Hydrocarbon-degrading Bacteria Research Articles

Halotolerant Bacillus velezensis sustainably enhanced oil recovery of low permeability oil reservoirs by producing biosurfactant and modulating the oil microbiome

Low permeability oil reservoirs are widely distributed in China and rich in oil resources. Microbial enhanced oil recovery (MEOR) is a promising technology for low permeability oil reservoirs, but its application is limited by the harsh reservoir conditions. In this study, we demonstrated that halotolerant strain Bacillus velezensis BSA1 could produce biosurfactant and modulate oil microbiome for enhancing oil recovery in low permeability oil reservoirs. B. velezensis BSA1 could produce biosurfactant which comprised of surfactin and was stable in harsh environments. The MEOR column experiments showed that the oil recovered by B. velezensis BSA1 was 21.6 ± 1.87 %. We conducted successful field application in a low permeability reservoir and the cumulative oil production was more than 500 m3 with the effective period of 12 months. After monitoring of microbial community in the reservoir for a year, it showed that B. velezensis BSA1 promoted the abundance of surfactant-producing bacteria and hydrocarbon-degrading bacteria increasing. The enrichment of the functions of surfactant synthesis, hydrocarbon degradation, and methane gas production contributed to the enhancement of oil recovery. Overall, results of this study showed that using biosurfactant-producing and halotolerant B. velezensis BSA1 was a promising microbial strategy for sustainable MEOR of low permeability oil reservoir.

Zavarzinia marina sp. nov., a novel hydrocarbon-degrading bacterium isolated from deep chlorophyll maximum layer seawater of the West Pacific Ocean and emended description of the genus Zavarzinia.

A Gram-stain-negative, motile, non-spore-forming, strictly aerobic and rod-shaped bacterial strain, Adcm-6AT, was isolated from a seawater sample collected from the deep chlorophyll maximum layer in the West Pacific Ocean. Strain Adcm-6AT grew at 20-37 °C (optimum, 28-32 °C), at pH 6-11 (pH 7) and in the presence of 0-6 % (1-2 %) NaCl (w/v). Phylogenetic analysis based on 16S rRNA gene sequences indicated that it belonged to the genus Zavarzinia and had 97.7 and 96.9 % sequence similarity to Zavarzinia compransoris DSM 1231T and Zavarzinia aquatilis JCM 32263T, respectively. Digital DNA-DNA hybridization and average nucleotide identity values between strain Adcm-6AT and the two type strains were 22.2-22.9 % and 79.7-80.4 %, respectively. The principal fatty acids were C19:0 cyclo ω8c, summed feature 8 (C18:1 ω6c and/or C18:1 ω7c) and C16:0. The predominant respiratory quinone was Q-10. The polar lipids were diphosphatidylglycerol, two phosphatidylethanolamines, two phosphatidyglycerols and an unidentified lipid. The genomic DNA G+C content of strain Adcm-6AT was 67.7 %. Based on phylogenetic analysis and genomic-based relatedness indices, as well as phenotypic and genotypic characteristics, strain Adcm-6AT represents a novel species within the genus Zavarzinia, for which the name Zavarzinia marina sp. nov. is proposed. The type strain is Adcm-6AT (=MCCC M24951T=KCTC 82849T).

Effects of different colored polyethylene mulching films on bacterial communities from soil during enrichment incubation

Studies have shown that mulching agricultural fields with plastic residues can influence microbial communities in the environment, but few studies have investigated the differences in the soil microbial communities in distinct areas under mulching with different colored plastic products. Thus, in this study, we explored how different colored polyethylene mulching films (PMFs) might affect soil bacterial communities during enrichment incubation. We found significant differences in the bacterial communities under different colored PMFs after incubation. Treatment with the same colored PMF obtained more similar bacterial community compositions. For instance, at the class level, Gammaproteobacteria and Bacteroidia were most abundant with black PMF, whereas Actinobacteria and Bacteroidia were most abundant with white PMF. The most abundant genera were Acinetobacter and Chryseobacterium with black PMF but Rhodanobacter and Paenarthrobacter with white PMF. Polyethylene- and hydrocarbon-degrading bacteria were the core members detected under both treatments, and the bacterial communities were predicted to have the potential for the biodegradation and metabolism of xenobiotics after enrichment culture according to the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) tool. In addition, the bacterial communities in soil from Xinjiang treated with white PMF and in soil from Yangling treated with black PMF were strongly correlated and stable. Our results suggest that the color of the PMF applied affected the soil bacterial communities, where plastics with the same color may have recruited similar species of microorganisms, although the origins of these microorganisms were not the same.

Interaction between indigenous hydrocarbon-degrading bacteria in reconstituted mixtures for remediation of weathered oil in soil

It has been demonstrated that biostimulation is necessary to investigate the interactions between indigenous bacteria and establish an approach for the bioremediation of soils contaminated with weathered oil. This was achieved by adjusting the carbon (C)/nitrogen (N)/phosphorus (P) ratio to 100/10/1 combined with the application of 0.8 mL/kg Tween-80. In addition, three indigenous bacteria isolated from the same soil were introduced solely or combined concomitantly with stimulation. Removal of n-alkanes and the ratios of n-heptadecane to pristane and n-octadecane to phytane were taken to indicate their biodegradation performance over a period of 16 weeks. One strain of Pseudomonas aeruginosa D7S1 improved the efficiency of the process of stimulation. However, another Pseudomonas aeruginosa, D5D1, inhibited the overall process when combined with other bacteria. One strain of Bacillus licheniformis D1D2 did not affect the process significantly. The Fourier transform infrared analysis of the residual hydrocarbons supported the conclusions pertaining to the biodegradation processes when probing the modifications in densities and stretching. The indigenous bacteria cannot mutually benefit from their metabolisms for bioremediation if augmented artificially. However, the strain Pseudomonas. aeruginosa D7S1 was able to perform better alone than in a consortium of indigenous bacteria.

Bacterial biofilms on medical masks disposed in the marine environment: a hotspot of biological and functional diversity

The present paper was aimed at investigating the role of disposable medical masks as a substrate for microbial biofilm growth and for the selection of specific microbial traits in highly impacted marine environments. In this view, we have immerged masks in a coastal area affected by a continuous input of artisanal fishery wastes and hydrocarbons pollution caused by intense maritime traffic. Masks maintained one month in the field were colonized by a bacterial community significantly different from that detected in the natural matrices from the same areas (seawater and sediments). The masks served as a viable substrate for the growth and enrichment of phototrophic microorganisms (Oxyphotobacteria), as well as Ruminococcaceae, Gracilibacteria, and Holophageae. In a follow-up investigation, masks previously colonized in the field were transferred in lab-scale microcosms which were supplemented with hydrocarbons and which contained also a piece of a virgin mask. After one month, a shift in the community composition, likely triggered by hydrocarbons addition, was observed in the previously colonized mask, with signatures characteristic of hydrocarbon-degrading microbial groups. Such hydrocarbon-degrading bacteria were also found to colonize the virgin mask. Remarkably, SEM micrographs provided indications of the occurrence of morphological modifications of the surface components of the virgin masks colonized by hydrocarbonoclastic bacteria. Overall, for the first time, we have demonstrated the potential risk for human and animal health determined by the uncorrected disposal of masks which are suitable substrates for pathogens colonization, permanence and spreading. Moreover, we have herein strengthened the knowledge on the role of hydrocarbon-degrading bacteria in the colonization and modification of fossil-based plastics in marine environment.

Even allocation of benefits stabilizes microbial community engaged in metabolic division of labor.

Microbial communities execute metabolic pathways to drive global nutrient cycles. Within a community, functionally specialized strains can perform different yet complementary steps within a linear pathway, a phenomenon termed metabolic division of labor (MDOL). However, little is known about how such metabolic behaviors shape microbial communities. Here, we derive a theoretical framework to define the assembly of a community that degrades an organic compound through MDOL. The framework indicates that to ensure community stability, the strains performing the initial steps should hold a growth advantage (m) over the "private benefit" (n) of the strain performing the last step. The steady-state frequency of the last strain is then determined by the quotient of n and m. Our experiments show that the framework accurately predicts the assembly of our synthetic consortia that degrade naphthalene through MDOL. Our results provide insights for designing and managing stable microbial systems for metabolic pathway optimization.

Study on the Breeding and Characterization of High-Efficiency Oil-Degrading Bacteria by Mutagenesis

In the present study, a high-efficiency petroleum hydrocarbon-degrading bacterium MX1 was screened from petrochemical wastewater sludge, and MX1 was identified using morphological, physiological, and biochemical experiments and combined with 16S rDNA. Results showed that the the MX1 strain belongs to Enterobacter sp. The degradation conditions were an incubation time of 18 days, temperature of 30 °C, pH of 7, and salinity of 2% (w/v), and the degradation proportion was 37.41% for 7 days. The combination of microwave and ultraviolet mutagenesis yielded the strain MXM3U2. The mutant strain had a petroleum hydrocarbon breakdown efficiency of 56.74% after 7 days of culture, and this value was 51.66% higher than the original strain. The number of strains and the rate of degradation of n-alkanes (C16, C24, C32, and C40) decreased steadily with the increase in carbon chains in the degradation test. GC/MS (Gas chromatography mass spectrometry) results showed that in the process of degrading crude oil, the hydrocarbons with carbon number C < 24 were degraded first, followed by hydrocarbons with carbon number C > 24. The strains had a good degradation effect on pristane, naphthalene, and phenanthrene. In this study, a high-efficiency petroleum hydrocarbon-degrading bacterium was screened via microwave-ultraviolet composite mutagenesis technology.

Identification and implications of a core bacterial microbiome in 19 clonal cultures laboratory-reared for months to years of the cosmopolitan dinoflagellate Karlodinium veneficum

Identification of a core microbiome (a group of taxa commonly present and consistently abundant in most samples of host populations) is important to capture the key microbes closely associated with a host population, as this process may potentially contribute to further revealing their spatial distribution, temporal stability, ecological influence, and even impacts on their host’s functions and fitness. The naked dinoflagellate Karlodinium veneficum is a cosmopolitan and toxic species, which is also notorious in forming harmful algal blooms (HABs) and causing massive fish-kills. Here we reported the core microbiome tightly associated with 19 strains of K. veneficum that were originally isolated from 6 geographic locations along the coast of China and from an estuary of Chesapeake Bay, United States, and have been maintained in the laboratory for several months to over 14 years. Using high-throughput metabarcoding of the partial 16S rRNA gene amplicons, a total of 1,417 prokaryotic features were detected in the entire bacterial microbiome, which were assigned to 17 phyla, 35 classes, 90 orders, 273 families, and 716 genera. Although the bacterial communities associated with K. veneficum cultures displayed heterogeneity in feature (sequences clustered at 100% sequence similarity) composition among strains, a core set of 6 genera were found persistent in their phycospheres, which could contribute up to 74.54% of the whole bacterial microbiome. Three γ-proteobacteria members of the “core,” namely, Alteromonas, Marinobacter, and Methylophaga, were the predominant core genera and made up 83.25% of the core bacterial microbiome. The other 3 core genera, Alcanivorax, Thalassospira, and Ponticoccus, are reported to preferably utilize hydrocarbons as sole or major source of carbon and energy, and two of which (Alcanivorax and Ponticoccus) are recognized as obligate hydrocarbonoclastic bacteria (OHCB). Since OHCB generally present in extremely low abundance in marine water and elevate their abundance mostly in petroleum-impacted water, our detection in K. veneficum cultures suggests that the occurrence of obligate and generalist hydrocarbon-degrading bacteria living with dinoflagellates may be more frequent in nature. Our work identified a core microbiome with stable association with the harmful alga K. veneficum and opened a window for further characterization of the physiological mechanisms and ecological implications for the dinoflagellate-bacteria association.

Microbial communities on plastic particles in surface waters differ from subsurface waters of the North Pacific Subtropical Gyre

The long-term fate of plastics in the ocean and their interactions with marine microorganisms remain poorly understood. In particular, the role of sinking plastic particles as a transport vector for surface microbes towards the deep sea has not been investigated. Here, we present the first data on the composition of microbial communities on floating and suspended plastic particles recovered from the surface to the bathypelagic water column (0-2000 m water depth) of the North Pacific Subtropical Gyre. Microbial community composition of suspended plastic particles differed from that of plastic particles afloat at the sea surface. However, in both compartments, a diversity of hydrocarbon-degrading bacteria was identified. These findings indicate that microbial community members initially present on floating plastics are quickly replaced by microorganisms acquired from deeper water layers, thus suggesting a limited efficiency of sinking plastic particles to vertically transport microorganisms in the North Pacific Subtropical Gyre.

Complete Genome Sequences of One Salt-Tolerant and Petroleum Hydrocarbon-Emulsifying Terribacillus saccharophilus Strain ZY-1.

Salt tolerance is one of the most important problems in the field of environmental governance and restoration. Among the various sources of factors, except temperature, salinity is a key factor that interrupts bacterial growth significantly. In this regard, constant efforts are made for the development of salt-tolerant strains, but few strains with salt tolerance, such as Terribacillus saccharophilus, were found, and there are still few relevant reports about their salt tolerance from complete genomic analysis. Furthermore, with the development of the economy, environmental pollution caused by oil exploitation has attracted much attention, so it is crucial to find the bacteria from T. saccharophilus which could degrade petroleum hydrocarbon even under high-salt conditions. Herein, one T. saccharophilus strain named ZY-1 with salt tolerance was isolated by increasing the salinity on LB medium step by step with reservoir water as the bacterial source. Its complete genome was sequenced, which was the first report of the complete genome for T. saccharophilus species with petroleum hydrocarbon degradation and emulsifying properties. In addition, its genome sequences were compared with the other five strains that are from the same genus level. The results indicated that there really exist some differences among them. In addition, some characteristics were studied. The salt-tolerant strain ZY-1 developed in this study and its emulsification and degradation performance of petroleum hydrocarbons were studied, which is expected to widely broaden the research scope of petroleum hydrocarbon-degrading bacteria in the oil field environment even in the extreme environment. The experiments verified that ZY-1 could significantly grow not only in the salt field but also in the oil field environment. It also demonstrated that the developed salt-tolerant strain can be applied in the petroleum hydrocarbon pollution field for bioremediation. In addition, we expect that the identified variants which occurred specifically in the high-salt strain will enhance the molecular biological understanding and be broadly applied to the biological engineering field.

Microbiome profile of soil and rhizosphere plants growing in traditional oil mining land in Wonocolo, Bojonegoro, Indonesia

Traditional oil mining poses negative effects on the environment through pollution with crude oil. One of the traditional mining sites in Wonocolo, Bojonegoro, Indonesia was reported to contaminate the surrounding area with a high level of crude oil. Therefore, this study aims to examine the microbiome profiles of contaminated soil and the rhizosphere of naturalized plants growing at the sites. It was conducted in Wonocolo, Bojonegoro to obtain an insight into the possible remediation efforts of using indigenous hydrocarbon-degrading bacteria and naturalized plants as in situ remediation agents. The results showed that the soil located close to the oil well-contained a high level of crude oil at 24.8%, and exhibited a distinct microbiome profile compared to those located further which had lower crude oil contamination of 14.15, 10.89, and 4.9%. Soil with the highest level of crude oil contamination had a comparatively higher relative abundance of assA, an anaerobic alkene-degrading gene. Meanwhile, the rhizosphere of the two naturalized plants, Muntingia calabura, and Pennisetum purpureum, exhibited indifferent microbiome profiles compared to the soil. They were found to contain less abundant hydrocarbon-degrading genes, such as C230, PAH-RHD-GP, nahAc, assA, and alkB suggesting that these naturalized plants might not be a suitable tool for in-situ remediation.

Plastic-Associated Microbial Communities in Aquaculture Areas

Microorganisms colonize plastics in the aquatic environment but their composition on plastics used in aquaculture remains poorly studied. Microorganisms play a significant role in aquaculture in terms of water quality and the health of cultivated species. In the current study, we explored the composition of microorganisms on floating plastics and their surrounding water collected from ponds and open aquaculture areas. Using scanning electron microscopy, the diversity of microbial communities, primarily diatoms, and bacteria were identified on the plastic surfaces. Additionally, epifluorescence microscopy revealed that prokaryotes were colonized on all plastic samples from 0.1 to 29.27×103 cells/cm2, with a high abundance found in open aquaculture areas compared to ponds. Bacterial communities were characterized by 16S rRNA sequencing which showed that bacterial communities on plastics were dominated by Proteobacteria, Cyanobacteria, Bacteroidetes, and Actinobacteria. The level of these microbial communities on the plastics differed from those found in the surrounding seawater samples and the abundance of potentially pathogenic bacteria was higher in plastics than in seawater samples. Moreover, hydrocarbon-degrading bacteria were more abundant in the investigated plastic samples than in the water samples. This study contributes to the knowledge regarding the plastisphere community in aquaculture.

A highly effective polycyclic aromatic hydrocarbon-degrading bacterium, Paracoccus sp. HPD-2, shows opposite remediation potential in two soil types

Bioaugmentation is an efficient and eco-friendly strategy for the bioremediation of polycyclic aromatic hydrocarbons (PAHs). Since the degrading abilities of soils can greatly alter the abilities of PAH-degrading bacteria, illustrating the potential and mechanism of highly efficient degrading bacteria in different soil environments is of great importance for bioremediation. A PAH-degrading bacterium, Paracoccus aminovorans HPD-2, and two soil types, red and paddy soils, with distinct PAH-degrading abilities, were selected for this study. A soil microcosm experiment was performed by adding pyrene (PYR) and benzo[a]pyrene (B[a]P). Illumina sequencing was used to examine bacterial community structure. The results showed that inoculation with HPD-2 significantly elevated PYR and B[a]P degradation rates by 44.7% and 30.7%, respectively, in the red soil, while it only improved the degradation rates by 1.9% and 11%, respectively, in the paddy soil. To investigate the underlying mechanism, the fate of strain HPD-2 and the response of the indigenous bacterial communities were determined. Strain HPD-2 occupied certain niches in both soils, and the addition of the bacterium changed the native community structure more noticeably in the red soil than in the paddy soil. The addition of PAHs and strain HPD-2 significantly changed the abundances of 7 phyla among the 15 detected phyla in the red soil. In the paddy soil, 5 of the 12 dominant phyla were significantly affected by PAHs and the inoculation of HPD-2, while 6 new phyla were detected in the low-abundance phyla (< 0.1%). The abundances of Massilia, Burkholderia , and Rhodococcus genera with PAH degradation efficiency were significantly increased by the inoculation of HPD-2 in the red soil during 42 d of incubation. Meanwhile, in the paddy soil, the most dominant effective genus, Massilia , was reduced by HPD-2 inoculation. This research revealed the remediation ability and inherent mechanism of the highly effective PAH-degrading strain HPD-2 in two different soil types, which would provide a theoretical basis for the application of degrading bacteria in different soils.

A mechanistic understanding of polyethylene biodegradation by the marine bacterium Alcanivorax

Polyethylene (PE) is one of the most recalcitrant carbon-based synthetic materials produced and, currently, the most ubiquitous plastic pollutant found in nature. Over time, combined abiotic and biotic processes are thought to eventually breakdown PE. Despite limited evidence of biological PE degradation and speculation that hydrocarbon-degrading bacteria found within the plastisphere is an indication of biodegradation, there is no clear mechanistic understanding of the process. Here, using high-throughput proteomics, we investigated the molecular processes that take place in the hydrocarbon-degrading marine bacterium Alcanivorax sp. 24 when grown in the presence of low density PE (LDPE). As well as efficiently utilising and assimilating the leachate of weathered LDPE, the bacterium was able to reduce the molecular weight distribution (Mw from 122 to 83 kg/mol) and overall mass of pristine LDPE films (0.9 % after 34 days of incubation). Most interestingly, Alcanivorax acquired the isotopic signature of the pristine plastic and induced an extensive array of metabolic pathways for aliphatic compound degradation. Presumably, the primary biodegradation of LDPE by Alcanivorax sp. 24 is possible via the production of extracellular reactive oxygen species as observed both by the material’s surface oxidation and the measurement of superoxide in the culture with LDPE. Our findings confirm that hydrocarbon-biodegrading bacteria within the plastisphere may in fact have a role in degrading PE.

ISOLATED BACTERIA FROM HOT SPRINGS ABLE TO USE HYDROCARBONS AS CARBON SOURCE

Petroleum derivates used in energy production are gravely pollutants for the ecosystem, especially for aquatic environments and human health. This study aimed to isolate hydrocarbons-degrading bacteria from hot springs. Three strains of hydrocarbondegrading bacteria strains, belonging to the Bacillus and one of the genus Lysinibacillus were isolated. These strains tolerate temperatures from 65 to 100 ºC and were able to degrade and grow on BH medium supplemented with gasoline and diesel. Strain M2-7 shared 100 % 16S rRNA identity with Bacillus licheniformis and was the only able to degrade pyrene and benzopyrene among these isolated strains. The results indicate that B. licheniformis M2-7 could degrade a wider range of hydrocarbons and some recalcitrant hydrocarbon components, which could be particularly helpful for the treatment and bioremediation of hydrocarbon-polluted systems.

Reduction of Surface Tension of Petroleum Using Hydrocarbon Degrading Bacterial Activity

Hydrocarbon degrading bacteria produce biosurfactants, which facilitate the biodegradation process. This is the first step towards getting access to interactions between the bacteria's hydrophilic surface and the hydrophobic surface of the hydrocarbons. Due to their amphipathic nature, biosurfactants facilitate this interaction. The focus of the study is to obtain hydrocarbon-degrading bacteria isolates and evaluate biosurfactant activity in reducing water surface tension. Hydrocarbon-degrading bacteria were isolated from contaminated marine sediment samples and consequently cultured on artificial seawater media with the addition of petroleum hydrocarbons as a carbon source. Surface tension reduction of biosurfactants was measured using a digital K20-EasyDyne tensiometer (KRÜSS: Hamburg, Germany). The study indicates that the isolates have biodegradation activity and reduced water surface tension by 22.14 mN/m, the data demonstrated that biosurfactant production was most effective on the third day of the exponential phase incubation. These studies demonstrate the effectiveness of hydrocarbon-degrading bacteria to produce biosurfactants as biodegradation agents to solve the problem of oil pollution.

Microbial community response to simulated diluted bitumen spills in coastal seawater and implications for oil spill response.

Oil spills in coastal waters can have devastating impacts on local ecosystems, from the microscopic base through to mammals and seabirds. Increasing transport of diluted bitumen has led to concerns about how this novel product might impact coastal ecosystems. A mesocosm study determined that the type of diluent and the season can affect the concentrations of hydrocarbons entering the water column from a surface spill. Those same mesocosms were sampled to determine whether diluent type and season also affected the microbial response to a surface spill. Overall, there were no differences in impacts among the three types of diluted bitumen, but there were consistent responses to all products within each season. Although microbial abundances with diluted bitumen rarely differed from unoiled controls, community structure in these organisms shifted in response to hydrocarbons, with hydrocarbon-degrading bacteria becoming more abundant. The relative abundance of heterotrophic eukaryotes also increased with diluted bitumen, with few photosynthetic organisms responding positively to oil. Overall shifts in the microbial communities were minimal relative to spills of conventional oil products, with low concentrations of hydrocarbons in the water column. Oil spill response should focus on addressing the surface slick to prevent sinking or stranding to minimize ecosystem impacts.

Characterization and Application of Biopolymer Producing Bacteria for Enhanced Oil Recovery

The objective of this research is to isolate and identify hydrocarbon-degrading bacteria for biopolymer synthesis and application in the augmentation of Nigerian heavy crude oil recovery. MEOR refers to the process of injecting either indigenous or non-indigenous microbes into hydrocarbon reserves. Injecting microorganisms with nutritional broth facilitate the formation of essential metabolites such as biosurfactants, biopolymers, and gases, resulting in decreased interfacial tension, viscosity modification, and mobility control. It is environmentally friendly, less expensive to implement, and requires minimal or no changes to the existing infrastructure. A soil sample from a hydrocarbon-contaminated site in Ogoniland was collected and sent to a laboratory for physicochemical and microbiological investigation. Bacillus sp, Pseudomonas sp, and Klebsiella sp were biochemically identified after screening three isolates for biopolymer production using Sudan black solution. To assess the ideal growth and biopolymer synthesis capability under reservoir conditions, a variety of pH, temperature, salinity, carbon, and nitrogen nutrition sources were applied to selected microorganisms. Peptone is the optimal nitrogen source for Bacillus sp, glucose is the optimal carbon source for Bacillus sp, and glycerol is the optimal carbon source for Pseudomonas sp and Klebsiella sp, as indicated by the results. In addition, the following are the ideal parameter ranges for the three microorganisms: pH 7–8, a temperature range between 25 and 350 degrees Celsius, and a salinity range between 0.5 and 5% are all desirable conditions for a body of water. After inoculation with microorganisms and the optimum nutrient source, an additional recovery range of 18.33% to 29.09% of the pore capacity was achieved. The post-recovery analysis uncovered a remarkable transformation of heavy crude to light hydrocarbon components by an average of 20.33 percent with glucose and 97.27 percent with peptone.

Pinisolibacter aquiterrae sp. nov., a novel aromatic hydrocarbon-degrading bacterium isolated from benzene-, and xylene-degrading enrichment cultures, and emended description of the genus Pinisolibacter.

Two Gram-reaction-negative strains, designated as B13T and MA2-2, were isolated from two different aromatic hydrocarbon-degrading enrichment cultures and characterized using a polyphasic approach to determine their taxonomic position. The two strains had identical 16S rRNA gene sequences and were most closely related to Pinisolibacter ravus E9T (97.36 %) and Siculibacillus lacustris SA-279T (96.33 %). Cells were facultatively aerobic rods and motile with a single polar flagellum. The strains were able to degrade ethylbenzene as sole source of carbon and energy. The assembled genome of strain B13T had a total length of 4.91 Mb and the DNA G+C content was 68.8 mol%. The predominant fatty acids (>5 % of the total) of strains B13T and MA2-2 were C18 : 1 ω7c/C18 : 1 ω6c, C16 : 1 ω7c/C16 : 1 ω6c and C16 : 0. The major ubiquinone of strain B13T was Q10, while the major polar lipids were phosphatidyl-N-methylethanolamine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and a phospholipid. Based on phenotypic characteristics and phylogenetic data, it is concluded that strains B13T and MA2-2 are members of the genus Pinisolibacter and represent a novel species for which the name Pinisolibacter aquiterrae sp. nov. is proposed. The type strain of the species is strain B13T (=LMG 32346T=NCAIM B.02665T).

Free and Immobilized Microbial Culture–Mediated Crude Oil Degradation and Microbial Diversity Changes Through Taxonomic and Functional Markers in a Sandy Loam Soil

Crude oil contamination of soil and water resources is a widespread issue. The present study evaluated the degradation of aliphatic hydrocarbons (C11–C36) in crude oil by 17 bacteria isolated from a crude oil–contaminated soil. The results suggested that Pseudomonas sp. and Bacillus amyloliquefaciens were the best hydrocarbon-degrading bacteria in the presence of surfactant Tween-80 (0.1% w/v). Based on the present investigation and a previous study, Pseudomonas sp. + B. amyloliquefaciens and fungus Aspergillus sydowii were identified as best oil degraders and were immobilized in alginate–bentonite beads, guargum–nanobenonite water dispersible granules (WDGs), and carboxy methyl cellulose (CMC)–bentonite composite. Sandy loam soil was fortified with 1, 2, and 5% crude oil, and total petroleum hydrocarbon (TPH) degradation efficiency of free cultures and bio-formulations was evaluated in sandy loam soils. Compared to a half-life (t1/2) of 69.7 days in the control soil (1% oil), free cultures of Pseudomonas sp. + B. amyloliquefaciens and A. sydowii degraded TPH with t1/2 of 10.8 and 19.4 days, respectively. Increasing the oil content slowed down degradation, and the t1/2 in the control and soils inoculated with Pseudomonas sp. + B. amyloliquefaciens and A. sydowii was 72.9, 14.7, and 22.2 days (2%) and 87.0, 23.4, and 30.8 days (5%), respectively. Supplementing soil with ammonium sulfate (1%) enhanced TPH degradation by Pseudomonas sp. + B. amyloliquefaciens (t1/2–10 days) and A. sydowii (t1/2–12.7 days). All three bio-formulations were effective in degrading TPH (1%), and the t1/2 was 10.7–11.9 days (Pseudomonas sp. + B. amyloliquefaciens and 14–20.2 days (A. sydowii) and were at par with free cultures. Microbial diversity analysis based on taxonomic markers and functional markers suggested that the bioaugmentation process helped keep soil in the active stage and restored the original microbial population to some extent. The present study concluded that bio-formulations of crude oil–degrading microbes can be exploited for its degradation in the contaminated environment.