Functional Analysis of Novel alkB Genes Encoding Long-Chain n-Alkane Hydroxylases in Rhodococcus sp. Strain CH91.

Rhodococcus sp. strain CH91 is capable of utilizing long-chain n-alkanes as the sole carbon source. Two new genes (alkB1 and alkB2) encoding AlkB-type alkane hydroxylase were predicted by its whole-genome sequence analysis. The purpose of this study was to elucidate the functional role of alkB1 and alkB2 genes in the n-alkane degradation of strain CH91. RT-qPCR analyses revealed that the two genes were induced by n-alkanes ranging from C16 to C36 and the expression of the alkB2 gene was up-regulated much higher than that of alkB1. The knockout of the alkB1 or alkB2 gene in strain CH91 resulted in the obvious reduction of growth and degradation rates on C16-C36 n-alkanes and the alkB2 knockout mutant exhibited lower growth and degradation rate than the alkB1 knockout mutant. When gene alkB1 or alkB2 was heterologously expressed in Pseudomonas fluorescens KOB2Δ1, the two genes could restore its alkane degradation activity. These results demonstrated that both alkB1 and alkB2 genes were responsible for C16-C36 n-alkanes' degradation of strain CH91, and alkB2 plays a more important role than alkB1. The functional characteristics of the two alkB genes in the degradation of a broad range of n-alkanes make them potential gene candidates for engineering the bacteria used for bioremediation of petroleum hydrocarbon contaminations.

Short-term microbial effects on n-alkane during the early phase degradation and consequential modification of biomarkers in a lowland subtropical rainforest in southern Taiwan: A litterbag experiment.

Accurate reconstructions of past environments are critical and urgent because they can help understand how modern environments might respond to current climatic and land-use changes. However, the effect of microbial degradation and consequential modification in plant-derived-biomarkers during the early degradation phase is not yet apparent, that might bias the paleoenvironmental investigation. In this regard, a litterbag experiment was conducted to reveal the microbial effects on n-alkane-associated biomarker changes associated with three habitats (ravine, windward, and leeward) in a lowland subtropical rainforest in southern Taiwan. Freshly collected leaves of plant species Iles rotunda, Ficus benjamina, and Castanopsis carlesii were distributed in the habitat leaf litterbag experiment for 15 and 75 days incubation, respectively. The results revealed that the average leaf decomposition rate was 19.4% ± 6.4% during the first 15 days and 39% ± 11% within 75 days incubation for all leaves. The overall leaf mass degradation of I. rotunda, F. benjamina and C. carlesii in the ravine after 75 days was 58%, 51% and 41%, respectively, which were higher than those in the windward (28%, 36% and 38%) and leeward habitats (35%, 26% and 42%, respectively) indicating higher decomposition rate in the ravine habitat than the others. The predominant n-alkanes in I. rotunda were C31 and C29, whereas in F. benjamina these were C31, C29, and C33, and in C. carlesii it was C31. After 75 days, the ravine habitat showed a 60% decrease in the total n-alkane concentration compared to windward and leeward habitats, suggesting the microbial community associated with the ravine habitat has a higher efficiency of degrading n-alkanes. However, the biomarkers such as carbon preference index (CPI), average carbon length (ACL) and the C31/C29 ratio did not show statistical difference in all habitats from 15 to 75 days incubation. The next-generation sequencing revealed that microbial communities changed significantly from 15 to 75 days in all habitats. The alkB gene-containing bacteria and their family lineages increased substantially during the first 15 days incubation in all habitats. Furthermore, several bacterial genera were exclusively present in the ravine habitat, whereas some were only in the leeward and windward habitats. Despite the heterogeneity of microbial proliferation, difference in biomass and n-alkane degradation among the three habitats, most of the n-alkane-associated biomarkers remained the same. Therefore, we concluded that the microbial effects on n-alkane degradation during the early phase in plant leaves had little influence on the results of most n-alkane biomarkers.

Activity of alkanmonooxygenase alkB gene in strains of hydrocarbon-oxidizing bacteria isolated from petroleum products.

Alkanmonooxygenase enzymes AlkB and Cyp153 are responsible for the aerobic degradation of n-alkanes of petroleum and petroleum products. To prove the usage of n-alkanes from oil and petroleum products by hydrocarbon-oxidizing bacteria isolated from aviation kerosene TS-1 and automobile gasoline AI-95, the detection of the key genes alkB, Alk1, Alk2, Alk3 and Cyp153 encoding alkanmonooxygenases AlkB and Cyp153 (responsible for the oxidation of hydrocarbons with a certain chain length) was carried out. It was found that bacterial strains isolated from TS-1 jet fuel, except Deinococcus sp. Bi7, had at least one of the studied n-alkane degradation genes. The strains Sphingobacterium multivorum Bi2; Alcaligenes faecalis Bi3; Rhodococcus sp. Bi4; Sphingobacterium sp. Bi5; Rhodococcus erythropolis Bi6 contained the alkB gene. In the strains of hydrocarbon-oxidizing bacteria isolated from gasoline AI- 95, this alkanmonooxygenase gene was not detected. Using the real-time PCR method, the activity of the alkB gene in all bacterial strains isolated from petroleum products was analyzed and the number of its copies was determined. By real-time PCR using a primer with a different sequence of nucleotides to detect the alkB gene, its activity was established in all bacterial strains isolated from gasoline AI-95; besides, the strain Paenibacillus agaridevorans Bi11 was assigned to the group with a high level of its activity (1290 copies/ml). According to the assessment of the growth of isolated hydrocarbon-oxidizing bacteria on a solid Evans mineral medium with the addition of the model mixture of hydrocarbons, the strains were divided into three groups. The distributions of strains of hydrocarbon-oxidizing bacteria in the groups based on the activity of the alkB gene and groups formed based on the growth ability and use of the model mixture of hydrocarbons and petroleum products were found to be consistent. The results obtained indicate that we need to use a complex of molecular and physiological methods for a comprehensive analysis of the distribution of the studied genes in bacteria and to assess their activity in the strains of hydrocarbon-oxidizing bacteria capable of biodegradation of petroleum hydrocarbons.

Impacts of groundwater level fluctuation on soil microbial community, alkane degradation efficiency and alkane-degrading gene diversity in the critical zone: Evidence from an accelerated water table fluctuation simulation.

Petroleum hydrocarbons are hazardous to ecosystems and human health, commonly containing n-alkanes and polycyclic aromatic hydrocarbons. Previous researches have studied alkane degraders and degrading genes under aerobic or anaerobic conditions, but seldom discussed them in the intermittent saturation zone which is a connective area between the vadose zone and the groundwater aquifer with periodic alteration of oxygen and moisture. The present study investigated the difference in alkane degradation efficiency, bacterial community, and alkane degrading gene diversity in aerobic, anaerobic, and aerobic-anaerobic fluctuated treatments. All biotic treatments achieved over 90% of n-alkane removal after 120 days of incubation. The removal efficiencies of n-alkanes with a carbon chain length from 16 to 25 were much higher in anaerobic scenarios than those in aerobic scenarios, explained by different dominant microbes between aerobic and anaerobic conditions. The highest removal efficiency was found in fluctuation treatments, indicating an accelerated n-alkane biodegradation under aerobic-anaerobic alternation. In addition, the copy numbers of the 16S rRNA gene and two alkB genes (alkB-P and alkB-R) declined dramatically when switched from aerobic to anaerobic scenarios and oppositely from anaerobic to aerobic conditions. This suggested that water level fluctuation could notably change the presence of aerobic alkane degrading genes. Our results suggested that alkane degradation efficiency, soil microbial community, and alkane-degrading genes were all driven by water level fluctuation in the intermittent saturation zone, helping better understand the effects of seasonal water table fluctuation on the biodegradation of petroleum hydrocarbons in the subsurface environment.

Degradation of long-chain n-alkanes by a novel thermal-tolerant Rhodococcus strain.

A novel bacterial strain, CH91, was isolated from a high-temperature oil reservoir. Morphological characterization, phylogenetic analyses of 16S rRNA gene sequence and genome relatedness indicated that the strain is a potential new species in the genus Rhodococcus. Strain CH91 could grow in the temperature range of 25-50 °C (optimally at 37 °C) and utilize a broad range of long-chain n-alkanes from hexadecane to hexatriacontane. The utilization of the n-alkanes mixture of strain CH91 revealed that the degradation rate was correlated to the length of the carbon chain. Two novel alkB genes encoding alkane 1-monooxygenase were found in the genome of this strain. The protein sequences of both alkane 1-monooxygenases showed a remarkable phylogenetic distance to other reported AlkB protein sequences. These results would help broaden our knowledge about alkane degradation by Rhodocuccus and its potential ecological role. The ability of the strain in the long-chain alkane degradation and thermal tolerance could also be further exploited for bioremediation of oil contaminations and microbial enhanced oil recovery.

Isolation and characterization of hydrocarbon-degrading bacteria from gas station leaking-contaminated groundwater in the Southern Amazon, Brazil / Isolamento e caracterização de bactérias degradadoras de hidrocarbonetos de água subterrânea contaminada por vazamentos em posto de combustível no sul da Amazônia, Brasil

Abstract Twenty-three hydrocarbon-degrading bacteria strains were isolated from gas station leaking-contaminated groundwater located in the Southern Amazon, Brazil. Based on hydrocarbon (diesel, hexadecane, benzene, toluene and xylene) degradation ability, two strains were selected for further study. The amplification and sequencing of the 16S rRNA gene showed that these two strains belonged to the genus Bacillus (Bacillus sp. L26 and Bacillus sp. L30). GC-MS analysis showed that strain L30 was the most effective in degrading n-alkane (C10-C27) from diesel after 7 days of cultivation in mineral medium. Both strains produced biosurfactants and showed emulsification activity, specially the strain L30. Alkane hydroxylase gene (group III), which is important for alkane biodegradation, was present in strains. As a result, this study indicated that these bacteria could have promising applications in hydrocarbon bioremediation.

Resumo Vinte e três linhagens bacterianas degradadoras de hidrocarbonetos foram isoladas de água subterrânea contaminada por vazamento em posto de combustível no sul da Amazônia, Brasil. Com base na habilidade de degradar hidrocarbonetos (diesel, hexadecano, benzeno, tolueno e xileno), duas linhagens foram selecionadas para estudos posteriores. A amplificação e sequenciamento do gene 16S rRNA demonstrou que essas linhagens pertencem ao gênero Bacillus (Bacillus sp. L26 and Bacillus sp. L30). Análises de GC-MS mostraram que a linhagem L30 foi mais eficiente em degradar n-alcanos (C10-C27) presentes no diesel, após 7 dias de cultivo em meio mineral. Ambas as linhagens produziram biossurfactantes e apresentaram atividade emulsificante, especialmente a linhagem L30. O gene alcano hidroxilase (grupo III), o qual é importante para degradação de alcanos, foram detectados nas linhagens. Como resultado, este estudo indicou que essas linhagens bacterianas podem ser promissoras se aplicadas em processos de biorremediação.

Enhanced crude oil depletion by constructed bacterial consortium comprising bioemulsifier producer and petroleum hydrocarbon degraders.

The aim of this work was to study the production of bioemulsifier by Rhodococcus erythropolis OSDS1, and the improvement of crude oil depletion efficiency using a consortium of petroleum hydrocarbon degraders and OSDS1. The results showed that R. erythropolis OSDS1 produced highly stable bioemulsifier under various salinity (0-35 g/L NaCl) and pH (5.0-9.0) conditions; more than 90% of the initial emulsification activity was retained after 168 h. Emulsification capacity of the bioemulsifier on different petroleum hydrocarbons was diesel > mineral oil/crude oil > gasoline. A mixed bacterial consortium combining OSDS1 and four other petroleum hydrocarbon degraders was constructed. GC-MS results revealed that the constructed consortium achieved 85.26% depletion efficiency of crude oil in 15 days, which was significantly higher than that of individual strains. During the process, alkane hydroxylase gene (alkB) was successfully amplified from the consortium, confirming presence of crude oil degrading enzymes.

Characteristics of crude oil-degrading bacteria Gordonia iterans isolated from marine coastal in Taean sediment.

Crude oil is a major pollutant of marine and coastal ecosystems, and it causes environmental problems more seriously. It is believed ultimate and complete degradation is accomplished mainly by microorganisms. In this study, we aim to search out for bacterial strains with high ability in degrading crude oil. From sediments contaminated by the petroleum spilled in 2007, an accident in Taean, South Korea, we isolated thirty-one bacterial strains in total with potential application in crude oil contamination remediation. In terms of removal percentage after 7 days, one of the strains, Co17, showed the highest removal efficiency with 84.2% of crude oil in Bushnell-Haas media. The Co17 strain even exhibited outstanding ability removing crude oil at a high salt concentration. Through the whole genome sequencing annotation results, many genes related with n-alkane degradation in the genome of Gordonia sp. Co17, revealed alkane-1-monooxygenase, alcohol dehydrogenase, and Baeyer-Villiger monooxygenase. Specially, for confirmation of gene-level, alkB gene encoding alkane hydroxylase (alkane-1-monooxygenase) was found in the strain Co17. The expression of alkB upregulated 125-fold after 18 hr accompany with the removal of n-alkanes of 48.9%. We therefore propose the strain Gordonia iterans Co17, isolated from crude oil-contaminated marine sediment, could be used to offer a new strategy for bioremediation with high efficiency.

Degradation of n-alkanes and PAHs from the heavy crude oil using salt-tolerant bacterial consortia and analysis of their catabolic genes.

In the present study, salt-tolerant strains, Dietzia sp. HRJ2, Corynebacterium variabile HRJ4, Dietzia cinnamea HRJ5 and Bacillus tequilensis HRJ6 were isolated from the Dagang oil field, China. These strains degraded n-alkanes and polycyclic aromatic hydrocarbons (PAHs) aerobically from heavy crude oil (HCO) in an experiment at 37 °C and 140 rpm. The GC/MS investigation for degradation of different chain lengths of n-alkanes (C8-C40) by individual strains showed the highest degradation of C8-C19 (HRJ5), C20-C30 (HRJ4) and C31-C40 (HRJ5), respectively. Moreover, degradation of 16 PAHs with individual strains demonstrated that the bicyclic and pentacyclic aromatic hydrocarbons (AHs) were mostly degraded by HRJ5, tricyclic and tetracyclic AHs by HRJ6 and hexacyclic AHs by HRJ2. However, the highest degradation of total petroleum hydrocarbons (TPHs), total saturated hydrocarbons (TSH), total aromatic hydrocarbons (TAH), n-alkanes (C8-C40) and 16 PAHs was achieved by a four-membered consortium (HRJ2 + 4 + 5 + 6) within 12 days, with the predominance of HRJ4 and HRJ6 strains which was confirmed by denaturing gradient gel electrophoresis. The abundance of alkB and nah genes responsible for catabolism of n-alkanes and PAHs was quantified using the qPCR. Maximum copy numbers of genes were observed in HRJ2 + 4 + 5 + 6 consortium (gene copies l-1) 2.53 × 104 (alkB) and 3.47 × 103 (nah) at 12 days, which corresponded to higher degradation rates of petroleum hydrocarbons. The superoxide dismutase (SOD) (total SOD (T-SOD), Cu2+Zn2+-SOD), catalase (CAT) and ascorbate peroxidase (APX) activities in Allium sativum and Triticum aestivum were lower in the HRJ2 + 4 + 5 + 6-treated HCO as compared to the plantlets exposed directly to HCO. The present results revealed the effective degradation of HCO-contaminated saline medium using the microbial consortium having greater metabolic diversity.

Isolation and characterization of hydrocarbon-degrading bacteria from gas station leaking-contaminated groundwater in the Southern Amazon, Brazil

Abstract Twenty-three hydrocarbon-degrading bacteria strains were isolated from gas station leaking-contaminated groundwater located in the Southern Amazon, Brazil. Based on hydrocarbon (diesel, hexadecane, benzene, toluene and xylene) degradation ability, two strains were selected for further study. The amplification and sequencing of the 16S rRNA gene showed that these two strains belonged to the genus Bacillus (Bacillus sp. L26 and Bacillus sp. L30). GC-MS analysis showed that strain L30 was the most effective in degrading n-alkane (C10-C27) from diesel after 7 days of cultivation in mineral medium. Both strains produced biosurfactants and showed emulsification activity, specially the strain L30. Alkane hydroxylase gene (group III), which is important for alkane biodegradation, was present in strains. As a result, this study indicated that these bacteria could have promising applications in hydrocarbon bioremediation.

Resumo Vinte e três linhagens bacterianas degradadoras de hidrocarbonetos foram isoladas de água subterrânea contaminada por vazamento em posto de combustível no sul da Amazônia, Brasil. Com base na habilidade de degradar hidrocarbonetos (diesel, hexadecano, benzeno, tolueno e xileno), duas linhagens foram selecionadas para estudos posteriores. A amplificação e sequenciamento do gene 16S rRNA demonstrou que essas linhagens pertencem ao gênero Bacillus (Bacillus sp. L26 and Bacillus sp. L30). Análises de GC-MS mostraram que a linhagem L30 foi mais eficiente em degradar n-alcanos (C10-C27) presentes no diesel, após 7 dias de cultivo em meio mineral. Ambas as linhagens produziram biossurfactantes e apresentaram atividade emulsificante, especialmente a linhagem L30. O gene alcano hidroxilase (grupo III), o qual é importante para degradação de alcanos, foram detectados nas linhagens. Como resultado, este estudo indicou que essas linhagens bacterianas podem ser promissoras se aplicadas em processos de biorremediação.

Enhanced removal of petroleum hydrocarbons using a bioelectrochemical remediation system with pre-cultured anodes.

Bioelectrochemical remediation (BER) systems such as microbial fuel cells (MFCs) have recently emerged as a green technology for the effective remediation of petroleum hydrocarbon contaminants (PH) coupled with simultaneous energy recovery. Recent research has shown that biofilms previously enriched for substrate degrading bacteria resulted in excellent performance in terms of substrate removal and electricity generation but the effects on hydrocarbon contaminant degradation were not examined. Here we investigate the differences between enriched biofilm anodes and freshly inoculated new anodes in diesel fed single chamber mediatorless microbial fuel cells (DMFC) using various techniques for the enhancement of PH contaminant remediation with concomitant electricity generation. An anodophilic microbial consortium previously selected for over a year through continuous culturing with a diesel concentration of about 800mgl(-1) and which now showed complete removal of this concentration of diesel within 30days was compared to that of a freshly inoculated new anode MFC (showing 83.4% removal of diesel) with a simultaneous power generation of 90.81mW/m(2) and 15.04mW/m(2) respectively. The behaviour of pre-cultured anodes at a higher concentration of PH (8000mgl(-1)) was also investigated. Scanning electron microscopy observation revealed a thick biofilm covering the pre-cultured anodic electrode but not the anode from the freshly inoculated MFC. High resolution imaging showed the presence of thin 60nm diametre pilus-like projections emanating from the cells. Anodic microbial community profiling confirmed that the selection for diesel degrading exoelectrogenic bacteria had occurred. Identification of a biodegradative gene (alkB) provided strong evidence of the catabolic pathway used for diesel degradation in the DMFCs.

Occurrence of diverse alkane hydroxylase alkB genes in indigenous oil-degrading bacteria of Baltic Sea surface water.

Formation of specific oil degrading bacterial communities in diesel fuel, crude oil, heptane and hexadecane supplemented microcosms of the Baltic Sea surface water samples was revealed. The 475 sequences from constructed alkane hydroxylase alkB gene clone libraries were grouped into 30 OPFs. The two largest groups were most similar to Pedobacter sp. (245 from 475) and Limnobacter sp. (112 from 475) alkB gene sequences. From 56 alkane-degrading bacterial strains 41 belonged to the Pseudomonas spp. and 8 to the Rhodococcus spp. having redundant alkB genes. Together 68 alkB gene sequences were identified. These genes grouped into 20 OPFs, half of them being specific only to the isolated strains. Altogether 543 diverse alkB genes were characterized in the brackish Baltic Sea water; some of them representing novel lineages having very low sequence identities with corresponding genes of the reference strains.