Anaerobic degradation of 1,3-propanediol by sulfate-reducing and by fermenting bacteria

Three strains of strictly anaerobic Gram-negative, non-sporeforming, motile bacteria were enriched and isolated from freshwater sediments with 1,3-propanediol as sole energy and carbon source. Strain OttPdl was a sulfate-reducing bacterium which grew also with lactate, ethanol, propanol, butanol, 1,4-butanediol, formate or hydrogen plus CO₂, the latter only in the presence of acetate. In the absence of sulfate, most of these substrates were fermented to the respective fatty acids in syntrophic cooperation with Methanospirillum hungatei. Sulfur, thiosulfate, or sulfite were reduced, nitrate not. The other two isolates degraded propanediol only in coculture with Methanospirillum hungatei. Strain OttGlycl grew in pure culture with acetoin and with glycerol in the presence of acetate. Strain WoAcl grew in pure culture only with acetoin. Both strains did not grow with other substrates, and did not reduce nitrate, sulfate, sulfur, thiosulfate or sulfite. The isolates were affiliated with the genera Desulfovibrio and Pelobacter. The pathways of propanediol degradation and the ecological importance of this process are discussed.     

Improved electrocompetence and metabolic engineering of Clostridium pasteurianum reveals a new regulation pattern of glycerol fermentation

Clostridium pasteurianum produces industrially valuable chemicals such as n‐butanol and 1,3‐propanediol from fermentations of glycerol and glucose. Metabolic engineering for increased yields of selective compounds is not well established in this microorganism. In order to study carbon fluxes and to selectively increase butanol yields, we integrated the latest advances in genome editing to obtain an electrocompetent Clostridium pasteurianum strain for further engineering. Deletion of the glycerol dehydratase large subunit (dhaB) using an adapted S. pyogenes Type II CRISPR/Cas9 nickase system resulted in a 1,3‐propanediol‐deficient mutant producing butanol as the main product. Surprisingly, the mutant was able to grow on glycerol as the sole carbon source. In spite of reduced growth, butanol yields were highly increased. Metabolic flux analysis revealed an important role of the newly identified electron bifurcation pathway for crotonyl‐CoA to butyryl‐CoA conversion in the regulation of redox balance. Compared to the parental strain, the electron bifurcation pathway flux of the dhaB mutant increased from 8 to 46% of the overall flux from crotonyl‐CoA to butyryl‐CoA and butanol, indicating a new, 1,3‐propanediol‐independent pattern of glycerol fermentation in Clostridium pasteurianum.     

Enrichment and isolation of Bacillus beveridgei sp. nov., a facultative anaerobic haloalkaliphile from Mono Lake, California, that respires oxyanions of tellurium, selenium, and arsenic

Mono Lake sediment slurries incubated with lactate and tellurite [Te(IV)] turned progressively black with time because of the precipitation of elemental tellurium [Te(0)]. An enrichment culture was established from these slurries that demonstrated Te(IV)-dependent growth. The enrichment was purified by picking isolated black colonies from lactate/Te(IV) agar plates, followed by repeated streaking and picking. The isolate, strain MLTeJB, grew in aqueous Te(IV)-medium if provided with a small amount of sterile solid phase material (e.g., agar plug; glass beads). Strain MLTeJB grew at high concentrations of Te(IV) (~8 mM) by oxidizing lactate to acetate plus formate, while reducing Te(IV) to Te(0). Other electron acceptors that were found to sustain growth were tellurate, selenate, selenite, arsenate, nitrate, nitrite, fumarate and oxygen. Notably, growth on arsenate, nitrate, nitrite and fumarate did not result in the accumulation of formate, implying that in these cases lactate was oxidized to acetate plus CO₂. Strain MLTeJB is a low G + C Gram positive motile rod with pH, sodium, and temperature growth optima at 8.5–9.0, 0.5–1.5 M, and 40°C, respectively. The epithet Bacillus beveridgei strain MLTeJB^T is proposed.     

Glycerol fermentation by a new 1,3-propanediol-producing microorganism:Enterobacter agglomerans

 According to their ability to synthesize 1,3-propanediol from glycerol, two species were isolated from the anoxic mud of a distillery waste-water digestor: Clostridium butyricum and Enterobacter agglomerans. The latter, a facultatively anaerobic gram-negative bacterium, is described for the first time as a microorganism producing 1,3-propanediol from glycerol. The products of glycerol conversion by E. agglomerans were identified using nuclear magnetic resonance. A 20-g/l glycerol solution was fermented mainly to 1,3-propanediol (0.51 mol/mol) and acetate (0.18 mol/mol). Ethanol, formate, lactate and succinate were formed as by-products. Gas production was very low; 1,3-propanediol production perfectly balanced the oxido-reduction state of the microorganism. Acetate was the predominant metabolite generating energy for growth. High-glycerol-concentration fermentations (71 g/l and 100 g/l) resulted in an increase of the 1,3-propanediol yield (0.61 mol/mol) at the expense of lactate and ethanol production. Specific rates of glycerol consumption and 1, 3-propanediol and acetate production increased whereas the growth rate decreased. The decrease in ATP yield was linearly correlated with the specific rate of 1,3-propanediol production. Incomplete glycerol consumption (about 40 g/l) was systematically observed when high glycerol concentrations were used. The unbalanced oxido-reduction state, the low carbon recovery and the detection of an unknown compound by HPLC observed in these cases indicate the formation of another metabolite, which is possibly an inhibitory factor. Received: 17 November 1994 / Accepted: 15 December 1994     

Engineered Saccharomyces cerevisiae that produces 1,3‐propanediol from d‐glucose

Aims: Saccharomyces cerevisiae is a safe micro‐organism used in fermentation industry. 1,3‐Propanediol is an important chemical widely used in polymer production, but its availability is being restricted owing to its expensively chemical synthesis. The aim of this study is to engineer a S. cerevisiae strain that can produce 1,3‐propanediol at low cost. Methods and Results: By using d ‐glucose as a feedstock, S. cerevisiae could produce glycerol, but not 1,3‐propanediol. In this study, we have cloned two genes yqhD and dhaB required for the production of 1,3‐propanediol from glycerol, and integrated them into the chromosome of S. cerevisiae W303‐1A by Agrobacterium tumefaciens‐mediated transformation. Both genes yqhD and dhaB functioned in the engineered S. cerevisiae and led to the production of 1,3‐propanediol from d ‐glucose. Conclusion: Saccharomyces cerevisiae can be engineered to produce 1,3‐propanediol from low‐cost feedstock d ‐glucose. Significance and Impact of the Study: To our knowledge, this is the first report on developing S. cerevisiae to produce 1,3‐propanediol by using A. tumefaciens‐mediated transformation. This study might lead to a safe and cost‐efficient method for industrial production of 1,3‐propanediol.     

Fermentation of glycerol by Anaerobium acetethylicum and its potential use in biofuel production

Growth of biodiesel industries resulted in increased coproduction of crude glycerol which is therefore becoming a waste product instead of a valuable ‘coproduct’. Glycerol can be used for the production of valuable chemicals, e.g. biofuels, to reduce glycerol waste disposal. In this study, a novel bacterial strain is described which converts glycerol mainly to ethanol and hydrogen with very little amounts of acetate, formate and 1,2‐propanediol as coproducts. The bacterium offers certain advantages over previously studied glycerol‐fermenting microorganisms. Anaerobium acetethylicum during growth with glycerol produces very little side products and grows in the presence of maximum glycerol concentrations up to 1500 mM and in the complete absence of complex organic supplements such as yeast extract or tryptone. The highest observed growth rate of 0.116 h^?1 is similar to that of other glycerol degraders, and the maximum concentration of ethanol that can be tolerated was found to be about 60 mM (2.8 g l^?1) and further growth was likely inhibited due to ethanol toxicity. Proteome analysis as well as enzyme assays performed in cell‐free extracts demonstrated that glycerol is degraded via glyceraldehyde‐3‐phosphate, which is further metabolized through the lower part of glycolysis leading to formation of mainly ethanol and hydrogen. In conclusion, fermentation of glycerol to ethanol and hydrogen by this bacterium represents a remarkable option to add value to the biodiesel industries by utilization of surplus glycerol.     

Alkaline iron(III) reduction by a novel alkaliphilic,halotolerant, <Emphasis Type="Italic">Bacillus</Emphasis> sp. isolated from salt flat sediments of Soap Lake

A halotolerant, alkaliphilic dissimilatory Fe(III)-reducing bacterium, strain SFB, was isolated from salt flat sediments collected from Soap Lake, WA. 16S ribosomal ribonucleic acid gene sequence analysis identified strain SFB as a novel Bacillus sp. most similar to Bacillus agaradhaerens (96.7% similarity). Strain SFB, a fermentative, facultative anaerobe, fermented various hexoses including glucose and fructose. The fructose fermentation products were lactate, acetate, and formate. Under fructose-fermenting conditions in a medium amended with Fe(III), Fe(II) accumulated concomitant with a stoichiometric decrease in lactate and an increase in acetate and CO₂. Strain SFB was also capable of respiratory Fe(III) reduction with some unidentified component(s) of Luria broth as an electron donor. In addition to Fe(III), strain SFB could also utilize nitrate, fumarate, or O₂ as alternative electron acceptors. Optimum growth was observed at 30°C and pH 9. Although the optimal salinity for growth was 0%, strain SFB could grow in a medium with up to 15% NaCl by mass. These studies describe a novel alkaliphilic, halotolerant organism capable of dissimilatory Fe(III) reduction under extreme conditions and demonstrate that Bacillus species can contribute to the microbial reduction of Fe(III) in environments at elevated pH and salinity, such as soda lakes.     

A new 3-hydroxybutyrate fermenting anaerobe,Ilyobacter polytropus,gen. nov. sp. nov., possessing various fermentation pathways

From marine anoxic mud, a new strictly anaerobic, Gram-negative, non-sporeforming bacterium was isolated with 3-hydroxybutyrate as substrate. 3-Hydroxybutyrate and crotonate were fermented to acetate and butyrate. Glycerol was fermented to 1,3-propanediol and 3-hydroxypropionate. Acetate and formate were the only products of pyruvate or citrate fermentation. Glucose and fructose were fermented to acetate, formate and ethanol. Malate and fumarate were fermented to acetate, formate and propionate. Neither sulfate, sulfur, nor nitrate was reduced. The DNA base ratio was 32.2±0.5 mol% guanine plus cytosine. Strain CuHbu1 is described as type strain of a new genus and species, Ilyobacter polytropus gen. nov. sp. nov., in the family Bacteroidaceae.     

一株新型同型产乙酸菌Clostridium sp.的自养和异养生长特性

【背景】同型产乙酸菌是一类利用乙酰辅酶A途径固定CO_2合成自身细胞物质并生成乙酸、乙醇等代谢产物的厌氧菌群,其分布广泛、种类繁多且代谢多样。深入研究同型产乙酸菌菌株的代谢能力及特性,对探索该种群的生理生化特性及其环境作用至关重要。【目的】研究一株同型产乙酸菌Clostridium sp. BXX的最适培养条件及其自养与异养生长特性。【方法】设置BXX菌株培养温度10-55°C、初始pH 6.0-9.0、NaCl浓度0-2.0%、不同氮源,测定菌体细胞含量和产物生成浓度,确定菌株最适培养条件。研究BXX菌株分别以H_2/CO_2、合成气、CO、葡萄糖、1,2-丙二醇、甲酸钠、乙二醇甲醚、甘油、丙酮酸和乳酸为底物时的底物消耗、产物生成、菌体细胞含量和pH等,探究其自养和异养生长特性。【结果】BXX菌株的最适培养温度为30°C,初始pH为7.0,NaCl浓度为1.0%,氮源为酵母粉。BXX菌株能以H2/CO2、合成气、葡萄糖、1,2-丙二醇、甲酸钠、乙二醇甲醚和甘油为底物生长,不能以CO、丙酮酸或乳酸为底物生长。【结论】BXX菌株既能自养生长产乙酸,又能异养生长产乙醇。BXX菌株是乙酸发酵的优良菌种资源,有较好的工业应用潜力。     

Characterization of and Ethanol Hyper-production by Clostridium thermocellum I-1-B

An ethanol hyper-producing clostridial strain, I-1-B, was isolated from Shibi hot spring, Kagoshima prefecture and identified as Clostridium thermocellum based on morphological and physiological proper­ ties. The carbohydrates used as energy sources were glucose, fructose, cellobiose, cellulose and esculin. Fermentation products were ethanol, lactate, acetate, formate, carbon dioxide, and hydrogen. The optimum, maximum, and minimum temperature for growth are about 60, 70, and 47°C, respectively. Optimum pH for growth is about 7.5, and growth occurs at starting pH between 6.0 and 9.0. I-1-B strain has strong tolerance for ethanol and hyper ethanol-productivity. Ethanol concentrations causing 50%. decrease of growth yield are 27 and 16g/liter for I-1-B and ATCC27405 of C. thermocellum, respectively. The organism was cultured on a medium containing 80 g/liter cellulose at 60°C for 156 h. The culture was fed with a vitamin mixture containing vitamin B₁₂ and mineral salts solution at intervals. In this culture the organism produced 23.6 g/liter (512mM) ethanol, 8.5 g/liter (94mM) lactate, 2.9 g/liter (48mM) acetate, and 0.9 g/liter (20mM) formate. The molar ratio of ethanol to total acidic products was 3.2. The ethanol productivity of the strain I-1-B is superior to any of the wild and mutant strains of C. thermocellum so far reported.     

Physiological characterization of strain DCB-1, a unique dehalogenating sulfidogenic bacterium.

Strain DCB-1 is an obligately anaerobic bacterium which carries out the reductive dehalogenation of halobenzoates and was previously known to grow only on pyruvate plus 20% ruminal fluid. When various electron acceptors were supplied, thiosulfate and sulfite were found to stimulate growth. Sulfide was produced from thiosulfate. Cytochrome c and desulfoviridin were detected. The mol% G+C was 49 (at the thermal denaturation temperature). Of 55 carbon sources tested, only pyruvate supported growth as the sole carbon source in mineral medium. Lactate, acetate, L- and D-malate, glycerol, and L- and D-arabinose stimulated growth when supplemented with 10% ruminal fluid and 20 mM thiosulfate. In mineral medium, pyruvate was converted to acetate and lactate, with small amounts of succinate and fumarate accumulating transiently. During growth with thiosulfate, all of these products accumulated transiently. Addition of excess hydrogen to pyruvate-grown cultures resulted in diversion of carbon to formate, lactate, and butyrate, which caused a decrease in cell yield. We conclude that strain DCB-1 is a new type of sulfidogenic bacterium.     

Physiological characterization of strain DCB-1, a unique dehalogenating sulfidogenic bacterium

Strain DCB-1 is an obligately anaerobic bacterium which carries out the reductive dehalogenation of halobenzoates and was previously known to grow only on pyruvate plus 20% ruminal fluid. When various electron acceptors were supplied, thiosulfate and sulfite were found to stimulate growth. Sulfide was produced from thiosulfate. Cytochrome c and desulfoviridin were detected. The mol% G+C was 49 (at the thermal denaturation temperature). Of 55 carbon sources tested, only pyruvate supported growth as the sole carbon source in mineral medium. Lactate, acetate, L- and D-malate, glycerol, and L- and D-arabinose stimulated growth when supplemented with 10% ruminal fluid and 20 mM thiosulfate. In mineral medium, pyruvate was converted to acetate and lactate, with small amounts of succinate and fumarate accumulating transiently. During growth with thiosulfate, all of these products accumulated transiently. Addition of excess hydrogen to pyruvate-grown cultures resulted in diversion of carbon to formate, lactate, and butyrate, which caused a decrease in cell yield. We conclude that strain DCB-1 is a new type of sulfidogenic bacterium.     

Eubacterium aggreganssp. nov., a New Homoacetogenic Bacterium from Olive Mill Wastewater Treatment Digestor

A strictly anaerobic, homoacetogenic, Gram-positive, non spore-forming bacterium, designated strain SR12^T(T=type strain), was isolated from an anaerobic methanogenic digestor fed with olive mill wastewater. Yeast extract was required for growth but could also be used as sole carbon and energy source. Strain SR12^Tutilized a few carbohydrates (glucose, fructose and sucrose), organic compounds (lactate, crotonate, formate and betaine), alcohols (methanol), the methoxyl group of some methoxylated aromatic compounds, and H₂+CO₂. The end-products of carbohydrate fermentation were acetate, formate, butyrate, H₂and CO₂. End-products from lactate and methoxylated aromatic compounds were acetate and butyrate. Strain SR12^Twas non-motile, formed aggregates, had a G+C content of 55 mol % and grew optimally at 35°C and pH 7.2 on a medium containing glucose. Phylogenetically, strain SR12^Twas related toEubacterium barkeri, E. callanderi, andE. limosumwithE. barkerias the closest relative (similarity of 98%) with which it bears little phenotypic similarity or DNA homology (60%). On the basis of its phenotypic, genotypic, and phylogenetic characteristics, we propose to designate strain SR12^TasEubacterium aggreganssp. nov. The type strain is SR12^T(=DSM 12183).     

Production of 1,3-propanediol by Klebsiella pneumoniae from glycerol broth

Broth containing 152 g glycerol l⁻¹ from Candida krusei culture was converted to 1,3-propanediol by Klebsiella pneumoniae. Residual glucose in the broth promoted growth of K. pneumoniae while acetate was inhibitory. After desalination treatment of glycerol broth by electrodialysis, the acetate in the broth was removed. A fed-batch culture with electrodialytically pretreated broth as␣substrate was developed giving 53 g 1,3- propanediol l⁻¹ with a yield of 0.41 g g⁻¹ glycerol and a productivity of 0.94 g l⁻¹ h⁻¹.     

Thermoanaerobacter mathranii sp. nov., an ethanol-producing, extremely thermophilic anaerobic bacterium from a hot spring in Iceland

The extremely thermophilic ethanol-producing strain A3 was isolated from a hot spring in Iceland. The cells were rod-shaped, motile, and had terminal spores; cells from the mid-to-late exponential growth phase stained gram-variable but had a gram-positive cell wall structure when viewed by transmission electron microscopy. Strain A3 used a number of carbohydrates as carbon sources, including xylan, but did not utilize microcrystalline cellulose. Fermentation end products were ethanol, acetate, lactate, CO₂, and H₂. The temperature optimum for growth was between 70 and 75° C, and growth occurred in the range of 50–75° C. The pH range for growth was 4.7–8.8, with an optimum at pH 7.0. Strain A3 was sensitive to tetracycline, chloramphenicol, penicillin G, neomycin, and vancomycin at 100 mg/l but was not sensitive to chloramphenicol and neomycin at 10 mg/l, which indicates that strain A3 belongs to the eubacteria. Addition of 50.66 kPa H₂ or 2% NaCl did not affect growth. The isolate grew in the presence of exogenously added 4% (w/v) ethanol. The G+C ratio was 37 mol%. 16S rDNA studies revealed that strain A3 belongs to the genus Thermoanaerobacter. Genotypic and phenotypic differences between strain A3 and other related species indicate that strain A3 can be assigned to a new species, and the name Thermoanaerobacter mathranii is proposed. Received: 7 October 1996 / Accepted: 14 March 1997     

Enhanced hydrogen and 1,3‐propanediol production from glycerol by fermentation using mixed cultures

The conversion of glycerol into high value products, such as hydrogen gas and 1,3‐propanediol (PD), was examined using anaerobic fermentation with heat‐treated mixed cultures. Glycerol fermentation produced 0.28 mol‐H₂/mol‐glycerol (72 mL‐H₂/g‐COD) and 0.69 mol‐PD/mol‐glycerol. Glucose fermentation using the same mixed cultures produced more hydrogen gas (1.06 mol‐H₂/mol‐glucose) but no PD. Changing the source of inoculum affected gas production likely due to prior acclimation of bacteria to this type of substrate. Fermentation of the glycerol produced from biodiesel fuel production (70% glycerol content) produced 0.31 mol‐H₂/mol‐glycerol (43 mL H₂/g‐COD) and 0.59 mol‐PD/mol‐glycerol. These are the highest yields yet reported for both hydrogen and 1,3‐propanediol production from pure glycerol and the glycerol byproduct from biodiesel fuel production by fermentation using mixed cultures. These results demonstrate that production of biodiesel can be combined with production of hydrogen and 1,3‐propanediol for maximum utilization of resources and minimization of waste. Biotechnol. Bioeng. 2009; 104: 1098–1106. © 2009 Wiley Periodicals, Inc.     

一株硫酸盐还原菌的分离鉴定和系统发育分析

从处理硫酸盐废水的厌氧折流板反应器中分离得到一株硫酸盐还原菌D11, 该菌株革兰氏反应阴性, 无芽孢, 菌体杆状稍有弯曲, 宽度在0.6 μm~0.8 μm, 长度在1.8 μm~3.3 μm之间, 有极生单鞭毛, 能运动, 接触酶阳性, 氧化酶阴性。菌株生长的pH范围介于6.0~8.0之间, 最适pH为7.0, 生长温度范围为25°C~37°C, 最适温度为30°C。能够以葡萄糖、蔗糖、乙酸、乳酸、乙醇和丙二醇为唯一碳源生长, 不能利用丙三醇、丁醇、琥珀酸和苹果酸。菌株DNA的G+C含量为62.7 mo     

Characterization of Desulfomicrobium escambium sp. nov. and proposal to assign Desulfovibrio desulfuricans strain Norway 4 to the genus Desulfomicrobium

A sulfate-reducing bacterium, designated strain ESC1, was isolated and found to be a new species. Strain ESC1 is a strictly anaerobic, gram-negative, non-sporeforming, motile, short, round-ended rod often occurring in pairs. Of 31 fermentative substrates tested, only pyruvate was utilized. Sulfate enhanced growth with pyruvate and allowed growth with ethanol, lactate, formate and hydrogen. Both sulfate and thiosulfate were reduced. Lactate was incompletely oxidized to acetate and CO₂. The strain was desulfoviridin negative. The G+C content is 59.9%. These data suggested placement of strain ESC1 in the genus Desulfomicrobium. Comparative 16S rRNA analysis showed that strain ESC1 shares 98% rRNA sequence similarity with Desulfomicrobium baculatum and Desulfovibrio desulfuricans strain Norway 4. The latter two strains shared greater than 99% 16S rRNA sequence similarity. Strain ESC1 has been designated as the new species Desulfomicrobium escambium. We also recommend that D. desulfuricans strain Norway 4 be considered for reclassification as a Desulfomicrobium species.     

Parameters Affecting Solvent Production by Clostridium pasteurianum

The effect of pH, growth rate, phosphate and iron limitation, carbon monoxide, and carbon source on product formation by Clostridium pasteurianum was determined. Under phosphate limitation, glucose was fermented almost exclusively to acetate and butyrate independently of the pH and growth rate. Iron limitation caused lactate production (38 mol/100 mol) from glucose in batch and continuous culture. At 15% (vol/vol) carbon monoxide in the atmosphere, glucose was fermented to ethanol (24 mol/100 mol), lactate (32 mol/100 mol), and butanol (36 mol/100 mol) in addition to the usual products, acetate (38 mol/100 mol) and butyrate (17 mol/100 mol). During glycerol fermentation, a completely different product pattern was found. In continuous culture under phosphate limitation, acetate and butyrate were produced only in trace amounts, whereas ethanol (30 mol/100 mol), butanol (18 mol/100 mol), and 1,3-propanediol (18 mol/100 mol) were the major products. Under iron limitation, the ratio of these products could be changed in favor of 1,3-propanediol (34 mol/100 mol). In addition, lactate was produced in significant amounts (25 mol/100 mol). The tolerance of C. pasteurianum to glycerol was remarkably high; growth was not inhibited by glycerol concentrations up to 17% (wt/vol). Increasing glycerol concentrations favored the production of 1,3-propanediol.     

Inactivation of <Emphasis Type="Italic">dhaD</Emphasis> and <Emphasis Type="Italic">dhaK</Emphasis> abolishes by-product accumulation during 1,3-propanediol production in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

1,3-Propanediol (1,3-PD) can be used for the industrial synthesis of a variety of compounds, including polyesters, polyethers, and polyurethanes. 1,3-PD is generated from petrochemical and microbial sources. 1,3-Propanediol is a typical product of glycerol fermentation, while acetate, lactate, 2,3-butanediol, and ethanol also accumulate during the process. Substrate and product inhibition limit the final concentration of 1,3-propanediol in the fermentation broth. It is impossible to increase the yield of 1,3-propanediol by using the traditional whole-cell fermentation process. In this study, dhaD and dhaK, the genes for glycerol dehydrogenase and dihydroxyacetone kinase, respectively, were inactivated by homologous recombination in Klebsiella pneumoniae. The dhaD/dhaK double mutant (designated TC100), selected from 5,000 single or double cross homologous recombination mutants, was confirmed as a double cross by using polymerase chain reaction. Analysis of the cell-free supernatant with high-performance liquid chromatography revealed elimination of lactate and 2,3-butanediol, as well as ethanol accumulation in TC100, compared with the wild-type strain. Furthermore, 1,3-propanediol productivity was increased in the TC100 strain expressing glycerol dehydratase and 1,3-PDO dehydrogenase regulated by the arabinose P_BAD promoter. The genetic engineering and medium formulation approaches used here should aid in the separation of 1,3-propanediol from lactate, 2,3-butanediol, and ethanol and lead to increased production of 1,3-propanediol in Klebsiella pneumoniae.