysate was used for butyric acid fermentation by in a FBB, which gave 42.6 and 53.0% higher butyric acid production from glucose and xylose, respectively, compared to free-cell fermentations. Fermentation with glucose and xylose mixture (1:1) produced 50.37 ± 0.04 g L butyric acid with a yield of 0.38 ± 0.02 g g and productivity of 0.34 ± 0.03 g L h. Batch fermentation with corn husk hydrolysate produced 21.80 g L butyric acid with a yield of 0.39 g g, comparable to those from glucose. Repeated-batch fermentations consistently produced 20.75 ± 0.65 g L butyric acid with an average yield of 0.39 ± 0.02 g g in three consecutive batches. An extractive fermentation process can be used to produce, separate, and concentrate butyric acid to > 30% (w/v) sodium butyrate at an economically attractive cost for application as an animal feed supplement.

Conclusion: A high concentration of total reducing sugars at ~ 50% (w/w) yield was obtained from corn husk after acid hydrolysis. Stable butyric acid production from corn husk hydrolysate was achieved in repeated-batch fermentation with immobilized in a FBB, demonstrating that corn husk can be used as an economical substrate for butyric acid production.
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http://dx.doi.org/10.1186/s13068-018-1165-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6003175PMC
June 2018

Biotransformation of soy flour isoflavones by Aspergillus niger NRRL 3122 β-glucosidase enzyme.

Nat Prod Res 2018 Oct 11;32(20):2382-2391. Epub 2017 Dec 11.

c William G. Lowrie Department of Chemical & Biomolecular Engineering , The Ohio State University , Columbus , OH , USA.

β-glucosidase enzyme produced from Aspergillus niger NRRL 3122 has been partially purified and characterised. Its molecular weight was 180 KDa. The optimal pH and temperature were 3.98 and 55 °C, respectively. It promoted the hydrolysis of soy flour isoflavone glycosides to their aglycone. Two-level Plackett-Burman design was applied and effective variables for genistein production were determined. Reaction time had a significant positive effect, and pH had a significant negative effect. They were further evaluated using Box-Behnken model. Accordingly, the optimal combination of the major reaction affecting factors was reaction time, 5 h and pH, 4. The concentration of genistein increased by 11.73 folds using this optimal combination. The antioxidant activity of the non-biotransformed and biotransformed soy flour extracts was determined by DPPH method. It was found that biotransformation increased the antioxidant activity by four folds.
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http://dx.doi.org/10.1080/14786419.2017.1413569DOI Listing
October 2018

Propionic acid production from soy molasses by Propionibacterium acidipropionici: Fermentation kinetics and economic analysis.

Bioresour Technol 2018 Feb 8;250:1-9. Epub 2017 Nov 8.

William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 151 West Woodruff Avenue, Columbus, OH 43210, USA. Electronic address:

Propionic acid (PA) is a specialty chemical; its calcium salt is widely used as food preservative. Soy molasses (SM), a low-value byproduct from soybean refinery, contains sucrose and raffinose-family oligosaccharides (RFO), which are difficult to digest for most animals and industrial microorganisms. The feasibility of using SM for PA production by P. acidipropionici, which has genes encoding enzymes necessary for RFO hydrolysis, was studied. With corn steep liquor as the nitrogen source, stable long-term PA production from SM was demonstrated in sequential batch fermentations, achieving PA productivity of >0.8 g/L h and yield of 0.42 g/g sugar at pH 6.5. Economic analysis showed that calcium propionate as the main component (63.5%) in the product could be produced at US $1.55/kg for a 3000-MT plant with a capital investment of US $10.82 million. At $3.0/kg for the product, the process offers attractive 40% return of investment and is promising for commercial application.
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http://dx.doi.org/10.1016/j.biortech.2017.11.016DOI Listing
February 2018

Comparative genomic analysis of Clostridium acetobutylicum for understanding the mutations contributing to enhanced butanol tolerance and production.

J Biotechnol 2017 Dec 16;263:36-44. Epub 2017 Oct 16.

William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 151 West Woodruff Ave., Columbus, OH 43210, USA. Electronic address:

Clostridium acetobutylicum JB200 is a hyper butanol tolerant and producing strain obtained from asporogenic C. acetobutylicum ATCC 55025 through mutagenesis and adaptation in a fibrous bed bioreactor. The complete genomes of both strains were sequenced by the Illumina Hiseq2000 technology and assembled using SOAPdenovo approach. Compared to the genomic sequence of the type strain ATCC 824, 143 single nucleotide polymorphisms (SNPs) and 17 insertion/deletion variations (InDels) were identified in the genome of ATCC 55025. Twenty-nine mutations were in genes involved in sporulation, solventogenesis and stress response. Compared to ATCC 55025, there were seven additional point mutations in the chromosome of JB200. Among them, a single-base deletion in cac3319 encoding an orphan histidine kinase caused protein C-terminal truncation. Disruption of this gene in ATCC 55025 and ATCC 824 resulted in significantly elevated butanol tolerance and production. This study provides genome-level information for the better understanding of solventogenic C. acetobutylicum in several key aspects of cell physiology and metabolism, which could help further metabolic engineering of Clostridium for butanol production.
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http://dx.doi.org/10.1016/j.jbiotec.2017.10.010DOI Listing
December 2017

Tailoring the Oxidative Stress Tolerance of Clostridium tyrobutyricum CCTCC W428 by Introducing Trehalose Biosynthetic Capability.

J Agric Food Chem 2017 Oct 27;65(40):8892-8901. Epub 2017 Sep 27.

Department of Chemical Engineering, The Ohio State University , Columbus, Ohio 43210, United States.

Fermentations employing anaerobes always suffer from the restriction of stringent anaerobic conditions during the production of bulk and fine chemicals. This work aims to improve the oxidative stress tolerance of C. tyrobutyricum CCTCC W428, an ideal butyric-acid-producing anaerobe, via the introduction of trehalose biosynthesis capability. Compared with the wild type, the engineered strain showed a wider substrate spectrum, an improved metabolic profile, and a significantly increased specific growth rate upon aeration and acid challenge. Molecular simulation experiments indicated that CoA transferase maintained its native folded state when protected by the trehalose system. Furthermore, qRT-PCR was combined assays for acid-related enzyme activities under various conditions to verify the effects of trehalose. These results demonstrate that introducing a trehalose biosynthetic pathway, which is redundant for the metabolism of C. tyrobutyricum, can increase the robustness of the host to achieve a better oxidative resistance.
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http://dx.doi.org/10.1021/acs.jafc.7b03172DOI Listing
October 2017

Enhanced robustness in acetone-butanol-ethanol fermentation with engineered Clostridium beijerinckii overexpressing adhE2 and ctfAB.

Bioresour Technol 2017 Nov 8;243:1000-1008. Epub 2017 Jul 8.

William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 151 West Woodruff Ave., Columbus, OH 43210, United States. Electronic address:

Clostridium beijerinckii CC101 was engineered to overexpress aldehyde/alcohol dehydrogenase (adhE2) and CoA-transferase (ctfAB). Solvent production and acid assimilation were compared between the parental and engineered strains expressing only adhE2 (CC101-SV4) and expressing adhE2, ald and ctfAB (CC101-SV6). CC101-SV4 showed an early butanol production from glucose but stopped pre-maturely at a low butanol concentration of ∼6g/L. Compared to CC101, CC101-SV6 produced more butanol (∼12g/L) from glucose and was able to re-assimilate more acids, which prevented "acid crash" and increased butanol production, under all conditions studied. CC101-SV6 also showed better ability in using glucose and xylose present in sugarcane bagasse hydrolysate, and produced 9.4g/L solvents (acetone, butanol and ethanol) compared to only 2.6g/L by CC101, confirming its robustness and better tolerance to hydrolysate inhibitors. The engineered strain of C. beijerinckii overexpressing adhE2 and ctfAB should have good potential for producing butanol from lignocellulosic biomass hydrolysates.
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http://dx.doi.org/10.1016/j.biortech.2017.07.043DOI Listing
November 2017

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

Biotechnol Biofuels 2017 24;10:105. Epub 2017 Apr 24.

Department of Zoology, University of the Punjab, Lahore, Pakistan.

Background: Biofuels 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.

Results: Present investigation deals with the valuation of ethanologenic potential of locally isolated moderate alkali-thermophilic fermentative bacterium, 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.

Conclusion: Effectual 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.
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http://dx.doi.org/10.1186/s13068-017-0785-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5402650PMC
April 2017

Metabolic engineering strategies for acetoin and 2,3-butanediol production: advances and prospects.

Crit Rev Biotechnol 2017 Dec 20;37(8):990-1005. Epub 2017 Apr 20.

d Department of Chemical and Biomolecular Engineering , Ohio State University , Columbus , OH , USA.

Acetoin and 2,3-butanediol (2,3-BD) have a large number of industrial applications. The production of acetoin and 2,3-BD has traditionally relied on oil supplies. Microbial production of acetoin and 2,3-BD will alleviate the dependence on oil. Acetoin and 2,3-BD are neighboring metabolites in the 2,3-BD metabolic pathway of bacteria. This review summarizes metabolic engineering strategies for improvement of microbial acetoin and 2,3-BD production. We also propose enhancements to current acetoin and 2,3-BD production strategies, by offering a metabolic engineering approach that is guided by systems biology and synthetic biology.
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http://dx.doi.org/10.1080/07388551.2017.1299680DOI Listing
December 2017

Butyric acid production from lignocellulosic biomass hydrolysates by engineered Clostridium tyrobutyricum overexpressing xylose catabolism genes for glucose and xylose co-utilization.

Bioresour Technol 2017 Jun 15;234:389-396. Epub 2017 Mar 15.

Bioprocessing Innovative Company, 4734 Bridle Path Ct., Dublin, OH 43017, USA.

Clostridium tyrobutyricum can utilize glucose and xylose as carbon source for butyric acid production. However, xylose catabolism is inhibited by glucose, hampering butyric acid production from lignocellulosic biomass hydrolysates containing both glucose and xylose. In this study, an engineered strain of C. tyrobutyricum Ct-pTBA overexpressing heterologous xylose catabolism genes (xylT, xylA, and xylB) was investigated for co-utilizing glucose and xylose present in hydrolysates of plant biomass, including soybean hull, corn fiber, wheat straw, rice straw, and sugarcane bagasse. Compared to the wild-type strain, Ct-pTBA showed higher xylose utilization without significant glucose catabolite repression, achieving near 100% utilization of glucose and xylose present in lignocellulosic biomass hydrolysates in bioreactor at pH 6. About 42.6g/L butyrate at a productivity of 0.56g/L·h and yield of 0.36g/g was obtained in batch fermentation, demonstrating the potential of C. tyrobutyricum Ct-pTBA for butyric acid production from lignocellulosic biomass hydrolysates.
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http://dx.doi.org/10.1016/j.biortech.2017.03.073DOI Listing
June 2017