Petroleum pollution is a major concern in Antarctica due to the persistent nature of its hydrocarbon components coupled with the region's extreme environmental conditions, which means that bioremediation approaches are largely inapplicable at present. The current study assessed the ability of the psychrotolerant phenol-degrader, Rhodococcus sp. strain AQ5-07, to assimilate diesel fuel as the sole carbon source. Factors expected to influence the efficiency of diesel degradation, including the initial hydrocarbon concentration, nitrogen source concentration and type, temperature, pH and salinity were studied. Strain AQ5-07 displayed optimal cell growth and biodegradation activity at 1% v/v initial diesel concentration, 1 g/L NH4Cl concentration, pH 7 and 1% NaCl during one-factor-at-a-time (OFAT) analyses. Strain AQ5-07 was psychrotolerant based on its optimum growth temperature being near 20 °C. In conventionally optimised media, strain AQ5-07 showed total petroleum hydrocarbons (TPH) mineralisation of 75.83%. However, the optimised condition for TPH mineralisation predicted through statistical response surface methodology (RSM) enhanced the reduction to 90.39% within a 2 days incubation. Our preliminary data support strain AQ5-07 being a potential candidate for real-field soil bioremediation by specifically adopting sludge-phase bioreactor system in chronically cold environments such as Antarctica. The study also confirmed the utility of RSM in medium optimisation.
A Rhodococcus sp. UKMP-5M isolate was shown to detoxify cyanide successfully, suggesting the presence of an intrinsic property in the bacterium which required no prior cyanide exposure for induction of this property. However, in order to promote growth, Rhodococcus sp. UKMP-5M was fully acclimatized to cyanide after 7 successive subcultures in 0.1 mM KCN for 30 days. To further shorten the lag phase and simultaneously increase the tolerance towards higher cyanide concentrations, the bacterium was induced with various nitrile compounds sharing a similar degradatory pathway to cyanide. Acetonitrile emerged as the most favored inducer and the induced cells were able to degrade 0.1 mM KCN almost completely within 18 h. With the addition of subsequent aliquots of 0.1 mM KCN a shorter period for complete removal of cyanide was required, which proved to be advantageous economically. Both resting cells and crude enzyme of Rhodococcus sp. UKMP-5M were able to biodegrade cyanide to ammonia and formate without the formation of formamide, implying the identification of a simple hydrolytic cyanide degradation pathway involving the enzyme cyanidase. Further verification with SDS-PAGE revealed that the molecular weight of the active enzyme was estimated to be 38 kDa, which is consistent with previously reported cyanidases. Since the recent advancement in the application of biological methods in treating cyanide-bearing wastewater has been promising, the discovery of this new bacterium will add value by diversifying the existing microbial populations capable of cyanide detoxification.
This study gives the first picture of whole RNA-Sequencing analysis of a PCB-degrading microbe, Rhodococcus jostii RHA1. Genes that were highly expressed in biphenyl-grown cells, compared with pyruvate-grown cells, were chosen based on the Reads Per Kilobase Million (RPKM) value and were summarized based on the criteria of RPKM ≥100 and fold change ≥2.0. Consequently, 266 total genes were identified as genes expressed particularly for the degradation of biphenyl. After comparison with previous microarray data that identified highly-expressed genes, based on a fold change ≥2.0 and p-value ≤0.05, 62 highly-expressed genes from biphenyl-grown cells were determined from both analytical platforms. As these 62 genes involve known PCB degradation genes, such as bph, etb, and ebd, the genes identified in this study can be considered as essential genes for PCB/biphenyl degradation. In the 62 genes, eleven genes encoding hypothetical proteins were highly expressed in the biphenyl-grown cells. Meanwhile, we identified several highly-expressed unannotated DNA regions on the opposite strand. In order to verify the encoded proteins, two regions were cloned into an expression vector. A protein was successfully obtained from one region at approximately 25 kDa from the unannotated strand. Thus, the genome sequence with transcriptomic analysis gives new insight, considering re-annotation of the genome of R. jostii RHA1, and provides a clearer picture of PCB/biphenyl degradation in this strain.
In the present study, locally isolated Rhodococcus strains were attempted as biological tools for methyl red removal, a mutagenic azo dye posing threat to the environment if left untreated. Rhodococcus strain UCC 0016 demonstrated superior methyl red-decolourizing activity of 100% after 24 h at static condition in comparison to Rhodococcus strain UCC 0008 which recorded 65% decolourization after 72 h. Optimization of physicochemical parameters at 30°C, pH 7 and supplementing glucose as the carbon source resulted in improved methyl red-decolourizing activity at static condition and reduced the time taken to achieve complete decolourization by 80%. Higher concentration of methyl red (5 g/L) was able to be decolourized completely within 10 h by adopting the technology of immobilization. The encapsulated cells of Rhodococcus strain UCC 0016 demonstrated higher substrate affinity (Km = 0.6995 g/L) and an accelerated rate of disappearance of methyl red (Vmax = 0.3203 g/L/h) compared to the free cells. Furthermore, the gellan gum beads could be reused up to nine batches without substantial loss in the catalytic activity indicating the economic importance of this protocol. Analysis of methyl red degradation products revealed no germination inhibition on Triticum aestivum and Vigna radiata demonstrating complete toxicity removal of the parent dye after biological treatment. The occurrence of new and altered peaks (UV-Vis and FTIR) further supported the notion that the removal of methyl red by Rhodococcus strain UCC 0016 was indeed through biodegradation. Therefore, this strain has a huge potential as a candidate for efficient bioremediation of wastewater containing methyl red.
The capability of the crude extract of Rhodococcus UKMP-5M was enhanced by adopting the technology of immobilization. Among the matrices screened to encapsulate the crude extract, gellan gum emerged as the most suitable immobilization material, exceeding the activity of cyanide-degrading enzyme by 61% and 361% in comparison to alginate carrier and non-immobilized crude extract, respectively. Improved bead mechanical strength which supported higher biocatalyst activity by 63% was observed when concentration of gellan gum, concentration of calcium chloride, number of beads and bead size were optimized. The immobilized crude extract demonstrated higher tolerance towards broad range of pH (5-10) and temperature (30°C-40°C), superior cyanide-degrading activity over time and improved storage stability by maintaining 76% of its initial activity after 30 days at 4°C. Furthermore, repeated use of the gellan gum beads up to 20 batches without substantial loss in the catalytic activity was documented in the present study, indicating that the durability of the beads and the stability of the enzyme are both above adequate. Collectively, the findings reported here revealed that the utilization of the encapsulated crude extract of Rhodococcus UKMP-5M can be considered as a novel attempt to develop an environmentally favourable and financially viable method in cyanide biodegradation.
Rhodococcus hoagii is a well-known zoonotic disease, especially in foals. Its occurrence in humans is uncommon and usually occurs in immunocompromised patients. We present a case of Rhodococcus hoagii infection resulting in necrotizing pneumonia in a patient with advanced retroviral disease who had defaulted treatment. Effective treatment of Rhodococcus hoagii infection requires a combination of antibiotics. We also highlighted the importance of effective communication between clinicians and microbiologists so that prompt treatment can be initiated to improve patient outcomes.
Poly-β-hydroxybutyrate (PHB) is a biodegradable polymer, synthesized as carbon and energy reserve by bacteria and archaea. To the best of our knowledge, this is the first report on PHB production by a rare actinomycete species, Rhodococcus pyridinivorans BSRT1-1. Response surface methodology (RSM) employing central composite design, was applied to enhance PHB production in a flask scale. A maximum yield of 3.6 ± 0.5 g/L in biomass and 43.1 ± 0.5 wt% of dry cell weight (DCW) of PHB were obtained when using RSM optimized medium, which was improved the production of biomass and PHB content by 2.5 and 2.3-fold, respectively. The optimized medium was applied to upscale PHB production in a 10 L stirred-tank bioreactor, maximum biomass of 5.2 ± 0.5 g/L, and PHB content of 46.8 ± 2 wt% DCW were achieved. Furthermore, the FTIR and 1H NMR results confirmed the polymer as PHB. DSC and TGA analysis results revealed the melting, glass transition, and thermal decomposition temperature of 171.8, 4.03, and 288 °C, respectively. In conclusion, RSM can be a promising technique to improve PHB production by a newly isolated strain of R. pyridinivorans BSRT1-1 and the properties of produced PHB possessed similar properties compared to commercial PHB.
In this work, we report on the isolation of a phenol-degrading Rhodococcus sp. with a high tolerance towards phenol. The isolate was identified as Rhodococcus sp. strain AQ5NOL 2, based on 16S rDNA analysis. The strain degraded phenol using the meta pathway, a trait shared by many phenol-degraders. In addition to phenol biodegradation, the strain was also capable of degrading diesel. Strain AQ5NOL 2 exhibited a broad optimum temperature for growth on phenol at between 20 °C and 35 °C. The best nitrogen sources were ammonium sulphate, glycine or phenylalanine, followed by proline, nitrate, leucine, and alanine (in decreasing efficiency). Strain AQ5NOL 2 showed a high tolerance and degradation capacity of phenol, for it was able to register growth in the presence of 2000 mg l(-1) phenol. The growth of this strain on phenol as sole carbon and energy source were modeled using Haldane kinetics with a maximal specific growth rate (μ(max)) of 0.1102 hr(-1), a half-saturation constant (K(s) ) of 99.03 mg l(-1) or 1.05 mmol l(-1), and a substrate inhibition constant (K(i)) of 354 mg l(-1) or 3.76 mmol l(-1). Aside from phenol, the strain could utilize diesel, 2,4-dinitrophenol and ρ-cresol as carbon sources for growth. Strain AQ5NOL 2 exhibited inhibition of phenol degradation by Zn(2+), Cu(2+), Cr(6+), Ag(+) and Hg(2+) at 1 mg l(-1).
With the progressive increase in human activities in the Antarctic region, the possibility of domestic oil spillage also increases. Developing means for the removal of oils, such as canola oil, from the environment and waste "grey" water using biological approaches is therefore desirable, since the thermal process of oil degradation is expensive and ineffective. Thus, in this study an indigenous cold-adapted Antarctic soil bacterium, Rhodococcus erythropolis strain AQ5-07, was screened for biosurfactant production ability using the multiple approaches of blood haemolysis, surface tension, emulsification index, oil spreading, drop collapse and "MATH" assay for cellular hydrophobicity. The growth kinetics of the bacterium containing different canola oil concentration was studied. The strain showed β-haemolysis on blood agar with a high emulsification index and low surface tension value of 91.5% and 25.14 mN/m, respectively. Of the models tested, the Haldane model provided the best description of the growth kinetics, although several models were similar in performance. Parameters obtained from the modelling were the maximum specific growth rate (qmax), concentration of substrate at the half maximum specific growth rate, Ks% (v/v) and the inhibition constant Ki% (v/v), with values of 0.142 h-1, 7.743% (v/v) and 0.399% (v/v), respectively. These biological coefficients are useful in predicting growth conditions for batch studies, and also relevant to "in field" bioremediation strategies where the concentration of oil might need to be diluted to non-toxic levels prior to remediation. Biosurfactants can also have application in enhanced oil recovery (EOR) under different environmental conditions.
Study of the potential of Antarctic microorganisms for use in bioremediation is of increasing interest due to their adaptations to harsh environmental conditions and their metabolic potential in removing a wide variety of organic pollutants at low temperature. In this study, the psychrotolerant bacterium Rhodococcus sp. strain AQ5-07, originally isolated from soil from King George Island (South Shetland Islands, maritime Antarctic), was found to be capable of utilizing phenol as sole carbon and energy source. The bacterium achieved 92.91% degradation of 0.5 g/L phenol under conditions predicted by response surface methodology (RSM) within 84 h at 14.8 °C, pH 7.05, and 0.41 g/L ammonium sulphate. The assembled draft genome sequence (6.75 Mbp) of strain AQ5-07 was obtained through whole genome sequencing (WGS) using the Illumina Hiseq platform. The genome analysis identified a complete gene cluster containing catA, catB, catC, catR, pheR, pheA2, and pheA1. The genome harbours the complete enzyme systems required for phenol and catechol degradation while suggesting phenol degradation occurs via the β-ketoadipate pathway. Enzymatic assay using cell-free crude extract revealed catechol 1,2-dioxygenase activity while no catechol 2,3-dioxygenase activity was detected, supporting this suggestion. The genomic sequence data provide information on gene candidates responsible for phenol and catechol degradation by indigenous Antarctic bacteria and contribute to knowledge of microbial aromatic metabolism and genetic biodiversity in Antarctica.
Bacterial adhesion on surfaces is an essential initial step in promoting bacterial mobilization for soil bioremediation process. Modification of the cell surface is required to improve the adhesion of bacteria. The modification of physicochemical properties by rhamnolipid to Pseudomonas putida KT2442, Rhodococcus erythropolis 3586 and Aspergillus brasiliensis ATCC 16404 strains was analysed using contact angle measurements. The surface energy and total free energy of adhesion were calculated to predict the adhesion of both bacteria strains on the A. brasiliensis surface. The study of bacterial adhesion was carried out to evaluate experimental value with the theoretical results. Bacteria and fungi physicochemical properties were modified significantly when treated with rhamnolipid. The adhesion rate of P. putida improved by 16% with the addition of rhamnolipid (below 1 CMC), while the increase of rhamnolipid concentration beyond 1 CMC did not further enhance the bacterial adhesion. The addition of rhamnolipid did not affect the adhesion of R. erythropolis. A good relationship has been obtained in which water contact angle and surface energy of fungal surfaces are the major factors contributing to the bacterial adhesion. The adhesion is mainly driven by acid-base interaction. This finding provides insight to the role of physicochemical properties in controlling the bacterial adhesion on the fungal surface to enhance bacteria transport in soil bioremediation.
We present a rare case of post-antiretroviral therapy (ART) paradoxically worsening of radiological findings in a patient with advanced HIV-infection on treatment for Rhodococcus pneumonia who was misdiagnosed with pulmonary tuberculosis. Despite clinical improvement, serial chest radiographs showed deteriorations a month after starting ART. This was attributed to Immune Reconstitution Inflammatory Syndrome (IRIS) which spontaneously resolved without any treatment.
Protein function depends greatly on its structure. Based on this principle, it is vital to study the
protein structure in order to understand its function. This study attempts to build the predicted
model of lipase gene in Rhodococcus sp. NAM81 using homology modelling method. The
predicted structure was then used to investigate the function of protein through several
bioinformatic tools. The DNA sequence of lipase gene was obtained from the Rhodococcus sp.
NAM81 genome scaffold. Blastx analysis showed 100% identity to the target enzyme andthe
appropriate template for homology modelling was determined using Blastp analysis. The 3D
protein structure was built using two homology modelling software, EsyPred3D and Swiss
Model Server. Both structures built obtained LGScore of greater than 4, which means they are
extremely good models according to ProQ validation criteria. Both structures also satisfied the
Ramachandran plot structure validation analysis. The predicted structures were 100% matched
with each other when superimposed with DaliLite pairwise. This shows that both structure
validation servers agreed on the same model. Structure analysis using ProFunc had found seven
motifs and active sites that indicate similar function of this protein with other known proteins.
Thus, this study has successfully produced a good 3D protein structure for the target enzyme.
Social bacteria use chemical communication to coordinate and synchronize gene expression via the quorum-sensing (QS) regulatory pathway. In Pectobacterium, a causative agent of the blackleg and soft-rot diseases on potato plants and tubers, expression of the virulence factors is collectively controlled by the QS-signals N-acylhomoserine lactones (NAHLs). Several soil bacteria, such as the actinobacterium Rhodococcus erythropolis, are able to degrade NAHLs, hence quench the chemical communication and virulence of Pectobacterium. Here, next-generation sequencing was used to investigate structural and functional genomics of the NAHL-degrading R. erythropolis strain R138. The R. erythropolis R138 genome (6.7 Mbp) contained a single circular chromosome, one linear (250 kbp) and one circular (84 kbp) plasmid. Growth of R. erythropolis and P. atrosepticum was not altered in mixed-cultures as compared with monocultures on potato tuber slices. HiSeq-transcriptomics revealed that no R. erythropolis genes were differentially expressed when R. erythropolis was cultivated in the presence vs absence of the avirulent P. atrosepticum mutant expI, which is defective for QS-signal synthesis. By contrast 50 genes (<1% of the R. erythropolis genome) were differentially expressed when R. erythropolis was cultivated in the presence vs absence of the NAHL-producing virulent P. atrosepticum. Among them, quantitative real-time reverse-transcriptase-PCR confirmed that the expression of some alkyl-sulfatase genes decreased in the presence of a virulent P. atrosepticum, as well as deprivation of organic sulfur such as methionine, which is a key precursor in the synthesis of NAHL by P. atrosepticum.
The cell-free extract of locally isolated Rhodococcus UKMP-5M strain was used as an alternative to develop greener and cost effective cyanide removal technology. The present study aims to assess the viability of the cell-free extract to detoxify high concentrations of cyanide which is measured through the monitoring of protein concentration and specific cyanide-degrading activity. When cyanide-grown cells were subjected to grinding in liquid nitrogen which is relatively an inexpressive and fast cell disruption method, highest cyanide-degrading activity of 0.63 mM min(-1) mg(-1) protein was obtained in comparison to enzymatic lysis and agitation with fine glass beads. The cell-free extracts managed to degrade 80% of 20 mM KCN within 80 min and the rate of cyanide consumption increased linearly as the concentration of protein was raised. In both cases, the addition of co-factor was not required which proved to be advantageous economically. The successful formation of ammonia and formate as endproducts indicated that the degradation of cyanide by Rhodococcus UKMP-5M proceeded via the activity of cyanidase and the resulting non-toxic products are safe for disposal into the environment. Further verification with SDS-PAGE revealed that the molecular weight of the active enzyme was estimated to be 38 kDa, which is consistent with previously reported cyanidases. Thus, the utilization of cell-free extracts as an alternative to live microbial in cyanide degradation offers numerous advantageous such as the potential to tolerate and degrade higher concentration of cyanide and total reduction in the overall cost of operation since the requirement for nutrient support is irrelevant.
Interest in the biodegradation of microplastics is due to their ubiquitous distribution, availability, high persistence in the environment and deleterious impact on marine biota. The present study evaluates the growth response and mechanism of polypropylene (PP) degradation by Bacillus sp. strain 27 and Rhodococcus sp. strain 36 isolated from mangrove sediments upon exposure to PP microplastics. Both bacteria strains were able to utilise PP microplastic for growth as confirmed by the reduction of the polymer mass. The weight loss was 6.4% by Rhodococcus sp. strain 36 and 4.0% by Bacillus sp. strain 27 after 40days of incubation. PP biodegradation was further confirmed using Fourier-transform infrared spectroscopy and scanning electron microscopy analyses, which revealed structural and morphological changes in the PP microplastics with microbial treatment. These analyses showed that the isolates can colonise, modify and utilise PP microplastics as carbon source.
Rhodococci are renowned for their great metabolic repertoire partly because of their numerous putative pathways for large number of specialized metabolites such as biosurfactant. Screening and genome-based assessment for the capacity to produce surface-active molecules was conducted on Rhodococcus sp. ADL36, a diesel-degrading Antarctic bacterium. The strain showed a positive bacterial adhesion to hydrocarbon (BATH) assay, drop collapse test, oil displacement activity, microplate assay, maximal emulsification index at 45% and ability to reduce water surface tension to < 30 mN/m. The evaluation of the cell-free supernatant demonstrated its high stability across the temperature, pH and salinity gradient although no correlation was found between the surface and emulsification activity. Based on the positive relationship between the assessment of macromolecules content and infrared analysis, the extracted biosurfactant synthesized was classified as a lipopeptide. Prediction of the secondary metabolites in the non-ribosomal peptide synthetase (NRPS) clusters suggested the likelihood of the surface-active lipopeptide production in the strain's genomic data. This is the third report of surface-active lipopeptide producers from this phylotype and the first from the polar region. The lipopeptide synthesized by ADL36 has the prospect to be an Antarctic remediation tool while furnishing a distinctive natural product for biotechnological application and research.
Rhodococcus jostii RHA1 (RHA1) degrades polychlorinated biphenyl (PCB) via co-metabolism with biphenyl. To identify the novel open reading frames (ORFs) that contribute to PCB/biphenyl metabolism in RHA1, we compared chromatin immunoprecipitation chip and transcriptomic data. Six novel ORFs involved in PCB/biphenyl metabolism were identified. Gene deletion mutants of these 6 ORFs were made and were tested for their ability to grow on biphenyl. Interestingly, only the ro10225 deletion mutant showed deficient growth on biphenyl. Analysis of Ro10225 protein function showed that growth of the ro10225 deletion mutant on biphenyl was recovered when exogenous recombinant Ro10225 protein was added to the culture medium. Although Ro10225 protein has no putative secretion signal sequence, partially degraded Ro10225 protein was detected in conditioned medium from wild-type RHA1 grown on biphenyl. This Ro10225 fragment appeared to form a complex with another PCB/biphenyl oxidation enzyme. These results indicated that Ro10225 protein is essential for the formation of the PCB/biphenyl dioxygenase complex in RHA1.
Actinobacteria from underexplored and unusual environments have gained significant attention for their capability in producing novel bioactive molecules of diverse chemical entities. Streptomyces is the most prolific Actinobacteria in producing useful molecules. Rapid decline effectiveness of existing antibiotics in the treatment of infections are caused by the emergence of multidrug-resistant pathogens. Intensive efforts are urgently required in isolating non-Streptomyces or rare Actinobacteria and understanding of their distribution in the harsh environment for new drug discovery. In this study, pretreatment of soil samples with 1.5% phenol was used for the selective isolation of Actinobacteria from Dee Island and Greenwich Island. A high number of non-Streptomyces (69.4%) or rare Actinobacteria was significantly recovered despite the Streptomyces (30.6%), including the genera Micromonospora, Micrococcus, Kocuria, Dermacoccus, Brachybacterium, Brevibacterium, Rhodococcus, Microbacterium and Rothia. Reduced diversity and shift of distribution were observed at the elevated level of soil pH. The members of genera Streptomyces, Micromonospora and Micrococcus were found to distribute and tolerate to a relatively high pH level of soil (pH 9.4-9.5), and could potentially be alkaliphilic Actinobacteria. The phylogenetic analysis had revealed some potentially new taxa members of the genera Micromonospora, Micrococcus and Rhodococcus. Principal Component Analysis of soil samples was used to uncover the factors that underlie the diversity of culturable Actinobacteria. Water availability in soil was examined as the principal factor that shaped the diversity of the Actinobacteria, by providing a dynamic source for microbial interactions and elevated diversity of Actinobacteria.
In this study, a total of 147 soil actinobacterial strains were screened for their ability to inhibit response of Chromobacterium violaceum CV026 to short chain N-acyl homoserine lactone (AHL) which is a quorum sensing molecule. Of these, three actinobacterial strains showed positive for violacein inhibition. We further tested these strains for the inhibition of Pseudomonas aeruginosa PAO1 quorum sensing-regulated phenotypes, namely, swarming and pyocyanin production. The three strains were found to inhibit at least one of the quorum sensing-regulated phenotypes of PAO1. Phylogenetic analysis of the 16S rRNA gene sequences indicated that these strains belong to the genera Micromonospora, Rhodococcus and Streptomyces. This is the first report presenting quorum quenching activity by a species of the genus Micromonospora. Our data suggest that Actinobacteria may be a rich source of active compounds that can act against bacterial quorum sensing system.