Impacts of rhizoremediation and biostimulation on soil microbial community, for enhanced degradation of petroleum hydrocarbons in crude oil-contaminated agricultural soils.

Hydrocarbonoclastic bacterial strains were isolated from rhizosphere of plants growing in crude oil-contaminated sites of Assam, India. These bacteria showed plant growth-promoting attributes, even when exposed to crude oil. Two independent pot trials were conducted to test the rhizodegradation ability of the bacterial consortium in combination of plants Azadirchta indica or Delonix regia in crude oil-contaminated soil. Field experiments were conducted at two crude oil-contaminated agricultural field at Assam (India), where plants (A. indica or D. regia) were grown with the selected bacterial consortium consisting of five hydrocarbonoclastic bacterial isolates (Gordonia amicalis BB-DAC, Pseudomonas aeruginosa BB-BE3, P. citronellolis BB-NA1, Rhodococcus ruber BB-VND, and Ochrobactrum anthropi BB-NM2), and NPK was added to the soil for biostimulation. The bacterial consortium-NPK biostimulation led to change in rhizosphere microbiome with enhanced degradation of petroleum hydrocarbons (PHs) in soils contaminated with crude oil. After 120 days of planting A. indica + consortium + NPK treatment, degradation of PHs was found to be up to 67%, which was 55% with D. regia with the same treatment. Significant changes in the activities of plant and soil enzymes were also noted. The shift is bacterial community was also apparent as with A. indica, the relative abundance of Proteobacteria, Actinobacteria, and Acidobacteria increased by 35.35%, 26.59%, and 20.98%, respectively. In the case of D. regia, the relative abundance of Proteobacteria, Actinobacteria, and Acidobacteria were increased by 39.28%, 35.79%, and 9.60%, respectively. The predicted gene functions shifted in favor of the breakdown of xenobiotic compounds. This study suggests that a combination of plant-bacterial consortium and NPK biostimulation could be a productive approach to bioengineering the rhizosphere microbiome for the purpose of commercial bioremediation of crude oil-contaminated sites, which is a major environmental issue faced globally.

De novogenomic analysis ofEnterobacter asburiaeEBRJ12, a plant growth-promoting rhizobacteria isolated from the rhizosphere of Phaseolus vulgarisL.

AIM: Environmental stresses such as water deficit induced stress are one of the major limiting factors in crop production. However, some plant growth-promoting rhizobacteria (PGPR) can promote plant growth in such adverse condition. Therefore, the objective was to isolate rhizospheric bacteria from Phaseolus vulgaris L. growing in a drought-affected soil and to analyze its plant growth promoting (PGP) efficacy to black gram (Vigna mungo L.) and Bhut jolokia (Capsicum chinense Jacq.). Whole-genome sequencing of the potential bacteria was targeted to analyze the genetic potential of the isolate as a plant growth-promoting agent. METHODS AND RESULTS: The isolate Enterobacter asburiae EBRJ12 was selected based on its PGP efficacy, which significantly improved plant growth and development. The genomic analysis revealed the presence of one circular chromosome of size 4.8 Mb containing 16 genes for osmotic stress regulation including osmotically inducible protein osmY, outer membrane protein A precursor ompA, aquaporin Z, and an operon for osmoprotectant ABC transporter yehZYXW. Moreover, the genome has a complete genetic cluster for biosynthesis of siderophore Enterobactin and siderophore Aerobactin.The PGP effects were verified with black gram and Bhut jolokia in pot experiments. The isolate significantly increased the shoot length by 35.0% and root length by 58.0% of black gram, while 41.0% and 57.0% of elevation in shoot and root length were observed in Bhut jolokia compared to non-inoculated plants. CONCLUSIONS: The EBRJ12 has PGP features that could improve the growth in host plants, and the genomic characterization revealed the presence of genetic potential for plant growth promotion.

The structure-function relationship of bacterial transcriptional regulators as a target for enhanced biodegradation of aromatic hydrocarbons.

The sheer persistence and dissemination of xenobiotic aromatic hydrocarbons contaminants demand sustainable solutions for degradation. Therefore, major pathways of microbial catabolism of aromatic hydrocarbons under aerobic conditions are reviewed and analysed to elicit enhanced biodegradation of aromatic hydrocarbons, via the structure-function relationship of bacterial transcriptional regulators. The initial step of the catabolism occurs via the incorporation of molecular oxygen into the aromatic ring by a multicomponent aromatic ring-hydroxylating-dioxygenase (RHD) enzyme system or monooxygenase system forming different central intermediates such as catechols, protocatechuates, gentisates, and (hydroxy)benzoquinols. The central or lower pathways involve the ring cleavage of central intermediates to tricarboxylic acids. These metabolic pathways are tightly regulated, where the inducer or substrate-specific transcriptional regulation of aromatic catabolic pathways depend on the specific regulatory proteins that acts on a specific promoter in response to a respective inducer signal. These regulatory systems have been grouped according to the regulatory proteins and their families, and identified based on their conserved motifs and their modes of DNA binding. Different regulators from protein families like AraC/XylS, LysR, XylR/NtrC, IclR, etc. have been identified, that are involved in aromatic hydrocarbon regulation. These regulatory proteins have different structures and have different mechanisms of regulation. The proteins of the XylS/AraC family have two domains structure: a highly conserved C-terminus that contains two HTH motifs and the N-terminus end containing the regulatory domain. The LysR type regulatory proteins (LTTRs) act as tetramers that have a helix-turn-helix (HTH) domain at the N terminus and a regulatory binding domain at the C terminus. The IclR regulatory proteins also have a helix-turn-helix DNA binding motif in the N-terminus domain-like LTTRs but include an effector binding motif in the C-terminus domain that is also involved in subunit multimerization. In contrast, the XylR-like regulatory proteins have three domain structures; one for effector sensing, another for ATP binding and hydrolysis, and a domain for DNA binding which contains an HTH motif. This review describes in depth and critical assessment of the aerobic bacterial degradation pathways of aromatic hydrocarbon pollutants with state of art information, underscores areas that are viable and others that require further development, with particular reference to metabolic engineering and synthetic biology applications.

Comparative Metagenomic Analysis of Two Alkaline Hot Springs of Madhya Pradesh, India and Deciphering the Extremophiles for Industrial Enzymes.

Hot springs are considered to be a unique environment with extremophiles, that are sources of industrially important enzymes, and other biotechnological products. The objective of this study was to undertake, analyze, and characterize the microbiome of two major hot springs located in the state of Madhya Pradesh explicitly, Chhoti Anhoni (Hotspring 1), and Badi Anhoni (Hotspring 2) to find out the inhabitant microbial population, and their functional characteristics. The taxonomic analysis of the microbiome of the hot springs revealed the phylum Proteobacteria was the most abundant taxa in both the hot-springs, however, its abundance in hot-spring 1 (~88%) was more than the hot-spring 2 (~52%). The phylum Bacteroides (~10-22%) was found to be the second most abundant group in the hot-springs followed by Spirocheates (~2-11%), Firmicutes (~6-8%), Chloroflexi (1-5%), etc. The functional analysis of the microbiome revealed different features related to several functions including metabolism of organics and degradation of xenobiotic compounds. The functional analysis showed that most of the attributes of the microbiome was related to metabolism, followed by cellular processes and environmental information processing functions. The functional annotation of the microbiomes at KEGG level 3 annotated the sequences into 279 active features that showed variation in abundance between the hot spring samples, where hot-spring 1 was functionally more diverse. Interestingly, the abundance of functional genes from methanogenic bacteria, was higher in the hot-spring 2, which may be related to the relatively higher pH and temperature than Hotspring 1. The study showed the presence of different unassigned bacterial taxa with high abundance which indicates the potential of novel genera or phylotypes. Culturable isolates (28) were bio-prospected for industrially important enzymes including amylase, protease, lipase, gelatinase, pectinase, cellulase, lecithinase, and xylanase. Seven isolates (25%) had shown positive results for all the enzyme activities whereas 23 isolates (82%) produced Protease, 27 isolates (96%) produced lipase, 27 isolates produced amylase, 26 isolates (92%) produced cellulase, 19 isolates (67%) produced pectinase, 19 isolates (67%) could produce lecithinase, and 13 isolates (46%) produced gelatinase. The seven isolates, positive for all the enzymes were analyzed further for quantitative analysis and identified through molecular characterization.

Paradigm shift in antibiotic-resistome of petroleum hydrocarbon contaminated soil.

The increasing prevalence of antibiotic-resistant microorganisms in both clinical and environmental samples is of great concern for public health. In the present study, environmental samples from seven different sites, heavily contaminated with petroleum hydrocarbons has been examined for the antimicrobial resistome through metagenomic approach. The soil samples were found to be contaminated with high concentration of total petroleum hydrocarbons (average 45 g/kg), polyaromatic hydrocarbons (average ∑16PAH = 280 mg/kg), and heavy metals, which shapes the microbial community and their function. Proteobacteria was found to be predominant phylum in the contaminated habitat with the highest diversity (55.91%) followed by Actinobacteria (9.86%). Although the taxonomical abundance of the non-contaminated sample was not significantly different from contaminated samples, the functional abundance of genes related to antibiotic resistance was found to be higher up to 2 fold in contaminated samples. The comparative metagenomic analysis revealed a higher abundance of different antibiotic resistance genes, especially genes for fluoroquinolones was found to be higher up to 10 fold in contaminated samples. Moreover, the study has shown a significant difference in total functional diversity and abundance, mainly genes for aromatic compound metabolism and genes for phages, mobile genetic elements. These higher abundances of well recognized antibiotic resistance genes, multidrug efflux pumps, and integrons, suggest that the petroleum hydrocarbon contaminated sites can act as reservoirs for development of antibiotic resistance genes (ARGs). From this study, a significant link between the presence of petroleum hydrocarbon and the development of antibiotic resistance in the microbiome of contaminated habitat has been established.

The genomic attributes of Cd-resistant, hydrocarbonoclastic Bacillus subtilis SR1 for rhizodegradation of benzo(a)pyrene under co-contaminated conditions.

Bacillus subtilis SR1 is a metal resistant, polyaromatic hydrocarbon-degrading bacterium isolated from petroleum contaminated sites. This study reports the characteristics of the genome of the isolate containing one circular chromosome (4,093,698 bp) annotated into 4155 genes and 4095 proteins. The genome analysis confirmed the presence of multiple catabolic genes: aromatic ring-hydroxylating dioxygenase (COG2146), aromatic ring hydroxylase (COG2368), catechol 2, 3 dioxygenase (COG2514), 4-hydroxybenzoate decarboxylase (COG0043), carboxymuconolactone decarboxylase (COG0599) responsible for the catabolism of aromatic hydrocarbons along with the genes for biosurfactant production and functional genes (czcD and cadA) for resistance to cadmium, zinc, and cobalt. Gas Chromatography-Mass spectroscopy analysis revealed up to 35% in-vitro degradation of benzo(a)pyrene after 21 days of growth along with the production of different intermediate metabolites. The pot trial analysis in the greenhouse condition validated the rhizodegradation of BaP, which was significantly higher in the presence of plant-microbe association (85%) than degradation in bulk soil (68%).

Difference in the rhizosphere microbiome of Melia azedarach during removal of benzo(a)pyrene from cadmium co-contaminated soil.

Benzo(a)pyrene (BaP) is a highly persistent biohazard polyaromatic hydrocarbon and often reported to be present in soils co-contaminated with heavy metals. The present study explains the rhizodegradation of BaP using bacterial consortium in the rhizosphere of Melia azedarach, along with a change in taxonomical and functional properties of the rhizosphere microbiome. The relative abundance of most dominant phylum Proteobacteria was 2% higher with BaP, while in the presence of both BaP and Cd, its abundance was 2.2% lower. Functional metagenome analysis also revealed the shifting of microbial community and functional gene abundance in the favor of xenobiotic compound degradation upon augmentation of bacterial consortium. Interestingly, upon the addition of BaP the range of functional abundance for genes of PAH degradation (0.165-0.19%), was found to be decreasing. However, augmentation of a bacterial consortium led to an increase in its abundance including genes for degradation of benzoate (0.55-0.64%), toluene (0.2-0.22%), naphthalene (0.25-0.295%) irrespective of the addition of BaP and Cd. Moreover, under greenhouse condition, the application of M. azedarach-bacterial consortium enhanced the degradation of BaP in the rhizosphere (88%) after 60 days, significantly higher than degradation in bulk soil (68.22%). The analysis also showed an increase in degradation of BaP by 15% with plant-native microbe association than in bulk soil. Therefore, the association of M. azedarach-bacterial consortium enhanced the degradation of BaP in soil along with the taxonomical and functional attributes of the rhizosphere microbiome.

Rhizosphere assisted biodegradation of benzo(a)pyrene by cadmium resistant plant-probiotic Serratia marcescens S2I7, and its genomic traits.

Melia azedarach-rhizosphere mediated degradation of benzo(a)pyrene (BaP), in the presence of cadmium (Cd) was studied, using efficient rhizobacterial isolate. Serratia marcescens S2I7, isolated from the petroleum-contaminated site, was able to tolerate up to 3.25 mM Cd. In the presence of Cd, the isolate S2I7 exhibited an increase in the activity of stress-responsive enzyme, glutathione-S-transferase. Gas Chromatography-Mass spectroscopy analysis revealed up to 59% in -vitro degradation of BaP after 21 days, while in the presence of Cd, the degradation was decreased by 14%. The bacterial isolate showed excellent plant growth-promoting attributes and could enhance the growth of host plant in Cd contaminated soil. The 52,41,555 bp genome of isolate S. marcescens S2I7 was sequenced, assembled and annotated into 4694 genes. Among these, 89 genes were identified for the metabolism of aromatic compounds and 172 genes for metal resistance, including the efflux pump system. A 2 MB segment of the genome was identified to contain operons for protocatechuate degradation, catechol degradation, benzoate degradation, and an IclR type regulatory protein pcaR, reported to be involved in the regulation of protocatechuate degradation. A pot trial was performed to validate the ability of S2I7 for rhizodegradation of BaP when applied through Melia azedarach rhizosphere. The rhizodegradation of BaP was significantly higher when augmented with S2I7 (85%) than degradation in bulk soil (68%), but decreased in the presence of Cd (71%).

Rhizosphere mediated biodegradation of benzo(A)pyrene by surfactin producing soil bacilli applied through Melia azadirachta rhizosphere.

Benzo(a)pyrene is a high-molecular-weight polycyclic aromatic hydrocarbon highly persistent in the environment as a biohazard. The present research emphasizes on rhizodegradation of BaP using bacterial isolates, Bacillus flexus S1I26 (NCBI accession no- KX692271), and Paenibacillus sp. S1I8 (KX602663) with plant Melia azadirachta. The isolates produced surfactin type bio-surfactant with high emulsification index that could solubilize BaP efficiently. The extracted crude bio-surfactants could solubilize BaP up to 24.41%, which was higher than the efficiency of synthetic surfactant SDS (9.7%) but less than other synthetic surfactant, tweens 80 (42.79%). The isolates showed excellent degradation of BaP after 21 days in laboratory conditions where B. flexus S2I26 showed degradation of BaP up to 70.7% and isolates Paenibacillus sp. S1I8 showed degradation rate of 76.76% in a liquid medium. Pot trial experiment showed efficient rhizodegradation of BaP in the soil after 60 days in the rhizosphere of plant Melia azadirachta. After application of S1I8 and S1I26, the rate of degradation was found to be much higher (87.42 and 86.08%) than in bulk (68.22%). Therefore, the results suggest that the bio-surfactant producing isolates could be a promising biodegradation tool for benzo(a)pyrene in soil and may be used for bioremediation of hydrocarbon contaminated sites.

The rhizosphere microbiome: Significance in rhizoremediation of polyaromatic hydrocarbon contaminated soil.

Microbial communities are an essential part of plant rhizosphere and participate in the functioning of plants, including rhizoremediation of petroleum contaminants. Rhizoremediation is a promising technology for removal of polyaromatic hydrocarbons based on interactions between plants and microbiome in the rhizosphere. Root exudation in the rhizosphere provides better nutrient uptake for rhizosphere microbiome, and therefore it is considered to be one of the major factors of microbial community function in the rhizosphere that plays a key role in the enhanced PAH biodegradation. Although the importance of the rhizosphere microbiome for plant growth has been widely recognized, the interactions between microbiome and plant roots in the process of rhizosphere mediated remediation of PAH still needs attention. Most of the current researches target PAH degradation by plant or single microorganism, separately, whereas the interactions between plants and whole microbiome are overlooked and its role has been ignored. This review summarizes recent knowledge of PAH degradation in the rhizosphere in the process of plant-microbiome interactions based on emerging omics approaches such as metagenomics, metatranscriptomics, metabolomics and metaproteomics. These omics approaches with combinations to bioinformatics tools provide us a better understanding in integrated activity patterns between plants and rhizosphere microbes, and insight into the biochemical and molecular modification of the meta-organisms (plant-microbiome) to maximize rhizoremediation activity. Moreover, a better understanding of the interactions could lead to the development of techniques to engineer rhizosphere microbiome for better hydrocarbon degradation.

Draft Genome Sequence of Heavy Metal-Resistant Soil Bacterium Serratia marcescens S2I7, Which Has the Ability To Degrade Polyaromatic Hydrocarbons.

Serratia marcescens S2I7 is a heavy metal-resistant, polyaromatic hydrocarbon-degrading bacterium isolated from petroleum-contaminated sites. The genome contains one circular chromosome (5,241,555 bp; GC content 60.1%) with 4,533 coding sequences. The draft genome sequence includes specific genetic elements for degradation of hydrocarbons and for heavy metal resistance.

Draft Genome Sequence of Alcaligenes faecalis BDB4, a Polyaromatic Hydrocarbon-Degrading Bacterium Isolated from Crude Oil-Contaminated Soil.

Alcaligenes fecalis BDB4 was isolated from crude oil-contaminated soil in India. The genome sequence of A. faecalis BDB4 revealed the presence of important genes required for polyaromatic hydrocarbon (PAH) metabolism and other associated functions, such as chemotaxis, membrane transport, and biofilm formation, giving insight into the complete PAH mineralization potential of this bacterium.

Draft Genome Sequence of Polyaromatic Hydrocarbon-Degrading Bacterium Bacillus subtilis SR1, Which Has Plant Growth-Promoting Attributes.

Bacillus subtilis SR1 is a heavy metal-resistant, polyaromatic hydrocarbon-degrading bacterium isolated from rhizospheric soil of contaminated sites. It has the ability to promote plant growth and utilize benzo[a]pyrene as a carbon source. This study reports the characteristics of the genome of B. subtilis SR1, which contains one circular chromosome (4,093,698 bp).

Draft Genome Sequence of Pseudomonas fragi Strain DBC, Which Has the Ability To Degrade High-Molecular-Weight Polyaromatic Hydrocarbons.

Pseudomonas fragi strain DBC was isolated from crude oil-contaminated soil. The genome of P. fragi DBC is comprised of 5,072,304 bp with 54.09% GC content. Genes for degradation of polyaromatic hydrocarbons were found in the genome, in addition to genetic elements for related physiological functions such as chemotaxis, detoxification, and quorum sensing.