Metagenomic analysis of soil microbial communities

Ramonda serbica i Ramonda nathaliae, retke biljke 'vaskrsnice' koje rastu na Balkanskom poluostrvu, u odgovoru na stres produkuju velike količine fenola. Bakterijske zajednice poreklom iz rizosfere ovih biljaka analizirane su metagenomskim pristupom. Fluorescentna 'in situ' hibridizacija (FISH) i DAPI bojenje pokazali su da u analiziranim zemljištima ima svega 5% metabolički aktivnih bakterija. Upotrebom prajmera specifičnih za bakterijsku DNK umnoženi su geni za 16S rDNK i konstruisane su dve genske biblioteke. Biblioteke su pretraživane uz pomoć RFLP metode. Od ukupno 192 klona dobijena iz uzorka rizosfere R. nathaliae identifikovano je 35 operativnih taksonomskih jedinica (OTJ), dok je iz uzorka rizosfere R. serbica dobijeno 13 OTJ od ukupno 80 klonova. Predstavnici svake OTJ su sekvencirani. Analizirane zajednice odlikuje veoma mali diverzitet i većina dobijenih sekvenci je pokazala malu sličnost sa DNK sekvencama do sada kultivisanih bakterija.Ramonda serbica and Ramonda nathaliae, rare resurrection plants growing in the Balkan Peninsula, produce a high amount of phenolic compounds as a response to stress. The composition and size of bacterial communities in two rhizosphere soil samples of these plants were analyzed using a metagenomic approach. Fluorescent in situ hybridization (FISH) experiments together with DAPI staining showed that the metabolically active bacteria represent only a small fraction, approximately 5%, of total soil bacteria. Using universal bacteria - specific primers 16S rDNA genes were amplified directly from metagenomic DNAs and two libraries were constructed. The Restriction Fragment Length Polymorphism (RLFP) method was used in library screening. Amongst 192 clones, 35 unique operational taxonomic units (OTUs) were determined from the rhizosphere of R. nathaliae, and 13 OTUs out of 80 clones in total from the library of R. serbica. Representative clones from each OTU were sequenced. The majority of sequences from metagenomes showed very little similarity to any cultured bacteria. In conclusion, the bacterial communities in the studied soil samples showed quite poor diversity

Four Bacillus sp. soil isolates capable of degrading phenol, toluene, biphenyl, naphthalene and other aromatic compounds exhibit different aromatic catabolic potentials

Two novel Bacillus sp. were isolated from a soil sample from a bank of the Tamiš river in close proximity to a petrochemical facility. They were capable of utilizing a broad range of aromatic compounds as a sole source of carbon and energy (including phenol, benzene, toluene, biphenyl, naphthalene). The isolates were designated as Bacillus sp. TN41 and TN42, based on their 16S rDNA sequence. Their catabolic potential was compared to two Bacillus sp. strains (PS1 and PS11) isolated from the rhizosphere of the endemorelict plant Ramonda serbica. Specific activities of phenol hydroxylase, catechol 1,2-dioxygenase and catechol 2,3-dioxygenase were analyzed from crude cell extracts of the isolates, as well as the temperature and pH effects on enzyme activity. Although all four isolates had the ability to degrade a similar range of aromatic compounds, the specific activities of the enzymes indicative of aromatic compound catabolism of TN isolates were 2 to 90-fold lower compared to the PS isolates. Phenol hydroxylase and catechol dioxygenases exhibited broad temperature (10°C-80°C) and pH (4-9) activity ranges in all four Bacillus isolates. While phenol inhibited both phenol hydroxylase and catechol dioxygenases in the TN strains, it was an inducer for phenol hydroxylase in the PS strains

Limited aromatic pathway genes diversity amongst aromatic compound degrading soil bacterial isolates

Identifikacija i karakterizacija novih gena koji pripadaju putevima mikrobiološke razgradnje aromatičnih jedinjenja je od velikog značaja, jer su se pokazali kao izuzetno dobri biokatalizatori. U ovoj studiji, korišćenjem PCR metodologije, analizirano je prisustvo pet različitih gena iz biodegradativnog puta aromatičnih jedinjenja među 19 sredinskih izolata sa sposobnošću razgradnje širokog spektra aromatičnih jedinjenja. U slučaju 4-oksalokrotonat tautomeraze i toluen dioksigenaze, koji su detektovani kod većine sredinskih izolata, sekvence fragmenata su ukazivale na veoma ograničen diverzitet ova dva gena i visoku homologiju sa već poznatim sekvencama opisanim kod vrsta roda Pseudomonas. Korišćenjem degenerisanih prajmera konstruisanih na osnovu poznatih katehol-i naftalendioksigenaznih gena vrlo mali broj fragmenata je amplifikovan kod sredinskih izolata. Samo dve katehol 2,3-dioksigenaze iz dva izolata roda Bacillus su sekvenciranjem ukazale na različitost u odnosu na poznate sekvence, a pokazale međusobnu sličnost od 80-90%. Potencijalno tri nove katehol 1,2-dioksigenaze su identifikovane kod Bacillus sp. TN102, Gordonia sp. TN103 i Rhodococcus sp. TN112. Visok stepen homologije tautomeraza i toluen dioksigenaza među sredinskim izolatima izolovanim iz zagađene sredine ukazuje na horizontalni transfer gena, dok je ograničen uspeh u detektovanju preostala tri gena ukazao na potencijal da se među ovim izolatima mogu naći nove varijante gena iz puteva razgradnje aromatičnih jedinjenja.Identification and characterization of novel genes belonging to microbial aromatic biodegradation pathway is of great importance as they have been proven versatile biocatalysts. In this study, the selection of 19 environmental bacterial isolates capable to degrade a wide range of aromatic compounds has been screened for the presence of five genes from the lower and the upper aromatic biodegradation pathway using PCR methodology. In the case of 4-oxalocrotonate tautomerase and toluene dioxygenases, although present in the most of environmental isolates, very limited diversity of the genes has been encountered. Highly conserved sequences of these genes in environmental samples revealed high homology with gene sequences of the characterized corresponding genes from Pseudomonas putida species. The screen using degenerate primers based on known catechol-and naphthalene dioxygenases sequences resulted in a limited number of amplified fragments. Only two catechol 2,3-dioxygenase from two Bacillus isolates were amplified and showed no significant similarities with dioxygenases from characterized organisms, but 80-90% identities with partial catechol 2,3-dioxygenase sequences from uncultured organisms. Potentially three novel catechol 1,2-dioxygenases were identified from Bacillus sp. TN102, Gordonia sp. TN103 and Rhodococcus sp. TN112. Highly homologous tautomerase and toluene dioxygenases amongst environmental samples isolated from the contaminated environment suggested horizontal gene transfer while limited success in PCR detection of the other three genes indicates that these isolates may still be a source of novel genes

Microbial diversity and isolation of multiple metal-tolerant bacteria from surface and underground pits within the copper mining and smelting complex Bor

The bacterial diversity of the surface and deep sediment of the Copper Mining and Smelting Complex Bor, Serbia, was investigated using culture-dependent and culture-independent approaches. Sequencing analysis of 16S rDNA libraries revealed greater bacterial diversity in the surface sediment of the mining complex (MS) in comparison to deeper mine sediment (MU). While in the MS sample members of seven different phylogenetic groups were detected, in the MU sample library representatives of only three different groups were detected. The use of a culture-dependent approach revealed the presence of only three bacterial groups in both samples: Actinobacteria, Firmicutes and Proteobacteria, while six isolates exhibiting the highest metal tolerance were members of Arthrobacter and Staphylococcus genera. The most promising isolate, MSI08, was able to grow in the presence of high concentrations of Cd2+ (535 μM), Ni2+(17 mM) and Cr6+ (38.5 mM) and as such this indigenous strain has potential in the bioremediation of the contaminated surrounds of the city of Bor

Limited Aromatic Pathway Genes Diversity Amongst Aromatic Compound Degrading Soil Bacterial Isolates

Identification and characterization of novel genes belonging to microbial aromatic biodegradation pathway is of great importance as they have been proven versatile biocatalysts. In this study, the selection of 19 environmental bacterial isolates capable to degrade a wide range of aromatic compounds has been screened for the presence of five genes from the lower and the upper aromatic biodegradation pathway using PCR methodology. In the case of 4-oxalocrotonate tautomerase and toluene dioxygenases, although present in the most of environmental isolates, very limited diversity of the genes has been encountered. Highly conserved sequences of these genes in environmental samples revealed high homology with gene sequences of the characterised corresponding genes from Pseudomonas putida species. The screen using degenerate primers based on known catechol-and naphthalene dioxygenases sequences resulted in a limited number of amplified fragments. Only two catechol 2,3-dioxygenase from two Bacillus isolates were amplified and showed no significant similarities with dioxygenases from characterized organisms, but 80-90% identities with partial catechol 2,3-dioxygenase sequences from uncultured organisms. Potentially three novel catechol 1,2-dioxygenases were identified from Bacillus sp. TN102, Gordonia sp. TN103 and Rhodococcus sp. TN112. Highly homologous tautomerase and toluene dioxygenases amongst environmental samples isolated from the contaminated environment suggested horizontal gene transfer while limited success in PCR detection of the other three genes indicates that these isolates may still be a source of novel genes

Supplementary data for article: Narančić, T.; Kadivojevic, J.; Jovanovic, P.; Francuski, D.; Bigović, M.; Maslak, V.; Savić, V.; Vasiljević, B.; O’Connor, K. E.; Nikodinović-Runić, J. Highly Efficient Michael-Type Addition of Acetaldehyde to Beta-Nitrostyrenes by Whole Resting Cells of Escherichia Coli Expressing 4-Oxalocrotonate Tautomerase. Bioresource Technology 2013, 142, 462–468. https://doi.org/10.1016/j.biortech.2013.05.074

Supplementary material for: [https://doi.org/10.1016/j.biortech.2013.05.074]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1377

Microbial Diversity and Isolation of Multiple Metal-Tolerant Bacteria from Surface and Underground Pits Within the Copper Mining and Smelting Complex Bor

The bacterial diversity of the surface and deep sediment of the Copper Mining and Smelting Complex Bor, Serbia, was investigated using culture-dependent and culture-independent approaches. Sequencing analysis of 16S rDNA libraries revealed greater bacterial diversity in the surface sediment of the mining complex (MS) in comparison to deeper mine sediment (MU). While in the MS sample members of seven different phylogenetic groups were detected, in the MU sample library representatives of only three different groups were detected. The use of a culture-dependent approach revealed the presence of only three bacterial groups in both samples: Actinobacteria, Firmicutes and Proteobacteria, while six isolates exhibiting the highest metal tolerance were members of Arthrobacter and Staphylococcus genera. The most promising isolate, MSI08, was able to grow in the presence of high concentrations of Cd2+ (535 mu M), Ni2+(17 mM) and Cr6+ (38.5 mM) and as such this indigenous strain has potential in the bioremediation of the contaminated surrounds of the city of Bor

Supplementary data for article: Narančić, T.; Kadivojevic, J.; Jovanovic, P.; Francuski, D.; Bigović, M.; Maslak, V.; Savić, V.; Vasiljević, B.; O’Connor, K. E.; Nikodinović-Runić, J. Highly Efficient Michael-Type Addition of Acetaldehyde to Beta-Nitrostyrenes by Whole Resting Cells of Escherichia Coli Expressing 4-Oxalocrotonate Tautomerase. Bioresource Technology 2013, 142, 462–468. https://doi.org/10.1016/j.biortech.2013.05.074

Supplementary material for: [https://doi.org/10.1016/j.biortech.2013.05.074]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1377

Metagenomic analysis of soil microbial communities

Ramonda serbica and Ramonda nathaliae, rare resurrection plants growing in the Balkan Peninsula, produce a high amount of phenolic compounds as a response to stress. The composition and size of bacterial communities in two rhizosphere soil samples of these plants were analyzed using a metagenomic approach. Fluorescent in situ hybridization (FISH) experiments together with DAPI staining showed that the metabolically active bacteria represent only a small fraction, approximately 5%, of total soil bacteria. Using universal bacteria - specific primers 16S rDNA genes were amplified directly from metagenomic DNAs and two libraries were constructed. The Restriction Fragment Length Polymorphism (RLFP) method was used in library screening. Amongst 192 clones, 35 unique operational taxonomic units (OTUs) were determined from the rhizosphere of R. nathaliae, and 13 OTUs out of 80 clones in total from the library of R. serbica. Representative clones from each OTU were sequenced. The majority of sequences from metagenomes showed very little similarity to any cultured bacteria. In conclusion, the bacterial communities in the studied soil samples showed quite poor diversity.

Recent developments in biocatalysis beyond the laboratory

Recent developments in biocatalysis, where implementation beyond the laboratory has been demonstrated, are explored: the use of transglutaminases to modify foods, reduce allergenicity and produce advanced materials, lipases for biodiesel production, and transaminases for biochemical production. The availability and application of enzymes at pilot and larger scale opens up possibilities for further improvements of biocatalyst-based processes and the development of new processes. Enzyme production, stability, activity, re-use, and product retrieval are common challenges for biocatalytic processes. We explore recent advances in biocatalysis within the process chain, such as protein engineering, enzyme expression, and biocatalyst immobilization, in the context of these challenges