Phylogenetic diversity of microbial communities of the Posolsk Bank bottom sediments,Lake Baikal

Massive parallel sequencing (the Roche 454 platform) of the 16S rRNA gene fragments was used to investigate microbial diversity in the sediments of the Posolsk Bank cold methane seep. Bacterial communities from all sediment horizons were found to contain members of the phyla Actinobacteria, Bacteroidetes, Deinococcus-Thermus, Firmicutes, Nitrospirae, Chloroflexi, Proteobacteria, and the candidate phyla Aminicenantes (OP8) and Atribacteria (OP9). Among Bacteria, members of the Chloroflexi and Proteobacteria were the most numerous (42 and 46%, respectively). Among archaea, the Thaumarchaeota predominated in the upper sediment layer (40.1%), while Bathyarchaeota (54.2%) and Euryarchaeota (95%) were predominant at 70 and 140 cm, respectively. Specific migration pathways of fluid flows circulating in the zone of gas hydrate stability (400 m) may be responsible for considerable numbers of the sequences of Chloroflexi, Acidobacteria, and the candidate phyla Aminicenantes and Atribacteria in the upper sediment layers and of the Deinococcus-Thermus phylum in deep bottom sediments.     

Phylogenetic and functional prokaryotic diversity in the Hoito-Gol mesothermal mineral spring (Eastern Sayan Mountains,Buryat Republic)

High-throughput sequencing was used for comparative analysis of microbial communities of the water and mat from the Hoito-Gol mesothermal mineral sulfide spring (Eastern Sayan Mountains, Buryat Republic). Activity of microbial communities was determined. While both spring biotopes were dominated by members of three bacterial phyla—Proteobacteria, Bacteroidetes, and Firmicutes—they differed drastically in the composition of predominant phylotypes (at the genus level). In the water, the organisms widespread in aquatic environments were predominant, mostly aerobic chemoorganotrophs of the genera Acinetobacter, Pedobacter, and Flavobacterium. In the microbial mat, the organisms actively involved in the sulfur cycle predominated, including sulfur-reducing bacteria Sulfurospirillum, sulfate-reducing deltaproteobacteria, sulfuroxidizing chemoautotrophic bacteria, anoxygenic phototrophic bacteria of the phyla Chloroflexi and Chlorobi, as well as purple bacteria belonging to the α-, ß-, and γ-Proteobacteria. Microbial mats of the spring exhibited higher phylogenetic diversity compared to high-temperature mats containing photosynthetic microorganisms.     

Impact of soil salinity on the microbial structure of halophyte rhizosphere microbiome

The rhizosphere microbiome plays a significant role in the life of plants in promoting plant survival under adverse conditions. However, limited information is available about microbial diversity in saline environments. In the current study, we compared the composition of the rhizosphere microbiomes of the halophytes Urochloa, Kochia, Salsola, and Atriplex living in moderate and high salinity environments (Khewra salt mines; Pakistan) with that of the non-halophyte Triticum. Soil microbiomes analysis using pyrosequencing of 16S rRNA gene indicated that Actinobacteria were dominant in saline soil samples whereas Proteobacteria predominated in non-saline soil samples. Firmicutes, Acidobacteria, Bacteriodetes and Thaumarchaeota were predominant phyla in saline and non-saline soils, whereas Cyanobacteria, Verrucomicrobia, Gemmatimonadetes and the unclassified WPS-2 were less abundant. Sequences from Euryarchaeota, Ignavibacteriae, and Nanohaloarchaeota were identified only from the rhizosphere of halophytes. Dominant halophilic bacteria and archaea identified in this study included Agrococcus, Armatimonadetes gp4, Halalkalicoccus, Haloferula and Halobacterium. Our analysis showed that increases in soil salinity correlated with significant differences in the alpha and beta diversity of the microbial communities across saline and non-saline soil samples. Having a complete inventory of the soil bacteria from different saline environments in Pakistan will help in the discovery of potential inoculants for crops growing on salt-affected land.     

Epiphytic bacterial communities of the alga <Emphasis Type="Italic">Fucus vesiculosus</Emphasis> in oil-contaminated water areas of the Barents Sea

Taxonomic compositions of epiphytic bacterial communities in water areas differing in levels of oil pollution were revealed. In total, 82 bacterial genera belonging to 16 classes and 11 phyla were detected. All detected representatives of epiphytic bacterial communities belonged to the phyla Actinobacteria, Bacteroidetes, Planctomycetes, Proteobacteria, Verrucomicrobia, Acidobacteria, Cyanobacteria, Firmicutes, and Fusobacteria and candidate division TM7. The ratio of the phyla in the communities varied depending on the levels of oil pollution. New data on taxonomic composition of uncultivated epiphytic bacterial communities of Fucus vesiculosus were obtained.     

Deep sequencing analysis of bacterial community structure of Soldhar hot spring,India

We investigated the bacterial community structure of Soldhar hot spring with extreme high temperature 95°C located in Uttarakhand, India using high throughput sequencing. Bacterial phyla Proteobacteria (88.8%), Deinococcus-Thermus (7.5%), Actinobacteria (2.3%), and Firmicutes (1.07%) were predominated in the sequencing survey, whereas Bacteroidetes, Verrucomicrobia, Aquificae and Acidobacteria were detected in relatively lower abundance in Soldhar hot spring. At the family level, Comamonadaceae (52.5%), Alteromonadaceae (15.9%), and Thermaceae (7.5%) were mostly dominated in the ecosystem followed by Chromatiaceae, Microbacteriaceae, and Cyclobacteriaceae. Besides, members of Rhodobacteraceae, Moraxellaceae, Xanthomonadaceae, Aquificaceae, Enterobacteriaceae, Bacillaceae, Methylophilaceae, etc. were detected as a relatively lower abundance. In the present study we discuss the overall microbial community structure and their relevance to the ecology of the Soldhar hot spring environment.     

Comparative analysis of prokaryotic diversity in solar salterns in eastern Anatolia (Turkey)

The prokaryotic communities of four salterns (Bingöl, Fadlum, Kemah, and Tuzlagözü) in Turkey were examined and compared using the cultivation and cultivation-independent methods [fluorescence in situ hybridization (FISH) and 454 pyrosequencing]. FISH analysis with universal probes revealed that feeding waters carried 1.6 × 10²–1.7 × 10³ cells mL^?1, while crystallization ponds carried 3.8 × 10⁶–2.0 × 10⁷ cells mL^?1 that were mostly haloarchaea, including square cells (except for Kemah). High-throughput 16S rRNA-based gene sequencing showed that the most frequent archaeal OTUs in Bingöl, Fadlum, Tuzlagözü, and Kemah samples were affiliated with Haloquadratum (76.8 %), Haloarcula (27.8 %), Halorubrum (49.6 %), and Halonotius (59.8 %), respectively. Bacteroidetes was the dominant bacterial phylum in Bingöl and Fadlum, representing 71.5 and 79.5 % of the bacterial OTUs (respectively), while the most abundant bacterial phylum found in the Kemah saltern was Proteobacteria (79.6 %). The majority of the bacterial OTUs recovered from Tuzlagözü belonged to the Cyanobacteria (35.7 %), Bacteroidetes (35.0 %), and Proteobacteria (25.5 %) phyla. Cultivation studies revealed that the archaeal isolates were closely related to the genera Halobacterium, Haloarcula, and Halorubrum. Bacterial isolates were confined to two phyla, Proteobacteria (Alphaproteobacteria and Gammaproteobacteria classes) and Bacteroidetes. Comparative analysis showed that members of the Euryarchaeota, Bacteroidetes, Proteobacteria, and Cyanobacteria phyla were major inhabitants of the solar salterns.     

Bacterial diversity obtained by culturable approaches in the gut of <Emphasis Type="Italic">Glossina pallidipes</Emphasis> population from a non sleeping sickness focus in Tanzania: preliminary results

Background

Glossina pallidipes is a haematophagous insect that serves as a cyclic transmitter of trypanosomes causing African Trypanosomiasis (AT). To fully assess the role of G. pallidipes in the epidemiology of AT, especially the human form of the disease (HAT), it is essential to know the microbial diversity inhabiting the gut of natural fly populations. This study aimed to examine the diversity of G. pallidipes fly gut bacteria by culture-dependent approaches.

Results

113 bacterial isolates were obtained from aerobic and anaerobic microorganisms originating from the gut of G. pallidipes. 16S rDNA of each isolate was PCR amplified and sequenced. The overall majority of identified bacteria belonged in descending order to the Firmicutes (86.6%), Actinobacteria (7.6%), Proteobacteria (5.5%)and Bacteroidetes (0.3%). Diversity of Firmicutes was found higher when enrichments and isolation were performed under anaerobic conditions than aerobic ones. Experiments conducted in the absence of oxygen (anaerobiosis) led to the isolation of bacteria pertaining to four phyla (83% Firmicutes, 15% Actinobacteria, 1% Proteobacteria and 0.5% Bacteroidetes, whereas those conducted in the presence of oxygen (aerobiosis) led to the isolation of bacteria affiliated to two phyla only (90% Firmicutes and 10% Proteobacteria). Phylogenetic analyses placed these isolates into 11 genera namely Bacillus, Acinetobacter, Mesorhizobium, Paracoccus, Microbacterium, Micrococcus, Arthrobacter, Corynobacterium, Curtobacterium, Vagococcus and Dietzia spp.which are known to be either facultative anaerobes, aerobes, or even microaerobes.

Conclusion

This study shows that G. pallidipes fly gut is an environmental reservoir for a vast number of bacterial species, which are likely to be important for ecological microbial well being of the fly and possibly on differing vectorial competence and refractoriness against AT epidemiology.

     

Diversity and dynamics of the bacterial community involved in pig manure biodegradation in a microbial fermentation bed system

Overproduction of livestock manures with unpleasant odors causes significant environmental problems. The microbial fermentation bed (MFB) system is considered an effective approach to recycling utilization of agricultural byproducts and pig manure (PM). To gain a better understanding of bacterial communities present during the degradation of PM in MFB, the PM bacterial community was evaluated at different fermentation stages using 16S rRNA high throughput sequencing technology. The heatmap plot clustered five samples into short-term fermentation stage of 0–10 days and long-term fermentation stage of 15–20 days. The most abundant OTUs at the phylum level were Firmicutes, Actinobacteria and Proteobacteria in the long-term fermentation stage of PM, whereas Firmicutes, Bacteroidetes, and Proteobacteria predominated in the short-term fermentation stage of PM. At the genus level, organic degradation strains, such as Corynebacterium, Bacillus, Virgibacillus, Pseudomonas, Actinobacteria, Lactobacillus, Pediococcus were the predominate genera at the long-term fermentation stage, but were found only rarely in the short-term fermentation stage. C/N ratios increased and the concentration of the unpleasant odor substance 3-hydroxy-5-methylisoxazole (3-MI) decreased with prolonged period of fermentation. Redundancy analysis (RDA) demonstrated that the relative abundance of Firmicutes, Actinobacteria, Acidobacteria and Proteobacteria had a close relationship with degradation of 3-MI and increasing C/N ratio. These results provide valuable additional information about bacterial community composition during PM biodegradation in animal husbandry.     

Composition of Microbial Communities in Sediments from Southern Baikal Containing Fe/Mn Concretions

Massive parallel sequencing of the 16S rRNA gene fragments was used to investigate the composition and diversity of microbial communities in sediments from Southern Baikal to a depth of 9 cm with 1-cm step. In the layers from the sediment surface to the lower border of oxygen penetration (2 cm), organotrophic bacteria with high similarity to the heterotrophic species Luteolibacter luojiensis constituted the largest fraction of the community. In the formation zone of Fe/Mn crusts (3–5 cm), Proteobacteria and Actinobacteria predominated in the community, while the share of Cyanobacteria was considerable. The lower reduced layers showed an increased contribution of the Bacteroidetes, while the shares of the taxa predominant in the higher layers remained significant. Analysis of archaeal 16S rRNA gene amplicons revealed predominance of the soil and aquatic Thaumarchaeota (Marine Group I lineage), which are involved in anaerobic ammonium oxidation, practically in all sediment layers. The buried oxidized layer (6–7 cm), where members of the uncultured Marine Benthic Group D lineage of the order Thermoplasmatales (Euryarchaeota) predominated, was an exception in this regard. Small numbers of archaea of the Baikal-1 lineages (below 1%) were observed in the communities from the 6–7 and 7–8 cm layers, while the archaea involved in anaerobic methane oxidation (including the ANME-2d group) were not detected.     

Identification of the di-<Emphasis Type="Italic">n</Emphasis>-butyl phthalate-biodegrading strains and the biodegradation pathway in strain LMB-1

DNA isolated from a greenhouse soil (Nanjing, Jiangsu Province, China) was suitable for PCR amplification of gene segment coding for the 16S rRNA. Diverse PCR products were characterized by cloning and sequencing, and analysis of bacterial colonies showed the presence over 26 phyla. The most bacteria belonged to Proteobacteria, Actinobacteria, Gemmatimonadetes, Acidobacteria and Planctomycetes. Furthermore, after the enrichment procedure of DBP-degrading microorganisms, 4 strains were isolated from the soil sample with di-n-butyl phthalate (DBP) biodegradability, and they were identified to be Rhizobium sp., Streptomyces sp., Pseudomonas sp. and Acinetobacter sp. Analysis of the degradation products by LC-MS led to identification of metabolites of DBP in strain LMB-1 (identified as Rhizobium sp.) which suggests that DBP was degraded through β-oxidation, demethylation, de-esterification and cleavage of aromatic ring.     

The first results of using the <Emphasis Type="Italic">Allium</Emphasis> test in estimating the chemical and radiation toxicity of bottom sediments in the Yenisei River

The first results of the use of the Allium test for estimation of toxicity of bottom sediments in the Yenisei River and the effect of external γ-radiation under laboratory conditions are presented. The effect of stimulation of the onion root growth, i.e., the absence of toxicity was discovered in toxicological experiments using bottom sediments and under external γ-radiation. The stimulating effect of radiation on the growth of onion roots limits the use of the Allium test for testing samples from the Yenisei River ecosystem in the zone subjected to the impact of radioactive discharges from the Mining and Chemical Combine.     

Dynamics and diversity of a microbial community during the fermentation of industrialized <Emphasis Type="Italic">Qingcai</Emphasis> paocai,a traditional Chinese fermented vegetable food,as assessed by Illumina MiSeq sequencing,DGGE and qPCR assay

Paocai is a traditional Chinese fermented food and typically produced via spontaneous fermentation. We have investigated the microbial community utilized for the fermentation of industrialized Qingcai paocai using the combination of Illumina MiSeq sequencing, PCR-mediated denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative PCR (qPCR) assay. Three main phyla, namely Firmicutes, Proteobacteria and Bacteroidetes, were identified by both MiSeq sequencing and PCR-DGGE. The dominant genera observed in the fermentation were Lactobacillus, Pseudomonas, Vibrio and Halomonas. Most genera affiliated with Proteobacteria or Bacteroidetes were detected more often during the earlier part of the fermentation, while Lactobacillus (affiliated with Firmicutes) was dominant during the later fermentation stages. Fungal community analysis revealed that Debaryomyces, Pichia and Kazachstania were the main fungal genera present in industrialized Qingcai paocai, with Debaryomyces being the most dominant during the fermentation process. The quantities of dominant genera Lactobacillus and Debaryomyces were monitored using qPCR and shown to be 10⁹–10¹² and 10⁶–10¹⁰ copies/mL, respectively. During the later fermentation process of industrialized Qingcai paocai, Lactobacillus and Debaryomyces were present at 10¹¹ and 10⁸ copies/mL, respectively. These results facilitate further understanding of the unique microbial ecosystem during the fermentation of industrialized Qingcai paocai and guide future improvement of the fermentation process.     

Distribution and characteristics of <Emphasis Type="Italic">Bacillus</Emphasis> bacteria associated with hydrobionts and the waters of the Peter the Great Bay,Sea of Japan

Bacilli of the species Bacillus subtilis, B. pumilus, B. mycoides, B. marinus and B. licheniformis (a total of 53 strains) were isolated from 15 invertebrate species and the water of the Vostok Bay, Peter the Great Bay, Sea of Japan. Bacilli were most often isolated from bivalves (22.7%) and sea cucumbers (18.9%); they occurred less frequently in sea urchins and starfish (13.2 and 7.5%, respectively). Most of bacilli strains were isolated from invertebrates inhabiting silted sediments. No Bacillus spp. strains were isolated from invertebrates inhabiting stony and sandy environments. The species diversity of bacilli isolated from marine objects under study was low. Almost all bacterial isolates were resistant to lincomycin. Unlike B. pumilus, B. subtilis isolates were mostly resistant to benzylpenicillin and ampicillin. Antibiotic sensitivity of B. licheniformis strains was variable (two strains were resistant to benzylpenicillin and oxacillin, while one was sensitive). A significant fraction of isolated bacilli contained pigments. Pigmented strains were more often isolated from seawater samples, while colorless ones predominated within hydrobionts. B. subtilis colonies had the broadest range of colors. In the Bacillus strains obtained, DNase, RNase, phosphatase, elastolytic, chitinase, and agarolytic activity was detected. Bacilli strains with hydrolytic activity occurred in invertebrates more often than in seawater.     

Diversity of Prokaryotes in Planktonic Communities of Saline Sol-Iletsk lakes (Orenburg Oblast,Russia)

Prokaryotic diversity was studied in the planktonic communities of six Sol-Iletsk lakes (Orenburg oblast, Russia) varying in salinity level using the Illumina technology of high-throughput sequencing. The extremely halophilic archaea of the phyla Euryarchaeota and Nanohaloarchaeota, as well as the bacterial phylum Bacteroidetes predominated in the communities of lakes with salinity of 285–300‰. Representatives of the phyla Bacteroidetes and Actinobacteria, as well as of the class Gammaproteobacteria were predominant in the lakes with salinity 110?180‰. A bloom of Cyanobacteria was observed in Bol’shoe Gorodskoe Lake (10‰ salinity). The dominant OTUs in the lakes with high salinity were represented by archaea Halonotius sp., uncultured Nanohaloarchaea, and bacteria Salinibacter sp. In the lakes with medium salinity level the dominants included gammaproteobacteria Spiribacter sp., alphaproteobacteria Roseovarius sp., flavobacteria Psychroflexus sp., unidentified archaea of the family Haloferacaceae, actinobacteria Pontimonas sp. and Rhodoluna sp. In the lake with low salinity level cyanobacteria of the genus Planktothrix were predominant. Effect of salinity on prokaryotic taxonomic richness, composition, and diversity in planktonic communities of the studied lakes was demonstrated.     

A new species of grayling <Emphasis Type="Italic">Thymallus svetovidovi</Emphasis> sp. nova (Thymallidae) from the Yenisei basin and its position in the genus <Emphasis Type="Italic">Thymallus</Emphasis>

The Upper Yenisei grayling Thymallus svetovidovi sp. nova that inhabits the headwaters of the Yenisei River in Mongolia is described. From the other representatives of the genus Thymallus, the species differs in the elements of body coloration, dorsal fin pattern, some morphometric characters, and genetic characteristics. Besides this species, most of the Yenisei basin is inhabited by Baikal grayling T. baicalensis Dyb., and its low reaches, by the Arctic grayling T. arcticus (Pall.).     

Characterization of bacterial composition and diversity in a long-term petroleum contaminated soil and isolation of high-efficiency alkane-degrading strains using an improved medium

Bacterial community and diversity in a long-term petroleum-contaminated soil of an oilfield were characterized using 16S rRNA gene-based Illumina MiSeq high-throughput sequencing. Results indicated that Proteobacteria (49.11%) and Actinobacteria (24.24%) were the most dominant phyla, and the most abundant genera were Pseudoxanthomonas (8.47%), Luteimonas (3.64%), Alkanindiges (9.76%), Acinetobacter (5.26%) and Agromyces (8.56%) in the soil. Meanwhile a series of cultivations were carried out for isolation of alkane degraders from petroleum-contaminated soil with gellan gum and agar as gelling agents. And the isolates were classified by their 16S rRNA genes. Nine of the isolates including Enterobacter, Pseudomonas,Acinetobacter, Rhizobium, Bacillus, Sphingomonas, Paenibacillus, Variovorax and Rhodococcus showed strong biodegradability of alkane mixture (C9–C30) in a wide range of chain-length, which could be potentially applied in enhancement of bioremediation.     

Benthic foraminifera in deep trenches of the Kara Sea and the relation of <Emphasis Type="Italic">Saccorhiza ramosa</Emphasis> (Brady) to the distribution of natural radionuclides

The abundance and structure of the foraminifera community have been investigated. The role of agglutinating and secreting species in bottom sediments of the deep trenches of the Kara Sea has been studied. It has been found that the abundance and dominance of the agglutinated foraminifera Saccorhiza ramosa depend on the depth of the sea and are related to the genetic type of origin of such sediments. A correlation has been revealed between the distribution of S. ramosa and the concentrations of natural radionuclides (²²⁶Ra, ²³²Th, and ²¹⁰Pb) in bottom sediments. The degree of dominance of S. ramosa in the community of benthic foraminifera increases along with natural radioactivity.     

Bacterial communities during the period of massive under-ice dinoflagellate development in Lake Baikal

Taxonomic diversity of Lake Baikal bacteria during the period of massive under-ice development of dinoflagellate Gymnodinium baicalense was studied. During the ice-covered period in 2013, both the abundance and biomass of G. baicalense were several orders of magnitude higher than the values for previous years, the maximum values were 8.9 × 10⁶ cells/L and 405 g/m³, respectively. The taxonomic structure of bacterial communities was determined using the data obtained by 454 pyrosequencing (Roche) with Mothur 1.19.0. Predominance of three phyla was revealed: Bacteroidetes, Proteobacteria, and Actinobacteria. Massive dinoflagellate development resulted in a considerable decrease in the richness and diversity of bacterial communities compared to the results of the earlier long-term studies.     

Structure of Microbial Communities of the Sediments of Alkaline Transbaikalia Lakes with Different Salinity

The goal of the work was to reveal the differences in the structure of microbial communities of Transbaikalia alkaline lakes stemming from the differences in their salinity and hydrochemical parameters. The lakes studied were Verkhnee Beloe (Buryat Republic, Russia), as well as Khilganta, Gorbunka, and Borzinskoe (Transbaikal krai, Russia) with salinity from 12.3 to 430 g/L, which differed in the mineral composition of the sediments and hydrochemical parameters. Lake sediments were found to contain 47 prokaryotic phyla (42 bacterial and 5 archaeal ones). The phyla Proteobacteria, Euryarchaeota, Bacteroides, Chloroflexi, Actinobacteria, and Firmicutes were predominant, comprising over 95% of the classified sequences. Comparative abundance of archaea increased with salinity from below 1% in Lake Verkhnee Beloe to 35% in Lake Borzinskoe. The most numerous bacterial OTUs belonged to gammaproteobacteria of the genus Halomonas (up to 15% of the number of classified sequences). The most numerous archaeal OTUs were identified at the genus level as members of the genera Halorubrum and Halohasta belonging to the family Halorubraceae, which comprises extremely halophilic Euryarchaeota.     

Distinctive features of the microbial diversity and the polyketide synthase genes spectrum in the community of the endemic Baikal sponge <Emphasis Type="Italic">Swartschewskia papyracea</Emphasis>

The diversity of the symbiotic community of the endemic Baikal sponge Swartschewskia papyracea was studied, and an analysis of the polyketide synthases genes spectrum in sponge-associated microorganisms was carried out. Six bacterial phyla were detected in the S. papyracea microbiome: Verrucomicrobia, Cyanobacteria, Actinobacteria, Bacteroidetes, Proteobacteria, and Planctomycetes. Unlike the microbial associations of other freshwater sponges, the community under study was dominated by the phylaVerrucomicrobia (42.1%) and Cyanobacteria (17.5%), while the proportion of the Proteobacteria was unusually low (9.7%). In the S. papyracea community metagenome, there were identified 18 polyketide synthases genes fragments, the closest homologues of which included the polyketide synthases of the microorganisms belonging to the bacterial phyla Cyanobacteria, Proteobacteria (classes Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria), and Acidobacteria as well as the eukaryotic algae of the phylum Heterokonta (class Eustigmatophyceae). Polyketide synthase sequences from S. papyracea formed three groups on the phylogenetic tree: a group of hybrid NRPS/PKS complexes, a group of cyanobacterial polyketide synthases, and a group of homologues of the eukaryotic alga Nannochloropsis gaditana. Notably, the identified polyketide synthase genes fragments showed only a 57–88% similarity to the sequences from the databases, which implies the presence of genes controlling the synthesis of the novel, still unstudied, polyketide compounds in the S. papyracea community. It was proposed that the habitat conditions of S. papyracea affect the taxonomic composition of the microorganisms associated with the sponge, including the diversity of the producers of secondary metabolites.