Minimal Agar Research Articles

Detection Time Distribution of Microcolonies Formed by Individual Heat-Injured Cells of Escherichia coli.

The microcolony formation at 30℃ on an enriched minimal salts agar plates by individual Escherichia coli cells heated at 50℃ was monitored with a time-lapse shadow image analysis system, MicroBio μ3DTM AutoScanner. While the time course of microcolony count detected every half an hour for the unheated cells seemingly demonstrated a normal distribution, that for the heated cell population demonstrated totally the growth delay probably resulting from cell injury and also interestingly distributed in its rather deformed pattern with a tailing. Those patterns of the cumulative counts of appearing microcolonies during the post-heating cultivation period were expressed in three different mathematical models. This approach may be proposed as a rapid cultivation method predictable for enumeration of viable and repairable injured cells in practical use.

ISOLATION AND CHARACTERIZATION OF PHENANTHRENE DEGRADATING BACTERIA FROM DAIRY WASTE SAMPLES

Phenanthrene is the commonly used hazardous chemical in various industries and the detoxification is a big problem till today. In this study, phenanthrene degrading bacteria were isolated from dairy waste samples, characterized by molecular techniques. A total of 10 samples were collected respectively from different spots of dairy industry. Minimum salt agar medium with phenanthrene composition was used to isolate the pure culture of resistant bacterium. Morphological, biochemical tests were done to identify the phenanthrene degrading bacteria. The colonies of the isolates were circular to irregular in dairy samples. Phenanthrene tolerant bacteria were isolated after the dairy waste soil samples was serially diluted and transferred onto M9 minimal agar medium amended with 10mg/l of phenanthrene. A concentration of 10 mg/l was chosen based on the prevailing concentration. Four Phenanthrene tolerant bacteria from dairy waste (HW2, HW1, HW3, HW5) were isolated. From the biochemical and 16srRNA PCR amplification, the bacterium identified was Micrococcus leuteus. The resistance to Phenanthrene by the isolate was tested with various concentrations of Phenanthrene from 0 to 10mM. The Micrococcus luteus showed a MTC value of 28Cfu/ml at 8mM and Bacillus cereus showed least.

Screening of Methionine-producing <i>Bacillus</i> Species from Nigerian Fermented Food Condiments and Effects of Some Cultural Parameters on Methionine Accumulation

Fermented food condiments are sources of proteins and vitamins and are added as spices or sauces to food for flavour or taste enhancers. The major fermenting microorganisms, Bacillus species, produce proteolytic enzymes that hydrolyze proteins and amino acids and peptides. Methionine is the most essential amino acid for chicken feeds and can be produced by Bacillus species. The screening of methionine-producing microorganisms from Nigerian fermented food condiments and the effect of some cultural parameters on methionine accumulation were conducted. Bacterial organisms isolated from fermented food condiments, ogiri and okpeye, were screened for methionine producers on minimal solid agar medium seeded with auxotrophic Escherichia coli and in submerged medium. The effects of medium to fermenter volume ratio, inoculum size, carbon and nitrogen sources on methionine accumulation by the isolates were investigated. Of the five methionine producers recovered, two of the active isolates identified as Bacillus pumilus and Bacillus amyloliquefaciens were used for methionine production. A 20.0ml fermentation medium and 5.0% inoculum size gave the highest methionine accumulation by the Bacillus species. At 8.0% glucose and 4.0% ammonium sulphate concentrations, methionine yields of 5.22mg/ml and 5.50mg/ml were accumulated in the broth cultures of B. pumilus and B. amyloliquefaciens respectively. B. pumilus and B. amyloliquefaciens recovered from Nigerian fermented food condiments were observed to produce methionine and the optimization of some cultural parameters enhanced methionine accumulation by the Bacillus species.

Characterization of Autochthonous Bacterial Isolates with The Potentiality to Degrade Spent Engine Oil from Contaminated Soil Area Enriched with Glycine max

This study was conducted to identify and characterize bacteria capable of degrading spent oil contaminated soil. The physicochemical parameters of used engine oil were analyzed according to existing standards, while the total heterotrophic plate counts (HBC) and hydrocarbon utilizing bacteria counts were ascertained with the pour plate methods using nutrient agar and minimal salt agar (MSA) media respectively. The results indicated a mean total HBC ranging from 2.86 ± 0.08 to 5.76 log10 CFU/g and mean hydrocarbon utilizing bacterial counts from 1.32 ± 0.09 to 3.82 ± 0.25 log10 CFU/g, with samples enriched with Glycine max (Soybean) recorded to have the highest bacterial counts. The phenotypic identification of the hydrocarbon utilizing bacteria as sources of carbon and energy showed the presence of two primary bacterial genera: Bacillus and Micrococcus.However, from the overall 50 counted colonies, the frequency of occurrence for Bacillus was 41 (82 %) whereas, the Micrococcus was (9) 18%. The obtained data, confirmed the breakdown capacity of autochthonous (indigenous) organisms notably; Bacillus in the reduction of pollutants linked with oil spillage. This provides for reliable and promising approach of ameliorating crude oil pollutants and its inherent threats. 
 Keywords: Soil, spent oil, Glycine max, degrading bacteria, isolation and bioremediation

Isolation and characterization of a novel paraffin wax and solid wax degrading endosymbiotic bacteria from Citrus mealy bug (Planococcus citri)

Bacterial isolates capable of degrading the paraffin wax and solid wax were isolated from the body of citrus mealy bug. In total nine isolates were isolated but among them only four showed degradation ability by formation of halo on Modified Davis Minimal Agar media and change in colour on Blue spirit agar media. These isolates were identified by 16srRNA and they were Bacillus cereus, Bacillus subtilis, Bacillus licheniformis and Pseudomonas putida. The morphological and biological profile of bacterial strains were also identified. The bacterial species showed reduction in residual amount of liquid paraffin and among them Bacillus subtilis (3.421g) becomes more efficient. The incubated samples containing strains viz., Bacillus cereus, Bacillus subtilisi, Bacillus licehniformis and Pseudomonas putida showed significant reduction in residual weight of solid wax. The highest reduction was observed by Bacillus cereus (66.53%). However, no difference was observed in the residual weight of solid wax in control even after 30 days (0%).

Application of Optimized and Validated Agar Overlay TLC-Bioautography Assay for Detecting the Antimicrobial Metabolites of Pharmaceutical Interest.

The agar overlay TLC-bioautography is one of the crucial methods for simultaneous in situ detection and separation of antimicrobial metabolites of pharmaceutical interest. The main focus of this research relies on the dereplication of an antimicrobial metabolite coriloxin derived from mycoendophytic Xylaria sp. NBRTSB-20 with a validation of agar overlay TLC-bioautography technique. This polyketide metabolite coriloxin was purified by column chromatography, and its purity was assessed by HPLC, UPLC-ESI-QTOF-MS, FT-IR and NMR spectral analysis. The antimicrobial capability of ethyl acetate extract and the purified compound coriloxin was determined by disc diffusion, minimal inhibitory concentration and agar overlay TLC-bioautography assay. The visible LOD of coriloxin antimicrobial activity was found at 10μg for Escherichia coli and 20μg for both Staphylococcus aureus and Fusarium oxysporum. Inter- and intra-day precision was determined as the relative standard deviation is less than 6.56%, which proved that this method was precise. The accuracy was expressed as recovery, and the values were found ranging from 91.18 to 108.73% with RSD values 0.94-2.30%, respectively. The overall findings of this investigation suggest that agar overlay TLC-bioautography assay is a suitable and acceptable method for the in situ determination of antimicrobial pharmaceuticals.

Comparative Evaluation on Tannase Production by Lasiodiplodia plurivora ACN-10 under Submerged Fermentation (SmF) and Solid State Fermentation (SSF)

Aim: Tannase (tannin acyl hydrolase, E.C. 3.1.1.20) catalyzes the hydrolysis of ester bonds from complex hydrolysable tannins with the production of gallic acid and glucose and possess broad applications in biotechnology. This study is aimed at the production of tannase by Lasiodiplodia plurivora ACN-10 in SmF and SSF using Terminalia cattapa (almond leaves) and Magnifera indica (mango leaves) as substrates.
 Study Design: The design adopted to evaluate the production of tannase is submerged (SmF) and solid state (SSF) fermentation.
 Study Area: Federal Institute of Industrial Research Oshodi, Lagos State Nigeria.
 Methodology: Fifteen different soil samples were indiscriminately collected within Oshodi, Lagos, Nigeria and were inoculated on PDA plates at 30°C for 3-5 days. A total of 30 isolates was screened on Czapek dox minimal agar incorporated with 1% tannic acid and plates were incubated for 96 h at 30°C. Fungal isolates which were able to disintegrate tannic acid produced a clear halo zone around the colony diameter and were selected to be positive for tannase activity. The best isolate was identified based on its morphological, microscopic and molecular characteristics. Thereafter production and extraction of tannase was carried out in SmF for 0-120 h and in SSF for 0-144 h using Terminalia cattapa (Almond leaves) and Magnifera indica (Mango leaves) as substrates.
 Results: The total fungal count ranged from 1.0×104 to 3.5×105 CFU/g. A total of 30 fungal isolates produced clear halo zones (ranging from 20 to 70 mm) around the colonies during the screening with tannic acid. Isolate ACN-10, which showed the highest tannic degradation was identified based on its morphological and microscopic characteristics. On the basis of 18S rRNA gene sequence studies, the isolate was identified as Lasiodiplodia plurivora strain ACN-10 and the sequence was submitted to the Genbank with the accession number: MG250374. Results obtained in this study indicated that both substrates can be used by the isolate for tannase production in both SmF and SSF. The result of our investigation on the use of Terminalia cattapa (almond leaves) and Magnifera indica (mango leaves) as substrates for tannase production showed that optimum yield (6.064 U/ml) was obtained at 120 h in SSF while optimum production (4.623 U/ml) was observed in SmF at 96 h using Terminalia cattapa as substrate.
 Conclusion: Results obtained from this study indicated higher tannase production in solid-state fermentation compared to submerged fermentation. This is the first report to the best of our knowledge that Lasiodiplodia plurivora strain is implicated in tannase secretion. The result also demonstrated high production of extracellular tannases from low cost substrates which can be optimized and scaled up for industrial processes.

Expression of an alcohol dehydrogenase gene in a heterotrophic bacterium induces carbon dioxide-dependent high-yield growth under oligotrophic conditions.

Sphingobium japonicum strain UT26, whose γ-hexachlorocyclohexane-degrading ability has been studied in detail, is a typical aerobic and heterotrophic bacterium that needs organic carbon sources for its growth, and cannot grow on a minimal salt agar medium prepared without adding any organic carbon sources. Here, we isolated a mutant of UT26 with the ability to grow to visible state on such an oligotrophic medium from a transposon-induced mutant library. This high-yield growth under oligotrophic conditions (HYGO) phenotype was CO2-dependent and accompanied with CO2 incorporation. In the HYGO mutant, a transposon was inserted just upstream of the putative Zn-dependent alcohol dehydrogenase (ADH) gene (adhX) so that the adhX gene was constitutively expressed, probably by the transposon-derived promoter. The adhX-deletion mutant (UT26DAX) harbouring a plasmid carrying the adhX gene under the control of a constitutive promoter exhibited the HYGO phenotype. Moreover, the HYGO mutants spontaneously emerged among the UT26-derived hypermutator strain cells, and adhX was highly expressed in these HYGO mutants, while no HYGO mutant appeared among UT26DAX-derived hypermutator strain cells, indicating the necessity of adhX for the HYGO phenotype. His-tagged AdhX that was expressed in Escherichia coli and purified to homogeneity showed ADH activity towards methanol and other alcohols. Mutagenesis analysis of the adhX gene indicated a correlation between the ADH activity and the HYGO phenotype. These results demonstrated that the constitutive expression of an adhX-encoding protein with ADH activity in UT26 leads to the CO2-dependent HYGO phenotype. Identical or nearly identical adhX orthologues were found in other sphingomonad strains, and most of them were located on plasmids, suggesting that the adhX-mediated HYGO phenotype may be an important adaptation strategy to oligotrophic environments among sphingomonads.

RNAi-Mediated Gene Silencing of Trcot1 Induces a Hyperbranching Phenotype in Trichoderma reesei.

Trichoderma reesei is the major filamentous fungus used to produce cellulase and there is huge interest in promoting its ability to produce higher titers of cellulase. Among the many factors affecting cellulase production in T. reesei, the mycelial phenotype is important but seldom studied. Herein, a close homolog of the Neurospora crassa COT1 kinase was discovered in T. reesei and designated TrCOT1, which is of 83.3% amino acid sequence identity. Functional disruption of Trcot1 in T. reesei by RNAi-mediated gene silencing resulted in retarded sporulation on potato dextrose agar and dwarfed colonies on minimal medium agar plates containing glucose, xylan, lactose, xylose, or glycerol as the sole carbon source. The representative mutant strain, SUS2/Trcot1i, also displayed reduced mycelia accumulation but hyperbranching in the MM glucose liquid medium, with hyphal growth unit length values decreased to 73.0 µm/tip compared to 239.8 µm/tip for the parent strain SUS2. The hyperbranching phenotype led to slightly but significantly increased cellulase secretion from 24 to 72 h in a batch culture. However, the cellulase production per unit of mycelial biomass was much more profoundly improved from 24 to 96 h.

Grey mould control by oxalate degradation using non-antifungal Pseudomonas abietaniphila strain ODB36

Grey mould is an important necrotrophic fungal pathogen that causes huge economic losses in agriculture. Many types of bacteria are used for biological control of grey mould via competition for space or nutrients and/or the production of antifungal metabolites. Oxalate is a key component of virulent necrotic fungal pathogens. In this study, we isolated non-antifungal oxalate-degrading bacteria (ODB) from the surfaces of oxalate-rich spinach and strawberries to investigate their ability to control necrotic fungal pathogens such as grey mould. A total of 36 bacteria grown on oxalate minimal (OM) agar plates were tested for oxalate-degrading activity. Five isolates exhibiting the highest oxalate degradation activity were subjected to molecular identification using 16S rRNA gene sequencing. Two isolates exhibiting non-antifungal activity were subjected to disease suppression assays using Arabidopsis–Botrytis systems. The isolate Pseudomonas abietaniphila ODB36, which exhibited significant plant protective ability, was finally selected for further investigation. Based on whole-genome information, the pseudomonad oxalate degrading (podA) gene, which encodes formyl-CoA transferase, was analysed. The podA− mutant did not inhibit Botrytis infection and oxalate toxicity; the defects were recovered by podA complementation. Purified PodA–His converted oxalate to formate and eliminated oxalate toxicity. These results indicate that P. abietaniphila ODB36 and PodA enzyme are associated with various aspects of grey mould disease inhibitory effects.

In vitro Antioxidant activity of extracellular L-glutaminase enzyme isolated from marine yeast Rhodotorula sp. DAMB1

Yeasts from marine ecosystem have been recognized as producers of several bioactive compounds and also for various enzyme productions. The present study mainly focuses on the extracellular production, medium optimization and in vitro antioxidant activity of L-glutaminase enzyme from marine yeasts isolated from marine water samples of the backwaters of Andaman-Nicobar islands, India. A total of 26 marine yeast isolates were recovered using yeast malt agar (YMA) medium. After primary screening using minimal glutamine agar, 4 marine yeast isolates DAMB1, DAMB2, DAMB3 and DAMB4 showed positive enzyme activity. Zones of hydrolysis for DAMB1 and DAMB3 were 40mm and 30mm respectively. Further, secondary screening was done for the two potential yeast isolates at pH 7.6 along with nesslerization for quantitative analysis. Yeast isolate DAMB1 showed highest L-glutaminase enzyme activity of 67%. This was followed by medium optimization for better production of L-glutaminase enzyme. The optimum temperature and pH was 37°C and pH 8 respectively. Optimum carbon source was arabinose and optimum nitrogen source was found to be casein. The potential yeast isolates showed good antioxidant activity too. DAMB1 showed highest DPPH activity of 62% and DAMB2 showed DPPH activity of 47%. The potential yeast isolate was identified as Rhodotorula sp. DAMB1 (Acc. No. MK968443) using 18s rRNA sequencing method.

Transition of the Bacterial Community and Culturable Chitinolytic Bacteria in Chitin-treated Upland Soil: From Streptomyces to Methionine-auxotrophic Lysobacter and Other Genera.

Chitin amendment is an agricultural management strategy for controlling soil-borne plant disease. We previously reported an exponential decrease in chitin added to incubated upland soil. We herein investigated the transition of the bacterial community structure in chitin-degrading soil samples over time and the characteristics of chitinolytic bacteria in order to elucidate changes in the chitinolytic bacterial community structure during chitin degradation. The addition of chitin to soil immediately increased the population of bacteria in the genus Streptomyces, which is the main decomposer of chitin in soil environments. Lysobacter, Pseudoxanthomonas, Cellulosimicrobium, Streptosporangium, and Nonomuraea populations increased over time with decreases in that of Streptomyces. We isolated 104 strains of chitinolytic bacteria, among which six strains were classified as Lysobacter, from chitin-treated soils. These results suggested the involvement of Lysobacter as well as Streptomyces as chitin decomposers in the degradation of chitin added to soil. Lysobacter isolates required yeast extract or casamino acid for significant growth on minimal agar medium supplemented with glucose. Further nutritional analyses demonstrated that the six chitinolytic Lysobacter isolates required methionine (Met) to grow, but not cysteine or homocysteine, indicating Met auxotrophy. Met auxotrophy was also observed in two of the five type strains of Lysobacter spp. tested, and these Met auxotrophs used d-Met as well as l-Met. The addition of Met to incubated upland soil increased the population of Lysobacter. Met may be a factor increasing the population of Lysobacter in chitin-treated upland soil.

Influence of Diclofenac on Activated Sludge Bacterial Communities in Fed-Batch Reactors.

SUMMARYResearch backgroundThe occurrence and environmental toxicity of pharmaceuticals have recently attracted increasing attention. Diclofenac is a highly consumed non-steroidal anti-inflammatory drug, which is often detected in wastewaters, but investigations of its influence on bacteria are scarce.Experimental approachWe investigated the influence of this pharmaceutical on bacterial community in activated sludge exposed to increasing concentrations of diclofenac in fed-batch reactors over 41 days. Nitrification activity of the activated sludge was measured and changes in bacterial community structure were followed using culture-independent molecular method (terminal restriction fragment length polymorphism, T-RFLP) and by the cultivation approach.Results and conclusionsNitrification activity was not detectably influenced by the addition of diclofenac, while the main change of the bacterial community structure was detected only at the end of incubation (after 41 days) when diclofenac was added to artificial wastewater as the only carbon source. Changes in community composition due to enrichment were observed using cultivation approach. However, taxonomic affiliation of isolates did not match taxons identified by T-RFLP community profiling. Isolates obtained from activated sludge used as inoculum belonged to five genera: Comamonas, Arthrobacter, Acinetobacter, Citrobacter and Aeromonas, known for their potential to degrade aromatic compounds. However, only Pseudomonas species were isolated after the last enrichment step on minimal agar plates with diclofenac added as the sole carbon source.Novelty and scientific contributionOur results suggest that the selected recalcitrant and commonly detected pharmaceutical does not strongly influence the sensitive and important nitrification process of wastewater treatment. Moreover, the isolated strains obtained after enrichment procedure that were able to grow on minimal agar plates with diclofenac added as the only carbon source could serve as potential model bacteria to study bacterial diclofenac degradation.

Safety of the live Escherichia coli vaccine Poulvac® E. coli in layer parent stock in a field trial

The safety of the live Escherichia coli vaccine Poulvac® E. coli was tested with a flock (10,000) of layer parents aged 30 weeks. Three and 7 days after vaccination, 60 whole unbroken eggs, the egg white and yolk of 60 eggs and 60 cloacal swabs were enriched in MacConkey broth. At both sampling times, 6 out of 60 whole eggs were found positive for coliform bacteria. None of the enriched samples of yolk + egg white were positive for coliform bacteria. Three and seven days after vaccination 44 and 37, respectively out of 60 swabs were positive for coliform bacteria in MacConkey broth. All coliform isolates collected from whole eggs and cloacal swabs were tested in parallel for growth on minimal agar and blood agar to identify the vaccine strain. Some isolates showed reduced growth on minimal agar compared to blood agar and they were tested further with a PCR for the aroA gene mutation and all were found with the wild type version of the gene. Only two isolates did not grow on minimal agar but grew on blood agar and they were tested both with PCR and PFGE. They also showed the wild type version of the aroA gene and their PFGE profile was different from the vaccine strain of Poulvac® E. coli. In conclusion, the Poulvac® E. coli vaccine strain of E. coli was not identified at the detection limit of one CFU on one egg or in the content of one egg or from a cloacal swab of one hen with at least 95 % probability on flock level. The use of the vaccine is safe for hens in lay with lack of survival of the vaccine strain and lack of negative effects on the hens including egg production.

Comparative 16S rDNA metagenomics study of two samples of cassava peel heap from Nigeria and India.

The microbiology of many cassava products and the wastes generated during the processing have been reported; however, majority of these reports used culture-dependent methods. This has resulted in a dearth of information on the bacterial diversity of cassava peels and peel heaps. Large amounts of cassava peels generated during the processing of cassava root are usually discharged on land or water as wastes and are allowed to rot in the open, especially in some developing countries. Culture-independent methods such as PCR-based amplification and sequencing of 16S rRNA genes, among others have been used in recent times to study the diversity of microbes in different environmental samples. In this study, bacterial isolates were screened for cellulase and xylanase enzyme activities on minimal agar and genomic DNA was isolated from cassava peel samples; metagenomics was carried out using MiSeq2 × 300 with primers specific for V3-V4 bacterial region. Samples collected from Nigeria (AAG) had more species compared with samples from India (JHA) with 793 and 525 observed OTUs (operational taxonomic units), respectively. Five bacterial isolates from cassava peel-heap samples obtained from Ogbomoso, Nigeria showed no ability to produce cellulase enzyme, seven isolates from the Nigeria samples and three from Jorhat samples were positive for xylanase production; the highest amylase activity was shown by isolate AG18 (10,055U/mL), while the lowest was recorded for isolate JA2 (2333U/mL) with a significant difference observed in the amylase activities of isolates (p ≤ 0.05). Comparing the most abundant taxonomy for each of the samples at different taxonomic levels, the most abundant for sample AAG were phylum Firmicutes (42.11%), class Bacilli (41.27%), order Lactobacillales (33.11%), family Acetobacteraceae (31.30%), genus Acetobacter (30.02%) and unclassified species of Acetobacter (29.88%), while sample JHA had Actinobacteria (47.47%) as the highest phylum and class, order Actinomycetales (47.47%), family Brevibacteriaceae (46.97%), genus Brevibacterium (46.97%) and unclassified species of Brevibacterium (46.89%). This study provides an insight into the vast diversity of the bacteria associated with cassava peel heaps and the ability of some of the bacteria to produce selected extracellular enzymes.

Cellulolytic enzyme-producing thermophilic Actinobacteria isolated from the soil of Cisolok Geysers, West Java, Indonesia

Abstract. Setyaningsih PP, Ningsih F, Rachmania MK, Syafitri WA, Sari DCAF, Yabe S, Yokota A, Oetari A, Sjamsuridzal W. 2019. Cellulolytic enzyme-producing thermophilic Actinobacteria isolated from the soil of Cisolok Geysers, West Java, Indonesia. Biodiversitas 20: 3134-3141. This study investigated 17 thermophilic Actinobacteria isolated from the soil of geysers in the Cisolok geothermal area, West Java, as potential producers of cellulase. Screening for cellulase was performed on minimal (Mm) agar medium with and without the addition of 1% (w/v) carboxymethylcellulose (CMC) and microcrystalline cellulose (MCC), then incubated at 45, 50, 55 and 60°C for up to 7 days. Formation of clear zones around colonies indicated cellulose hydrolysis. The results showed that 15, 14, 4, and 3 isolates showed cellulolytic activity on CMC agar medium at 45, 50, 55, and 60°C, respectively, after 7 days of incubation. Three potential isolates showed cellulolytic activity on MCC agar medium after being incubated for 7 days at 45°C. Molecular identification based on the 16S rRNA gene was performed for three isolates with positive cellulolytic activity at 60°C. The results showed that the three isolates are closely related to Actinomadura keratinilytica WCC-2265T with 99.93-100% sequence similarities. A phylogenetic tree based on 16S rRNA gene sequences confirmed that the three isolates were clustered together with Actinomadura keratinilytica WCC-2265T with 100% bootstrap value. The tree also showed that cellulase producers and non-cellulase producers in Thermomonosporaceae are grouped into different clades.

Effect of the Structural and Regulatory Heat Shock Proteins on Hydrocarbon Degradation by Rhodococcus pyridinivorans 5Ap

The role of heat shock proteins in ability of Rhodococcus pyridinivorans 5Ap to degrade hydrocarbons at different temperatures was studied. The presence of the Сpn60.1–Сpn10 chaperons and of the Hrc regulatory protein was found to be required for hexadecane degradation at 42°C. When genetic determinants responsible for synthesis of these proteins were inactivated, the efficiency of hexadecane degradation decreased 1.7 and 2.7 times, respectively. Mutations in the cpn and hrcA genes did not affect the viability of R. pyridinivorans 5Ар: the original strain and the mutants exhibited the same growth rates at all temperatures in the minimal medium with succinate and in full-strength medium. In the absence of the Сpn60.1–Сpn10 heat shock proteins, the growth rate at 42°C decreased in the case of minimal agar media with kerosene, diesel fuel, acetone, naphthalene, 2-methylnaphthalene, or phenanthrene.

Isolation and Characterization of Chlorpyrifos Degrading Bacteria from Contaminated Agricultural Lands

Previous literature has confirmed that pesticides are posing serious threats to the lives of human beings and animals alike because of their use in agricultural crops. Pesticides have also increased pollution in aquatic and terrestrial environment. In this study, chlorpyriphos degrading bacterial strains were isolated from pesticide contaminated agricultural soils by the selective enrichment method. Bacterial isolates were biochemically characterized for the strain identification. The effect of different environmental factors on optimum growth was also checked. These factors included pH, temperature, and concentration of pesticide. Isolated strains were also ribotyped for identification. Metal resistance profiling was performed for different heavy metals, that is, ZnSO4.7H2O (Zinc sulphate), CdCl2.H2O (Cadmium chloride), CuSO4 (Copper sulphate), and K2CrO4 (Potassium chromate). It was found that strains NW3O and NW3T were sensitive to cadmium except NR2 and NR4, which were resistant at the concentration of 100 and 200 µg ml-1. NR4, NW4 and NW3G were resistant to chromium upto the concentration of 100 µgml-1, while NR2, NW3O and NW3T tolerated 200 µgml-1 of the metal. All the strains were resistant to zinc at different concentrations. NR2 showed maximum resistance to copper by showing growth on all concentrations of the metal. Thin layer chromatography was performed for the detection of different intermediates formed during the degradation of chlorpyrifos. Minimum inhibitory concentration of pesticide was estimated in M9 medium containing chlorpyriphos. The current study resulted in the isolation of efficient chlorpyrifos degrading strains with a wide range of pH and temperature tolerance that can utilize chlorpyrifos upto 700mg/L during lab scale degradation tests (growth on chlorpyrifos supplemented minimal agar and broth).

Bioprospecting of polyhydroxyalkanoates-producing bacteria from Indonesian marine environment

Abstract. Tan WA, Wijaya I, Purwadaria T. 2019. Bioprospecting of polyhydroxyalkanoates-producing bacteria from Indonesian marine environment. Biodiversitas 20: 1309-1315. Polyhydroxyalkanoates (PHA) are potential alternates to conventional synthetic plastics. PHA production in bacteria involves PHA synthase gene encoded by phaC. In this study, we isolated PHA-producing bacteria from the coastline and 1 mile from the coastline of three beaches in Indonesia. Further phaC detection and characterization of PHA production were conducted. The isolates were subjected to phylogenetic analysis based on 16S rDNA. Red Nile staining on minimal agar revealed that twenty-three isolates showed orange fluorescent, which indicated that they accumulated PHA in their cells. PCR detection showed the presence of PHA synthase class I-encoding gene phaC in twelve isolates. One representative amplicon was sequenced to verify its identity, in which it shared 86% similarity with the PHA synthase class I-encoding gene from an uncultured bacterium. Interestingly, the production of PHA in isolate ST.PA.75, which was closely related to Vibrio sp., was 2.1-fold higher than that in the Ralstonia eutropha JMP134 control. Three isolates showed similarity with bacterial genera and/or species for which PHA producing phenotypes had never been described before TP.SWC.20, which was closely related to Microbacterium arborescens, as well as TP.SWC.33 and TP.SWC.85, which were similar to Psychrobacter spp. Phylogenetic analysis showed that the PHA producing isolates were clustered into three phyla: γ-Proteobacteria, Actinomycetes, and Bacilli. A majority of the isolates (75%) were related to γ-Proteobacteria. In this study, we uncovered diverse novel promising strains for use in the production of PHA as a more environmentally-friendly alternative to hydrocarbon-based plastics.

Suppression of substrate inhibition in phenanthrene-degrading Mycobacterium by co-cultivation with a non-degrading Burkholderia strain.

In natural environments contaminated by recalcitrant organic pollutants, efficient biodegradation of such pollutants has been suggested to occur through the cooperation of different bacterial species. A phenanthrene-degrading bacterial consortium, MixEPa4, from polluted soil was previously shown to include a phenanthrene-degrading strain, Mycobacterium sp. EPa45, and a non-polycyclic aromatic hydrocarbon (PAH)-degrading strain, Burkholderia sp. Bcrs1W. In this study, we show that addition of phenanthrene to rich liquid medium resulted in the transient growth arrest of EPa45 during its degradation of phenanthrene. RNA-sequencing analysis of the growth-arrested cells showed the phenanthrene-dependent induction of genes that were predicted to be involved in the catabolism of this compound, and many other cell systems, such as a ferric iron-uptake, were up-regulated, implying iron deficiency of the cells. This negative effect of phenanthrene became much more apparent when using phenanthrene-containing minimal agar medium; colony formation of EPa45 on such agar was significantly inhibited in the presence of phenanthrene and its intermediate degradation products. However, growth inhibition was suppressed by the co-residence of viable Bcrs1W cells. Various Gram-negative bacterial strains, including the three other strains from MixEPa4, also exhibited varying degrees of suppression of the growth inhibition effect on EPa45, strongly suggesting that this effect is not strain-specific. Growth inhibition of EPa45 was also observed by other PAHs, biphenyl and naphthalene, and these two compounds and phenanthrene also inhibited the growth of another mycobacterial strain, M. vanbaalenii PYR-1, that can use them as carbon sources. These phenomena of growth inhibition were also suppressed by Bcrs1W. Our findings suggest that, in natural environments, various non-PAH-degrading bacterial strains play potentially important roles in the facilitation of PAH degradation by the co-residing mycobacteria.