Mohamed Chamkha

Isolation and characterization of Halomonas sp. strain C2SS100, a hydrocarbon-degrading bacterium under hypersaline conditions

Aims:  To isolate and characterize an efficient hydrocarbon‐degrading bacterium under hypersaline conditions, from a Tunisian off‐shore oil field.

Simultaneous hydrocarbon biodegradation and biosurfactant production by oilfield-selected bacteria

Aims:  To study the bacterial diversity associated with hydrocarbon biodegradation potentiality and biosurfactant production of Tunisian oilfields bacteria.

Isolation of a thermophilic and halophilic tyrosol-degrading Geobacillus from a Tunisian high-temperature oil field.

An aerobic, thermophilic, halotolerant and Gram-positive bacterium, designated strain C5, was isolated from a high-temperature oil field, located in Sfax, Tunisia, after enrichment on tyrosol. Strain C5 grew between 25 and 70 degrees C and optimally at 50 degrees C. It grew in the presence of 0-12% (w/v) NaCl, with optimum growth at 3% (w/v) NaCl. Strain C5 was able to degrade tyrosol aerobically, in the presence of 30 g L(-1) NaCl and under warm conditions (55 degrees C). The degradation of tyrosol proceeded via p-hydroxyphenylacetic and 3,4-dihydroxyphenylacetic acids. The products were confirmed by HPLC and GC-MS analyses. Strain C5 was also found to degrde a wide range of other aromatic compounds, including benzoic, p-hydroxybenzoic, protocatechuic, vanillic, p-hydroxyphenylacetic, 3,4-dihydroxyphenylacetic, cinnamic and ferulic acids, phenol and m-cresol. Moreover, strain C5 was grown on diesel and crude oil as sole carbon and energy sources. Strain C5 was also able to utilize several carbohydrates. Phenotypic characteristics and phylogenetic analysis of the 16S rRNA gene sequence of strain C5 revealed that it was related to members of the genus Geobacillus, being most closely related to the type strain of G. pallidus (99% sequence similarity). In addition, we report on growth of the type strain of G. pallidus on different aromatic compounds and hydrocarbons.

Characterization of a novel biosurfactant produced by Staphylococcus sp. strain 1E with potential application on hydrocarbon bioremediation.

A biosurfactant‐producing bacterium (Staphylococcus sp. strain 1E) was isolated from an Algerian crude oil contaminated soil. Biosurfactant production was tested with different carbon sources using the surface tension measurement and the oil displacement test. Olive oil produced the highest reduction in surface tension (25.9 dynes cm–1). Crude oil presented the best substrate for 1E biosurfactant emulsification activity. The biosurfactant produced by strain 1E reduced the growth medium surface tension below 30 dynes cm–1. This reduction was also obtained in cell‐free filtrates. Biosurfactant produced by strain 1E showed stability in a wide range of pH (from 2 to 12), temperature (from 4 to 55 °C) and salinity (from 0 to 300 g l–1) variations. The biosurfactant produced by strain 1E belonged to lipopeptide group and also constituted an antibacterial activity againt the pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis. Phenanthrene solubility in water was enhanced by biosurfactant addition. Our results suggest that the 1E biosurfactant has interesting properties for its application in bioremediation of hydrocarbons contaminated sites. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Isolation of a cinnamic acid-metabolizing Clostridium glycolicum strain from oil mill wastewaters and emendation of the species description

A strictly anaerobic, gram-positive, motile, sporulated bacterium, designated strain CIN5, was isolated from olive mill wastewaters after enrichment on cinnamic acid. The rod-shaped cells were slightly curved (0.4-1.1 x 2.0-15 microm) and occurred singly or in pairs. Strain CIN5 utilized a limited number of carbohydrates (glucose, fructose, maltose, sorbitol), grew optimally at 37 degrees C and at pH 7.3-7.5 and had a DNA G+C content of 29.1+/-0.3 mol%. Strain CIN5 was very closely related to Clostridium glycolicum DSM 1288T. Both strain CIN5 and the type strain of C. glycolicum transformed cinnamic acid to hydrocinnamic acid and a wide range of other cinnamic acid derivatives, including o-, m- and p-coumaric, o-, m- and p-methoxycinnamic, p-methylcinnamic, caffeic, ferulic and isoferulic acids, to their corresponding 3-phenylpropionic acids by reducing the double bond of the side chain. Glucose supplementation increased the rate of conversion markedly. The emendation of the description of C. glycolicum is proposed to include these new characteristics.

Isolation and characterization of Klebsiella oxytoca strain degrading crude oil from a Tunisian off-shore oil field

A facultatively anaerobic, Gram‐negative, mesophilic, moderately halotolerant, non‐motile, and non‐sporulated bacterium, designated strain BSC5 was isolated from an off‐shore “Sercina” oil field, located near the Kerkennah island, Tunisia. Yeast extract was not required for growth. Phenotypic characteristics and phylogenetic analysis of the 16S rRNA gene sequence of strain BSC5 revealed that it was related to members of the genus Klebsiella, being most closely related to the type strain of K. oxytoca (99% sequence similarity). Strain BSC5 was capable of using aerobically the crude oil as substrate growth. The growth of strain BSC5 on crude oil was followed by measuring the OD600 nm and by enumeration of viable cells at different cultures time. GC‐MS analysis showed that strain BSC5 was capable of degrading a wide range of aliphatic hydrocarbons from C13 to C30. The biodegradation rate for n ‐alkanes reached 44% and 75%, after 20 and 45 days of incubation, respectively. Addition of the synthetic surfactant, Tween 80, accelerated the crude oil degradation. The biodegradation rate for n ‐alkanes reached 61% and 98%, after 20 and 45 days of incubation, respectively. Moreover, three aromatic compounds, p ‐hydroxybenzoate, protocatechuate and gentisate, were metabolized completely by strain BSC5 after 24 h, under aerobic conditions. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Microbial diversity in Tunisian olive fermentation brine as evaluated by small subunit rRNA - Single strand conformation polymorphism analysis.

The microbial diversity of a Tunisian olive fermentation brine was analysed using a culture-independent approach based on the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). SSCP patterns show a remarkably simple microbial community but higher for bacterial community than for the eukaryotic community. This study did not show the presence of archaeal populations. After PCR amplification, two small subunit (SSU) rRNA clone libraries of Bacteria and Eucarya populations were established. Three bacteria and only one eukaryotic phylotype were identified. Two dominant bacteria showed 100% phylogenetic similarity to the 16S rRNA sequences of Lactobacillus plantarum and Lactobacillus collinoides and represent 85% of bacterial community. The third bacteria phylotype was phylogenetically close related to the 16S rRNA sequence of a moderately halophilic bacterium belonging to the class gamma Proteobacteria. The dominant eukaryotic phylotype was identified as a Pichia membranaefaciens.

Metabolism of cinnamic acids by some clostridiales and emendation of the descriptions of Clostridium aerotolerans, Clostridium celerecrescens and Clostridium xylanolyticum

The ability of Clostridium aerotolerans DSM 5434T, Clostridium celerecrescens DSM 5628T, Clostridium methoxybenzovorans DSM 12182T, Clostridium stercorarium ATCC 35414T, Clostridium subterminale DSM 2636, Clostridium termitidis DSM 5398T, Clostridium thermolacticum DSM 2910T, Clostridium thermopalmarium DSM 5974T and Clostridium xylanolyticum DSM 6555T to metabolize cinnamic acid and various derivatives, with or without glucose supplementation, was examined. Only C aerotolerans DSM 5434T and C. xylanolyticum DSM 6555T, closely related species, transformed cinnamic acid to 3-phenylpropionic acid. Both species also reduced a wide range of cinnamic acid derivatives, including o-, m- and p-coumaric, o-, m- and p-methoxycinnamic, p-methylcinnamic, caffeic, ferulic, isoferulic and 3,4,5-trimethoxycinnamic acids to their corresponding 3-phenylpropionic acid derivatives. C. aerotolerans DSM 5434T, however, also decarboxylated p-coumaric acid into 4-vinylphenol, which was then reduced to 4-ethylphenol. C. celerecrescens was grouped with C. aerotolerans and C. xylanolyticum in subcluster XIVa of the Clostridiales. C. celerecrescens DSM 5628T only metabolized m- and p-methoxycinnamic and p-methylcinnamic acids to their corresponding 3-phenylpropionic acid derivatives, reducing the double bond in the C3 aliphatic side chain. Addition of glucose markedly increased the yield of the biotransformations by these three species. An emendation of the descriptions of C. aerotolerans, C. celerecrescens and C. xylanolyticum is proposed, based on these observations.

Isolation from a shea cake digester of a tannin-degrading Streptococcus gallolyticus strain that decarboxylates protocatechuic and hydroxycinnamic acids, and emendation of the species.

A facultatively anaerobic, mesophilic, non-motile, non-sporulating bacterium, designated strain B7, was isolated from an anaerobic digester fed with shea cake rich in tannins and aromatic compounds, after enrichment on tannic acid. The coccoid cells (less than 2 microm in diameter) occurred in pairs or short chains and stained gram-positive. Strain B7 fermented a wide range of carbohydrates (cellobiose, fructose, galactose, glucose, lactose, maltose, mannitol, melibiose, raffinose and trehalose), grew optimally at pH 7.0 and had a G+C content of 40.4+/-0.3 mol%. Strain B7 was closely related to Streptococcus gallolyticus ACM 3611T, a member of the Streptococcus bovis rRNA cluster, with a sequence similarity of 98% and a DNA hybridization value of 86 mol%. Isolate B7 hydrolysed tannic acid and decarboxylated gallic acid to pyrogallol, traits also observed in S. gallolyticus ACM 3611T. In addition, both strains decarboxylated protocatechuic acid to catechol, p-coumaric acid to 4-vinylphenol, caffeic acid to 4-vinylcatechol and ferulic acid to 4-vinylguaiacol. An unsubstituted para-hydroxyl group on the benzene ring was required for decarboxylation. Glucose addition markedly increased the conversion rate. As these traits were not described previously, emendation of the description of the species Streptococcus gallolyticus is proposed.

Isolation from a shea cake digester of a tannin-tolerant Escherichia coli strain decarboxylating p-hydroxybenzoic and vanillic acids.

A facultatively anaerobic, mesophilic, Gram-negative, non-motile, non-sporulated bacterium, designated strain C2, was isolated from an anaerobic digester fed with shea cake rich in tannins and aromatic compounds and previously inoculated with anaerobic sludge from the pit of a slaughterhouse, after enrichment on tannic acid. The straight rods occurred singly or in pairs. Strain C2 fermented numerous carbohydrates (fructose, galactose, glucose, lactose, mannose, maltose, melibiose, raffinose, rhamnose, ribose, saccharose, sorbitol, trehalose, and xylose) and peptides (Biotrypcase, Casamino acids, and yeast extract), producing acid and gas, and had a G + C content of 51.6 ± 0.1 mol %. Strain C2 was very closely related to Escherichia coli (= DSM 30083T) phylogenetically (similarity of 99%), genotypically (DNA homology of 79%), and phenotypically. The isolate tolerated tannic acid (hydrolyzable tannin) and decarboxylated by non-oxidative decarboxylation only p-hydroxybenzoic and vanillic acids to their corresponding phenol and guaicol, under anaerobic and aerobic conditions without further degradation. Adding glucose increased growth and the rate of conversion. High concentrations of p-hydroxybenzoic acid or vanillic acid inhibited growth, and decarboxylation could not occur completely, suggesting phenol toxicity. In contrast, the type strain of E. coli cannot metabolize p-hydroxybenzoic and vanillic acids, anaerobically or aerobically, with or without glucose added.