Biodegradation of deltamethrin and its hydrolysis product 3-phenoxybenzaldehyde by a newly isolated Streptomyces aureus strain HP-S-01

A newly isolated actinomycete strain HP-S-01 from activated sludge could effectively degrade deltamethrin and its major hydrolysis product 3-phenoxybenzaldehyde. Based on the morphological, cultural, physio-biochemical characteristics, and 16S rDNA sequence analysis, strain HP-S-01 was identified as Streptomyces aureus. Strain HP-S-01 was also found highly efficient in degrading cyfluthrin, bifenthrin, fenvalerate, fenpropathrin, permethrin, and cypermethrin. Strain HP-S-01 rapidly degraded deltamethrin without a lag phase over a wide range of temperature (18~38°C) and pH (5~10), and metabolized to produce α-hydroxy-3-phenoxy-benzeneacetonitrile and 3-phenoxybenzaldehyde by hydrolysis of the carboxylester linkage. The 3-phenoxybenzaldehyde was further oxidized to form 2-hydroxy-4-methoxy benzophenone resulting in its detoxification. No persistent accumulative product was detected by gas chromatography-mass spectrometry (GC/MS) analysis. Response surface methodology was used to optimize degradation conditions. Strain HP-S-01 completely removed 50~300 mg L⁻¹ deltamethrin within 7 days under the optimal degradation conditions. Furthermore, the biodegradation kinetics corresponded with the first-order model. Therefore, strain HP-S-01 is suitable for the efficient and rapid bioremediation of pyrethroid-contaminated environment.     

Bioremediation of β-cypermethrin and 3-phenoxybenzaldehyde contaminated soils using Streptomyces aureus HP-S-01

Using laboratory and field experiments, the ability of Streptomyces aureus HP-S-01 to eliminate β-cypermethrin (β-CP) and its metabolite 3-phenoxybenzaldehyde (3-PBA) in soils was investigated. In the laboratory, 80.5% and 73.1% of the initial dose of β-CP and 3-PBA (50 mg kg⁻¹) was removed in sterilized soils within 10 days, respectively, while in the same period, disappearance rate of β-CP and 3-PBA in non-sterilized soils was higher and reached 87.8% and 79.3%, respectively. Furthermore, the disappearance process followed the first-order kinetics and the half-life (T _1/2) for β-CP and 3-PBA reduced by 20.3–52.9 and 133.7–186.8 days, respectively, as compared to the controls. The addition of sucrose to the soils enhanced the ability of strain HP-S-01 to eliminate β-CP and 3-PBA. Similar results were observed in the field experiments. The introduced strain HP-S-01 quickly adapted to the environment and rapidly removed β-CP and 3-PBA without any lag phases in the field experiments. Compared with the controls, 47.9% and 67.0% of applied dose of β-CP and 3-PBA was removed from the soils without extra carbon sources and 52.5% and 73.3% of β-CP and 3-PBA was eliminated in soils supplemented with sucrose within 10 days, respectively. Analysis of β-CP degradation products in soil indicated that the tested strain transform β-CP to 3-PBA and α-hydroxy-3-phenoxy-benzeneacetonitrile. However, both intermediates were transient and they disappeared after 10 days. Therefore, the selected actinomyces strain HP-S-01 is suitable for the efficient and rapid bioremediation of β-CP contaminated soils.     

Isolation of a Xanthobacter sp. degrading dichloromethane and characterization of the gene involved in the degradation

A bacterial strain able to degrade dichloromethane (DCM) as the sole carbon source was isolated from a wastewater treatment plant receiving domestic and pharmaceutical effluent. 16S rDNA studies revealed the strain to be a Xanthobacter sp. (strain TM1). The new isolated strain when grown aerobically on DCM showed Luong type growth kinetics, with μ_max of 0.094 h⁻¹ and S _m of 1,435 mg l⁻¹. Strain TM1 was able to degrade other aromatic and aliphatic halogenated compounds, such as halobenzoates, 2-chloroethanol and dichloroethane. The gene for DCM dehalogenase, which is the key enzyme in DCM degradation, was amplified through PCR reactions. Strain TM1 contains type A DCM dehalogenase (dcmAa), while no product could be obtained for type B dehalogense (dcmAb). The sequence was compared against 12 dcmAa from other DCM degrading strains and 98% or 99% similarity was observed with all other previously isolated DCM dehalogenase genes. This is the first time a Xanthobacter sp. is reported to degrade DCM.     

Studies revealing bioremediation potential of the strain Burkholderia sp. GB-01 for abamectin contaminated soils

Burkholderia sp. GB-01 strain was used to study different factors affecting its growth for inoculum production and then evaluated for abamectin degradation in soil for optimization under various conditions. The efficiency of abamectin degradation in soil by strain GB-01 was seen to be dependent on soil pH, temperature, initial abamectin concentration, and inoculum size along with inoculation frequency. Induction studies showed that abamectin depletion was faster when degrading cells were induced by pre-exposure to abamectin. Experiments performed with varying concentrations (2–160 mg Kg⁻¹) of abamectin-spiked soils showed that strain GB-01 could effectively degrade abamectin over the range of 2–40 mg Kg⁻¹. The doses used were higher than the recommended dose for an agricultural application of abamectin, taking in account the over-use or spill situations. A cell density of approximately 10⁸ viable cells g⁻¹ dry weight of soil was found to be suitable for bioremediation over a temperature range of 30–35°C and soil pH 7.5–8.5. This is the first report on bacterial degradation of abamectin in soil by a Burkholderia species, and our results indicated that this bacterium may be useful for efficient removal of abamectin from contaminated soils.     

Isolation of a buprofezin co-metabolizing strain of <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. DFS35-4 and identification of the buprofezin transformation pathway

Buprofezin is a widely used insecticide that has caused environmental pollution in many areas. However, biodegradation of buprofezin by pure cultures has not been extensively studied, and the transformation pathway of buprofezin remains unclear. In this paper, a buprofezin co-metabolizing strain of DFS35-4 was isolated from a buprofezin-polluted soil in China. Strain DFS35-4 was preliminarily identified as Pseudomonas sp. based on its morphological, physiological, and biochemical properties, as well as 16S rRNA gene analysis. In the presence of 2.0 g l⁻¹ sodium citrate, strain DFS35-4 degraded over 70% of 50 mg l⁻¹ buprofezin in 3 days. Strain DFS35-4 efficiently degraded buprofezin in the pH range of 5.0–10.0 and at temperatures between 20 and 30°C. Three metabolites, 2-imino-5-phenyl-3-(propan-2-yl)-1,3,5-thiadiazinan-4-one, 2-imino-5-phenyl-1,3,5-thiadiazinan-4-one, and methyl(phenyl) carbamic acid, were identified during the degradation of buprofezin using gas chromatography–mass spectrometry (GC–MS) and tandem mass spectrometry (MS/MS). A partial transformation pathway of buprofezin in Pseudomonas sp. DFS35-4 was proposed based on these metabolites.     

Biodegradation of malathion by <Emphasis Type="Italic">Brevibacillus</Emphasis> sp. strain KB2 and <Emphasis Type="Italic">Bacillus cereus</Emphasis> strain PU

We report here the degradation of a pesticide, malathion, by Brevibacillus sp. strain KB2 and Bacillus cereus strain PU, isolated from soil samples collected from malathion contaminated field and an army firing range respectively. Both the strains were cultured in the presence of malathion under aerobic and energy-limiting conditions. Both strains grew well in the medium having malathion concentration up to 0.15%. Reverse phase HPLC–UV analysis indicated that Strain KB2 was able to degrade 72.20% of malaoxon (an analogue of malathion) and 36.22% of malathion, while strain PU degraded 87.40% of malaoxon and 49.31% of malathion, after 7 days of incubation. The metabolites mal-monocarboxylic acid and mal-dicarboxylic acid were identified by Gas chromatography/mass spectrometry. The factors affecting biodegradation efficiency were investigated and effect of malathion concentration on degradation rate was also determined. The strain was analyzed for carboxylesterase activity and maximum activity 210 ± 2.5 U ml⁻¹ and 270 U ± 2.7 ml⁻¹ was observed for strains KB2 and PU, respectively. Cloning and sequencing of putative malathion degrading carboxylesterase gene was done using primers based PCR approach.     

Hydrolytic Dechlorination of Chlorothalonil by Ochrobactrum sp. CTN-11 Isolated from a Chlorothalonil-Contaminated Soil

A bacterial strain, designated as CTN-11, capable of degrading chlorothalonil (CTN), was isolated from a chlorothalonil-contaminated soil in China. Based on the morphological, biochemical characteristics and comparative analysis of the 16S rRNA genes, strain CTN-11 was identified as Ochrobactrum sp. Strain CTN-11 could degrade 50 mg l⁻¹ CTN to a non-detectable level within 48 h, and efficiently degrade CTN in a relatively broad range of temperatures from 20 to 40°C and initial pH values from 6.0 to 9.0. The new isolate differed from those previously reported CTN co-metabolic degraders by transforming CTN in the absence of other carbon sources. A glutathione S-transferase (GST) coding gene, which showed 88% sequence similarity with that from Ochrobactrum anthropi SH35B, was cloned from strain CTN-11. However, the gene was not functionally expressed in the presence of glutathione, indicating that CTN was not reductively dechlorinated by thiolytic substitution catalyzed by GST in strain CTN-11. The metabolite hydroxyl-trichloroisophthalonitrile (CTN-OH) produced during CTN anaerobic degradation was identified based on tandem MS/MS, confirming that hydrolytic dechlorination was involved in the CTN degradation. The removal of CTN by strain CTN-11 in sterile and non-sterile soils was also studied. In both soil samples, 50 mg kg⁻¹ CTN could be degraded to an undetectable level within 3 days. This study highlights an important potential use of strain CTN-11 for the cleanup of CTN-contaminated sites and presents a hydrolytic dechlorination reaction of CTN by a pure culture.     

Biodegradation of diesel oil and production of fatty acid esters by a newly isolated Pseudomonas citronellolis KHA

The ability of a newly isolated Pseudomonas citronellolis KHA to degrade diesel oil and to synthesize fatty acid esters has been screened in aerobic batch cultures. The microorganism was able to grow with diesel oil at initial concentrations up to 126 g/l, with optimal growth at 25 g/l. Strain KHA has produced compounds showing strong emulsifying properties (E₂₄ = 75% at the end of the exponential growth phase). The crude extract reduces the surface tension of water from 72 mN m⁻¹ down to 35 mN m⁻¹ with a corresponding minimal concentration value of 60 mg/l. GC and GC–MS analysis of crude product show that the major components are those of hexadecanoic acid propyl ester and octadecanoic acid propyl ester, which have potential for applications in cosmetics, pharmaceutical and foods industries. In addition, strain KHA represents a valuable source of compounds with surface-active properties and potential for the application in clean up of the sites contaminated with hydrocarbons.     

Characterization of a strain of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> isolated from agricultural soil that degrades cadusafos (an organophosphorus pesticide)

Bacteria capable of degrading the pesticide, cadusafos, were isolated from agricultural soil using an enrichment method. In this way, five distinct cadusafos-degrading strains of Pseudomonas putidia were isolated, and were characterized using morphological and biochemical analysis, as well as 16S rRNA sequencing. Strain PC1 exhibited the greatest cadusafos degradation rate and was consequently selected for further investigation. Degradation of cadusafos by strain PC1 was rapid at 20 and 37°C, but was greatly reduced (~1.5-fold) by the presence of carbon sources. Strain PC1 was able to effectively degrade cadusafos in sterilized soil using low inoculum levels. The maximum degradation rate of cadusafos (V _max ) was calculated as 1.1 mg l⁻¹ day⁻¹, and its saturation constant (K _s ) was determined as 2.5 mg l⁻¹. Bacteria such as strain PC1, that use cadusafos as a carbon source, could be employed for the bioremediation of sites contaminated with pesticides.     

Growth promotion effects of the endophyte <Emphasis Type="Italic">Acinetobacter johnsonii</Emphasis> strain 3-1 on sugar beet

An endophytic bacteriumn identified as Acinetobacter johnsonii strain 3–1 was isolated from surface-sterilized roots of Beta vulgaris. Its effect on sugar beet seedling growth was studied using pot assays and field experiments. This strain promoted beet seedling growth following seed inoculation by seed dipping. Plant height and dry weight of beet increased by 19% and 69%, respectively, compared with controls. Strain 3–1 exhibited the ability to increase absorption of N, P, K, and Mg elements from soil and increase the content of vitamins B and C, and protein within beet. In addition, the strain also produced a phytohormone-auxin, produced nearly twice as much IAA as that produced by strain 2–2, and was able to solubilize phosphates. The concentration of dissolved P in the medium was 180.5 mg L⁻¹ after 4 days of incubation. In field experiments, strain 3–1 significantly increased the content of sucrose, fructose, and the yield of the beet. The growth-promoting properties of Acinetobacter johnsonii strain 3–1 indicates that this promising isolate merits further investigation into its symbiosis with beet plants and its potential application in agriculture.     

Enrichment, isolation, and characterization of 4-chlorophenol-degrading bacterium Rhizobium sp. 4-CP-20

The objectives of this research were to monitor the variations of species in mixed cultures during the enrichment period, isolate species and identify and characterize the pure 4-chlorophenol (4-CP) degrading strains from enriched mixed cultures. Strain Rhizobium sp. 4-CP-20 was isolated from the acclimated mixed culture. The DGGE result indicated that strain Rhizobium sp. 4-CP-20 was undetectable at the beginning but detectable after 2 weeks of enrichment. The optimum growth temperatures for Rhizobium sp. 4-CP-20 were both 36°C using 350 mg l⁻¹ glucose or sodium acetate as the substrate. The optimum pH range for degrading 100 mg l⁻¹ 4-CP was between 6.89 and 8.20. Strain Rhizobium sp. 4-CP-20 could degrade 4-CP completely within 3.95 days, as the initial 4-CP concentration was 100 mg l⁻¹. If the initial 4-CP concentration was higher than 240 mg l⁻¹, the growth of bacterial cells and the activity of degrading 4-CP were both inhibited.     

Biodegradation of methyl parathion and p-nitrophenol by a newly isolated Agrobacterium sp. strain Yw12

Strain Yw12, isolated from activated sludge, could completely degrade and utilize methyl parathion as the sole carbon, phosphorus and energy sources for growth in the basic salt media. It could also completely degrade and utilize p-nitrophenol as the sole carbon and energy sources for growth in the minimal salt media. Phenotypic features, physiological and biochemical characteristics, and phylogenetic analysis of 16S rRNA sequence showed that this strain belongs to the genus of Agrobacterium sp. Response surface methodology was used to optimize degradation conditions. Under its optimal degradation conditions, 50 mg l⁻¹ MP was completely degraded within 2 h by strain Yw12 and the degradation product PNP was also completely degraded within 6 h. Furthermore, strain Yw12 could also degrade phoxim, methamidophos, chlorpyrifos, carbofuran, deltamethrin and atrazine when provided as the sole carbon and energy sources. Enzymatic analysis revealed that the MP degrading enzyme of strain Yw12 is an intracellular enzyme and is expressed constitutively. These results indicated that strain Yw12 might be used as a potential and effective organophosphate pesticides degrader for bioremediation of contaminated sites.     

Isolation and characterization of an abamectin-degrading <Emphasis Type="Italic">Burkholderia cepacia</Emphasis>-like GB-01 strain

Abamectin is widely used in agriculture as an insecticide and in veterinary as an anti-parasitic agent, and has caused great environmental pollution by posing potential risk to non-target soil invertebrates and nearby aquatic systems. A bacterium designated GB-01, which was capable of degrading abamectin, was isolated from soil by enrichment culture method. On the basis of morphological, physiological and biochemical characteristics, combined with phylogenetic analysis of 16S rRNA gene, the bacterium GB-01 was identified as Burkholderia cepacia-like species. The bacterium GB-01 was able to utilize abamectin as its sole carbon source for growth, and could degrade more than 90% of abamectin at initial concentrations of 50 and 100 mg l⁻¹ in mineral salt medium in 30 and 36 h, respectively. The longer degradation cycle was observed with abamectin concentrations higher than 100 mg l⁻¹. Optimal growth temperatures and pH values with highest degradation rate were 30–35°C and 7–8, respectively. Two new degradation products were identified and characterized by high performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS) based mass spectral data and a plausible partial degradation pathway of abamectin was proposed. This is the first report in which an abamectin-degrading Burkholderia species isolated from soil was identified and characterized.     

Chlorothalonil degradation by <Emphasis Type="Italic">Ochrobactrum lupini</Emphasis> strain TP-D1 and identification of its metabolites

Chlorothalonil (2, 4, 5, 6-tetrachloroisophthalonitrile, TPN) has been widely used as a wide-spectrum fungicide in China and other countries, and is considered to be an important soil and water contaminant. Here we report the isolation and characterization of a novel TPN-degrading bacterial strain TP-D1 from a heavily TPN-polluted soil in Henan Province, China, and identified it as a strain of Ochrobactrum lupini based on 16S rRNA gene sequence analysis and its morphological, biochemical, and physiological characteristics. Strain TP-D1 could degrade 90.4 and 99.7% of TPN after 4- and 7-day incubation in mineral salt broth with 50 mg TPN l⁻¹ and in autoclaved soil with 50 μg TPN g⁻¹, respectively. Two new metabolites, methyl 2, 5, 6-trichloro-3-cyano-4-methoxy-benzoate (metabolite C) and methyl 3-cyano-2, 4, 5, 6-tetrachlorobenzoate (metabolite D), were detected besides previously reported 4-hydroxy-2, 5, 6-trichloroisophthalonitrile (TPN-OH, metabolite A). This result suggests that the cyano-group in TPN could be converted into amide groups by strain TP-D1, and reveal the biodegradation mechanism of TPN in soil.     

Isolation and characterization of a fungus <Emphasis Type="Italic">Aspergillus</Emphasis> sp. strain F-3 capable of degrading alkali lignin

A fungus strain F-3 was selected from fungal strains isolated from forest soil in Dalian of China. It was identified as one Aspergillus sp. stain F-3 with its morphologic, cultural characteristics and high homology to the genus of rDNA sequence. The budges or thickened node-like structures are peculiar structures of hyphae of the strain. The fungus degraded 65% of alkali lignin (2,000 mg l⁻¹) after day 8 of incubation at 30°C at pH 7. The removal of colority was up to 100% at 8 days. The biodegradation of lignin by Aspergillus sp. F-3 favored initial pH 7.0. Excess acid or alkali conditions were not propitious to lignin decomposing. Addition of ammonium l-tartrate or glucose delayed or repressed biodegradation activities. During lignin degradation, manganese peroxidase (28.2 U l⁻¹) and laccase (3.5 U l⁻¹)activities were detected after day 7 of incubation. GC-MS analysis of biodegraded products showed strain F-3 could convert alkali lignin into small molecules or other utilizable products. Strain F-3 may co-culture with white rot fungus and decompose alkali lignin effectively.     

Biodegradation of phenol and 4-chlorophenol by the yeast <Emphasis Type="Italic">Candida tropicalis</Emphasis>

Biodegradation of phenol and 4-chlorophenol (4-cp) using a pure culture of Candida tropicalis was studied. The results showed that C. tropicalis could degrade 2,000 mg l⁻¹ phenol alone and 350 mg l⁻¹ 4-cp alone within 66 and 55 h, respectively. The capacity of the strain to degrade phenol was obviously higher than that to degrade 4-cp. In the dual-substrate system, 4-cp intensely inhibited phenol biodegradation. Phenol beyond 800 mg l⁻¹ could not be degraded in the presence of 350 mg l⁻¹ 4-cp. Comparatively, low-concentration phenol from 100 to 600 mg l⁻¹ supplied a sole carbon and energy source for C. tropicalis in the initial phase of biodegradation and accelerated the assimilation of 4-cp, which resulted in the fact that 4-cp biodegradation velocity was higher than that without phenol. And the capacity of C. tropicalis to degrade 4-cp was increased up to 420 mg l⁻¹ with the presence of 100–160 mg l⁻¹ phenol. In addition, the intrinsic kinetics of cell growth and substrate degradation were investigated with phenol and 4-cp as single and mixed substrates in batch cultures. The results illustrated that the models proposed adequately described the dynamic behaviors of biodegradation by C. tropicalis.     

Use of Acadian marine plant extract powder from Ascophyllum nodosum in tissue culture of Kappaphycus varieties

Three varieties of Kappaphycus alvarezii (Kapilaran, KAP), Tambalang purple (PUR), Adik-adik (AA), and one variety of Kappaphycus striatum var. sacol (green sacol (GS) were used to determine the efficiency of Acadian marine plant extract powder (AMPEP) as a culture medium at different concentrations, for the regeneration of young plants of Kappaphycus varieties, using tissue culture techniques for the production of seed stock for nursery and outplanting purposes for the commercial cultivation of carrageenophytes. A shorter duration for shoot formation was observed when the explant was treated with AMPEP + Plant Growth Regulator (PGR = PAA + zeatin at 1 mg L⁻¹) compared to AMPEP when used singly. However, four explants responded differently to the number of days required for shoot formation. The KAP variety took 46 days to form shoots at 3–4 mg L⁻¹ AMPEP + PGR; while PUR required 21 days at 3–5 mg L⁻¹ AMPEP and 3–4 mg L⁻¹ AMPEP + PGR. AA required 17 days at 3–5 mg L⁻¹ AMPEP and AMPEP + PGR; and GS 25 days at 1 mg L⁻¹ AMPEP + PGR. It was observed that among the four explants used, PUR and AA initiated shoot formation with the use of AMPEP only at higher concentrations (3–5 mg L⁻¹) after a shorter period. Only PUR responded positively to ESS/2 for shoot initiation. The use of AMPEP alone and/or in combination with PGR as a culture medium in the propagation of microplantlets using tissue culture technique is highly encouraging.     

Acidianus manzaensis sp. nov., a Novel Thermoacidophilic Archaeon Growing Autotrophically by the Oxidation of H2 with the Reduction of Fe3+

A novel thermoacidophilic iron-reducing Archaeon, strain NA−1, was isolated from a hot fumarole in Manza, Japan. Strain NA-1 could grow autotrophically using H₂ or S⁰ as an electron donor and Fe³⁺ as an electron acceptor, and also could grow heterotrophically using some organic compounds. Fe³⁺ and O₂ served as electron acceptors for growth. However, S⁰, NO₃ ⁻, NO₂ ⁻, SO₄ ²⁻, Mn⁴⁺, fumarate, and Fe₂O₃ did not serve as electron acceptors. The ranges of growth temperature and pH were 60–90°C (optimum: 80°C) and pH 1.0–5.0 (optimum: pH 1.2–1.5), respectively. Cells were nearly regular cocci with an envelope comprised of the cytoplasmic membrane and a single outer S-layer. The crenarchaeal-specific quinone (cardariellaquinone) was detected, and the genomic DNA G + C content was 29.9 mol%. From 16S rDNA analysis, it was determined that strain NA-1 is closely related to Acidianus ambivalens (93.1%) and Acidianus infernus (93.0%). However, differences revealed by phylogenetic and phenotypic analyses clearly show that strain NA-1 represents a new species, Acidianus manzaensis, sp. nov., making it the first identified thermoacidophilic iron-reducing microorganism (strain NA-1^T = NBRC 100595 = ATCC BAA 1057). Strain NA-1 has been deposited in the culture collections of the National Institute of Technology and Evolution (NBRC 100595) and American Type Culture Collection (ATCC BAA 1057). The 16S rDNA sequence has been deposited at GenBank under accession number AB182498.     

Continuous butanol production with reduced byproducts formation from glycerol by a hyper producing mutant of <Emphasis Type="Italic">Clostridium pasteurianum</Emphasis>

Butanol, a four-carbon primary alcohol (C₄H₁₀O), is an important industrial chemical and has a good potential to be used as a superior biofuel. Bio-based production of butanol from renewable feedstock is a promising and sustainable alternative to substitute petroleum-based fuels. Here, we report the development of a process for butanol production from glycerol, which is abundantly available as a byproduct of biodiesel production. First, a hyper butanol producing strain of Clostridium pasteurianum was isolated by chemical mutagenesis. The best mutant strain, C. pasteurianum MBEL_GLY2, was able to produce 10.8 g l⁻¹ butanol from 80 g l⁻¹ glycerol as compared to 7.6 g l⁻¹ butanol produced by the parent strain. Next, the process parameters were optimized to maximize butanol production from glycerol. Under the optimized batch condition, the butanol concentration, yield, and productivity of 17.8 g l⁻¹, 0.30 g g⁻¹, and 0.43 g l⁻¹ h⁻¹ could be achieved. Finally, continuous fermentation of C. pasteurianum MBEL_GLY2 with cell recycling was carried out using glycerol as a major carbon source at several different dilution rates. The continuous fermentation was run for 710 h without strain degeneration. The acetone–butanol–ethanol productivity and the butanol productivity of 8.3 and 7.8 g l⁻¹ h⁻¹, respectively, could be achieved at the dilution rate of 0.9 h⁻¹. This study reports continuous production of butanol with reduced byproducts formation from glycerol using C. pasteurianum, and thus could help design a bioprocess for the improved production of butanol.