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A method named sequence-specific capture of oligonucleotide probes (SCOPE) was developed for quantification of microbial rRNA molecules in a multiplex manner. In this method, a molecular weight cutoff membrane (MWCOM) was used for the separation of fluorescence-labeled oligonucleotide probes hybridized with rRNA from free unhybridized probes. To demonstrate proof of concept, probes targeting bacteria or archaea at different taxonomic levels were prepared and were hybridized with rRNAs. The hybridization stringency was controlled by adjusting reaction temperature and urea concentration in the mixture. Then, the mixture was filtered through the MWCOM. The rRNA and hybridized probes collected on the MWCOM were recovered and quantified using a spectrophotometer and fluorospectrometer, respectively. The method showed high accuracy in detecting specific microbial rRNA in a defined nucleic acid mixture. Furthermore, the method was capable of simultaneous detection and quantification of multiple target rRNAs in a sample with sensitivity up to a single-base mismatch. The SCOPE method was tested and benchmarked against reverse transcription-quantitative PCR (RT-qPCR) for the quantification of Bacteria , Archaea , and some key methanogens in anaerobic sludge samples. It was observed that the SCOPE method produced more reliable and coherent results. Thus, the SCOPE method allows simple and rapid detection and quantification of target microbial rRNAs for environmental microbial population analysis without any need for enzymatic reactions. IMPORTANCE Microorganisms play integral roles in the Earth's ecosystem. Microbial populations and their activities significantly affect the global nutrient cycles. Quantification of key microorganisms provides important information that is required to understand their roles in the environment. Sequence-based analysis of microbial population is a powerful tool, but it provides information only on relative abundance of microorganisms. Hence, the development of a simpler and quick method for the quantification of microorganisms is necessary. To address the shortcomings of a variety of molecular methods reported so far, we developed a simple, rapid, accurate, and multiplexed microbial rRNA quantification method to evaluate the abundance of specific microbial populations in complex ecosystems. This method demonstrated high specificity, reproducibility, and applicability to such samples. The method is useful for quantitative detection of particular microbial members in the environment.

The plastic film is extensively applied with limited recycling, leading to the long-run residue accumulation in soil, which offers a distinctive habitat for microorganisms, and creates a plastisphere. In this study, traditional low-density polyethylene (LDPE) plastic film and biodegradable polybutylene adipate terephthalate (PBAT) plastic film materials were selected to test their effects on soil microbial ecology. Based on high-throughput sequencing, compared to the soil environment, the alpha-diversity of bacterial communities in plastisphere was lower, and the abundance of Actinobacteria increased. Plastic film residues, as bacterial habitats, exhibited greater heterogeneity and harbor unique bacterial communities. The communities were distinguished between plastisphere and soil environment by means of a random-forest (RF) machine-learning model. Prominent distinctions emerged among bacterial functions between soil environment and plastisphere, especially regarding organics degradation. The neutral model and null model indicated that the constitution of bacterial communities was dominated by random processes except in LDPE plastisphere. The bacterial co-occurrence network of the plastisphere exhibited higher complexity and modularity. This study contributes to our comprehending of characteristics of plastisphere bacterial communities in soil environment and the associated ecological risks of plastic film residues accumulation., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

A pilot-scale experiment of natural rubber processing wastewater treatment was conducted using a combination system consisting of a two-stage up-flow anaerobic sludge blanket (UASB) and a down-flow hanging sponge (DHS) reactor for more than 10 months. The system achieved a chemical oxygen demand (COD) removal efficiency of 95.7% ± 1.3% at an organic loading rate of 0.8 kg COD/(m(3).d). Bacterial activity measurement of retained sludge from the UASB showed that sulfate-reducing bacteria (SRB), especially hydrogen-utilizing SRB, possessed high activity compared with methane-producing bacteria (MPB). Conversely, the acetate-utilizing activity of MPB was superior to SRB in the second stage of the reactor. The two-stage UASB-DHS system can reduce power consumption by 95% and excess sludge by 98%. In addition, it is possible to prevent emissions of greenhouse gases (GHG), such as methane, using this system. Furthermore, recovered methane from the two-stage UASB can completely cover the electricity needs for the operation of the two-stage UASB-DHS system, accounting for approximately 15% of the electricity used in the natural rubber manufacturing process.

Objective: Heterotrophic manganese (Mn)-oxidizing microorganisms responsible for biogenic manganese oxide (Bio-MnOx) production are fastidious. Their enrichment is not easily accomplished by merely adding a soluble organic substrate to non-sterile mixed cultures. The objective of this study was to evaluate polycaprolactone (PCL), an aliphatic polyester, as an effective solid organic substrate for the enrichment of marine Mn-oxidizing microorganisms., Results: We successfully obtained marine microbial enrichment with the capacity for dissolved Mn removal and MnOx production using PCL as a solid organic substrate. The removal of dissolved Mn by the Mn-oxidizing enrichment culture followed first-order kinetics with a rate constant of 0.014 h -1 . 16S rRNA gene amplicon sequencing analysis revealed that the Mn-oxidizing enrichment culture was highly dominated by operational taxonomic units related to the bacterial phyla Cyanobacteria, Planctomycetes, and Proteobacteria., Conclusions: Our data demonstrate that PCL can serve as a potential substrate to enrich Mn-oxidizing microorganisms with the ability to produce MnOx under marine conditions.

Aims: This study aimed to develop an efficient, cost-effective and eco-friendly bacterial consortium to degrade petroleum sludge., Methods and Results: Four bacterial strains belonging to genera Acinetobacter and Pseudomonas were selected to constitute three different consortia based on their initial concentration. The highest degradation rate (78%) of 1% (v/v) crude oil after 4 weeks of incubation was recorded when the concentration of biosurfactant (BS) producing isolate was high. Genes, such as alkB, almA, cyp153, pah-rhdGN, nah, phnAC and cat23 were detected using the polymerase chain reaction method and their induction levels were optimal at pH 7·0. A crude oil sludge was artificially constituted, and its bacterial composition was investigated using 16S rRNA gene amplicon sequencing. The results showed that the soil bacterial community was dominated by plant growth-promoting bacteria (PGPB) after crude oil treatment., Conclusions: Our findings indicate the decontamination of the crude oil contaminated soil was more effective in the presence of both the constituted consortium and PGPB compared to the presence of PGPB alone., Significance and Impact of the Study: This study showed that the PGPB (Taibaiella) present in petroleum uncontaminated soil can promote the soil decontamination. The addition of both efficient hydrocarbon-degrading and BS producing bacteria is also necessary to improve the decontamination., (© 2020 The Society for Applied Microbiology.)

The petrochemicals produced from refining oil have become a large part of human life, making oil a valuable and expensive commodity. As a non-renewable resource, extraction and transport efforts have intensified to keep up with the demand, increasing the occurrences of oil spills. Such accidents have devastating impacts on the environment, the health of organisms, and a country's economy like the Philippines, and thus, need to be resolved immediately. One way to deal with oil spills is through bioremediation but the process is still facing several challenges. For one, the toolbox for bioremediation is limited. About 79 genera of bacteria were observed to degrade oil but there are only a small number of bacterial species and/or strains that have been recognized as useful for bioremediation. Second is that most oil-degrading bacteria found have low oil degradation efficiencies. Another challenge is keeping the bacteria alive to carry out the process. Fortunately, progress has been made in solving these challenges. Researchers are now testing different consortia, including bacteria--bacteria, bacteria--fungi, bacteria--microalgae, that can complement each other such as biosurfactant-producing bacteria with different oil-degrading microorganisms or microalgae or fungi that enhance the growth of oil-degrading bacteria. A consortium like this improves the survivability of each microorganism and enhances the oil-degrading efficiency. Moreover, the search for additional oil-degrading and biosurfactant-producing bacteria and other microorganisms to add to the bioremediation toolbox has been improved with the emergence of high-throughput sequencing. Aside from microorganisms, seaweeds have shown potential for bioremediation. The seaweed Caulerpa prolifera has been demonstrated to degrade diesel up to a certain concentration with the help of the bacteria growing on its surface. Bioremediation has a long way to go, but recent developments have shown promise and it remains to be the cheapest, most environment-friendly, and most effective way of dealing with oil spills. [ABSTRACT FROM AUTHOR]

Tanjung Emas Port is the entry and exit point for trade commodities, both regional and international filled with many ships. This condition makes biofouling a very massive process in that place by various types of marine bacteria. The initial formation of a biofilm is relevant to bacterial diversity, colonization and adhesion. The objective of the study was assessing bacterial diversity in relation to with biofouling within Tanjung Mas Port Semarang, by using 16S rRNA eDNA metabarcoding Next-Generation Sequencing (NGS). Seawater samples from aquatic sites of Tanjung Mas harbor was used for DNA extraction and amplification of the 16S rRNA V-3-V4 hypervariable region, followed by sequencing and library construction of eDNA Metabarcoding. Sequence processing and analysis was performed in QIIME 2 and RStudio using DADA2 for advanced sequencing processing and Phyloseq. The results of this research showed that bacteria is the predominant taxon constituting 100% of the community. The taxon consists of Proteobacteria (49.38%), Bacteroidota (8.67%), and Firmicutes (8.88%). Alphaproteobacteria (20.92%) and Gammaproteobacteria (12.39%) dominate at the Class level, emphasizing their versatility and ecological influence. At the Order and Family levels reveals the prevalence of Rhodobacterales (10.04%), Chitinophagales (2.53%), Rhizobiales (3.61%), Rhodobacteraceae (5.67%), Saprospiraceae (3.63%), and Rhizobiaceae (2.15%). It was found that the unculturable taxa dominance in Tanjung Mas Port was 44.66%. These taxonomic entities contribute significantly to the taxonomic and functional diversity of the microbial community, influencing nutrient cycling, organic matter degradation, ecosystem stability and biofilm formation. [ABSTRACT FROM AUTHOR]

The oceans cover over 70% of our planet, hosting a biodiversity of tremendous wealth. Sponges are one of the major ecosystem engineers on the seafloor, providing a habitat for a wide variety of species to be considered a good source of bioactive compounds. In this study, a metataxonomic approach was employed to describe the bacterial communities of the sponges collected from Faro Lake (Sicily) and Porto Paone (Gulf of Naples). Morphological analysis and amplification of the conserved molecular markers, including 18S and 28S (RNA ribosomal genes), CO1 (mitochondrial cytochrome oxidase subunit 1), and ITS (internal transcribed spacer), allowed the identification of four sponges. Metataxonomic analysis of sponges revealed a large number of amplicon sequence variants (ASVs) belonging to the phyla Proteobacteria, Cloroflexi, Dadabacteria, and Poribacteria. In particular, Myxilla (Myxilla) rosacea and Clathria (Clathria) toxivaria displayed several classes such as Alphaproteobacteria, Dehalococcoidia, Gammaproteobacteria, Cyanobacteria, and Bacteroidia. On the other hand, the sponges Ircinia oros and Cacospongia mollior hosted bacteria belonging to the classes Dadabacteriia, Anaerolineae, Acidimicrobiia, Nitrospiria, and Poribacteria. Moreover, for the first time, the presence of Rhizobiaceae bacteria was revealed in the spongeM. (Myxilla) rosacea, which was mainly associated with soil and plants and involved in biological nitrogen fixation. [ABSTRACT FROM AUTHOR]

The textile industry generates significant amounts of wastewater containing high concentrations of azo dyes. An important point in the process of purification of azo dyes is their influence on the activated sludge (AS) in wastewater treatment plants. Azo dyes, such as amaranth, play the role of xenobiotics. This article seeks to answer the question of how organisms manage to respond to xenobiotics remains very important and open, i.e., how they will react to toxic conditions. The aim of this research was to study how these changes are expressed in terms of the different trophic levels of AS. In our experiment, it was found that the dominant trophic units are significantly changed due to the xenobiotic entering the system. The data reveal the significant development of the bacterial segment (genus Pseudomonas and azo-degrading bacteria) at times of large amaranth removal. In the most active phase of amaranth biodetoxification (48 h), the culturable bacteria of the genus Pseudomonas change by about 40%, while the azo-degrading bacteria change by about 2%. Fauna organisms have a sharp change in the dominant groups—from attached and crawling ciliates and testate amoebas to the mass development of small and large flagellates. This is of great importance because micro- and metafauna play an important role in the detoxification process by ingesting some of the xenobiotics. This role is expressed in the fact that after dying, macro-organisms release this xenobiotic in small portions so that it can then be effectively degraded by adapting to the amaranth biodegradation bacteria. In this study, it is clear that all these events lead to a decline in the quality of AS. But on the other hand, these allow AS to survive as a microbial community, and the fauna segment does not disappear completely. [ABSTRACT FROM AUTHOR]

Marine oil spills are a significant concern worldwide, destroying the ecological environment and threatening the survival of marine life. Various oil-degrading bacteria have been widely reported in marine environments in response to marine oil pollution. However, little information is known about culturable oil-degrading bacteria in cold seep of the deep-sea environments, which are rich in hydrocarbons. This study enriched five oil-degrading consortia from sediments collected from the Haima cold seep areas of the South China Sea. Parvibaculum, Erythrobacter, Acinetobacter, Alcanivorax, Pseudomonas, Marinobacter, Halomonas, and Idiomarina were the dominant genera. Further results of bacterial growth and degradation ability tests indicated seven efficient alkane-degrading bacteria belonging to Acinetobacter, Alcanivorax, Kangiella, Limimaricola, Marinobacter, Flavobacterium, and Paracoccus, whose degradation rates were higher in crude oil (70.3-78.0%) than that in diesel oil (62.7-66.3%). From the view of carbon chain length, alkane degradation rates were medium chains > long chains > short chains. In addition, Kangiella aquimarina F7, Acinetobacter venetianus F1, Limimaricola variabilis F8, Marinobacter nauticus J5, Flavobacterium sediminis N3, and Paracoccus sediminilitoris N6 were first identified as oil-degrading bacteria from deep-sea environments. This study will provide insight into the bacterial community structures and oil-degrading bacterial diversity in the Haima cold seep areas, South China Sea, and offer bacterial resources to oil bioremediation applications. [ABSTRACT FROM AUTHOR]

Five strains of psychrotrophic hydrocarbon-oxidizing bacteria were isolated from the bottom sediments of Peter the Great Bay of the Sea of Japan. They were classified into the following species: Rhodococcus erythropolis, Rhodococcus sp., Sphingomonas sp., Pseudomonas sp., and Alcanivorax sp. All studied bacteria showed high oxidizing ability in relation to the decomposition of n-alkanes (C9–C27), phytane, pristane, and polycyclic aromatic hydrocarbons at 5 and 22°C. At the same time, the degradation of hydrocarbons was more intense at 5°C. Despite the different taxonomic affiliations of the obtained microorganisms, all strains primarily utilized short- (C9–C13) and long-chain (C21–27) alkanes, as well as polycyclic aromatic hydrocarbons. The highest hydrocarbon-oxidizing activity was shown by the strain Rhodococcus erythropolis AP_291. The latter utilized more than 50% of all hydrocarbons in the model mixture during the first week of the experiment at 5°C. [ABSTRACT FROM AUTHOR]

The present work provides the first knowledge about the diversity and functionality of the microorganisms occurring in oily bilge wastes, from the port of Mar del Plata, Argentina. We used a culture independent approach, based on massive sequencing of the 16S rRNA gene (Illumina), to analyze the microbiome structure from different oily bilge wastes. To gain insight into functional biomarkers for hydrocarbon (HC) degradation in these wastes, the occurrence of alkB genes in the degrading bacteria communities was also investigated trough PCR-DGGE method. The microbial compositions were determined in bilge waste oily phase (BWOP) samples from a deep-sea, a fishing ship, a dredge ship, a small coastal vessel, and an open pool, where bilge wastes from Mar del Plata's vessels are deposited. A core microbiome with OTUs classified as bacterial types associated with HC biodegradation was found across all samples. Seven highly predominant and stable bacterial types accounted for 82% of the relative composition determined in all the BWOP samples. These results indicate that oily bilge is a strong selector for bacteria having common taxonomical and functional features, independent of the vessel of collection. Alkane degradation marker gene alkB PCR-DGGE fingerprinting showed distinct diversity patterns among the samples. Our analysis showed that there is a well-defined bacterial assemblage able to withstand and thrive in the harsh conditions of the oily bilge wastes. Also, a well-established aliphatic biodegradation potential marker was detected, indicating that it could promptly be used for the generation of new bioremediation technologies. [ABSTRACT FROM AUTHOR]

Marine hydrocarbon-degrading bacteria play an important role in natural petroleum biodegradation processes and were initially associated with man-made oil spills or natural seeps. There is no full clarity though on what, in the absence of petroleum, their natural niches are. Few studies pointed at some marine microalgae that produce oleophilic compounds (alkanes, long-chain fatty acids, and alcohols) as potential natural hosts of these bacteria. We established Dansk crude oil-based enrichment cultures with photobioreactor-grown marine microalgae cultures Pavlova lutheri and Nannochloropsis oculata and analyzed the microbial succession using cultivation and SSU (16S) rRNA amplicon sequencing. We found that petroleum enforced a strong selection for members of Alpha- and Gamma-proteobacteria in both enrichment cultures with the prevalence of Alcanivorax and Marinobacter spp., well-known hydrocarbonoclastic bacteria. In total, 48 non-redundant bacterial strains were isolated and identified to represent genera Alcanivorax , Marinobacter , Thalassospira , Hyphomonas , Halomonas , Marinovum , Roseovarius , and Oleibacter , which were abundant in sequencing reads in both crude oil enrichments. Our assessment of public databases demonstrated some overlaps of geographical sites of isolation of Nannochloropsis and Pavlova with places of molecular detection and isolation of Alcanivorax and Marinobacter spp. Our study suggests that these globally important hydrocarbon-degrading bacteria are associated with P. lutheri and N. oculata. [ABSTRACT FROM AUTHOR]

Vinasse is a by-product from the ethanol industry and can be used for methane production through anaerobic digestion process driven by microbial consortia. The microorganisms involved must be known to obtain an ideal methane production. The present work evaluated the production of methane and byproducts from different ratios substrate/biomass (S0/X0), using sludge from an effluent treatment of the vegetable oil industry as inoculum in media containing vinasse. Also, the microbial community of the best methane production bioassay was characterized by high-throughput DNA sequencing. The following chemical parameters were evaluated: methanogenic activity, chemical oxygen demand, carbohydrate consumption, and production of volatile fatty acids. The highest methane production occurred at S0/X0 ratio of 1.5, which produced 59.78 mmol CH4 L−1. A great variety of microorganism genera was identified by high-throughput DNA sequencing, showing differences in the microbial consortia of the initial and final sampling times. At the final sampling point, the classes Bacteroidia (Porphyromonadaceae—OTU genera unknown 42.26% and Bacteroides genus 10.58%) and the class Betaproteobacteria (Proteobacteria-Comamonadaceae OTU) were identified as the dominant bacteria. The most abundant archaeal genera in the bioassay were Methanosaeta, Methanomassiliicoccaceae OTU vadinCA11, and Methanobacterium. The identification of the microorganisms of consortia involved in anaerobic digestion can collaborate on technologies to increase methane production through microbial isolation, bioaugmentation, and co-cultures. [ABSTRACT FROM AUTHOR]

This study examined impacts of Macondo MC252 oil from the Deepwater Horizon spill on oil-degrading bacteria in fresh and oligohaline marshes dominated by the common reed Phragmites australis (Cav.) Trin. ex Steud. and correlated microbial changes to soil variables. We hypothesized that indigenous oil-degrading bacteria and soil characteristics in the marshes effectively respond to Macondo oil. We tested this hypothesis with a greenhouse mesocosm study. Weathered and emulsified Macondo oil was applied to P. australis sods at different rates (0, 4, 8, 12, and 16 L m−2). Populations of oil degrading bacteria containing alkane monooxygenase and PAH-ring hydroxylating dioxygenase alpha subunit (PAH-RHDα) genes, the expression of these genes, soil respiration rate, residual total petroleum hydrocarbon, redox potential (Eh), as well as dissolved organic carbon (DOC) and its aromaticity index of SUVA254 were measured in soils 2 months after oil addition. Oil exposure generally increased oil-degrading bacterial populations, in particular Gram-positive (GP) PAH-degraders, soil respiration rate, DOC concentration, and aromaticity of DOC, but decreased Eh values. GP PAH-RHDα genes accounted for approximately 98% of total detected genes, and expression of these genes increased by a factor of 3 to 5 at various oil dosages. Both abundance and expression of GP PAH-RHDα genes significantly correlated to SUVA254 (P < 0.05). The study revealed that indigenous oil-degrading bacteria effectively responded to weathered Macondo oil in the P. australis marsh soil wherein GP bacteria with PAH-RHDα genes played a major role in biodegradation of PAH-enriched petroleum hydrocarbons. [ABSTRACT FROM AUTHOR]

Field data from the first several days after an oil spill is rare but crucial for our understanding of a spill’s impact on marine microbiota given their short generation times. Field data collected within days of the Texas City “Y” oil spill showed that exposure to crude oil can rapidly imbalance populations of marine microbiota, which leads to the proliferation of more resistant organisms. Vibrionales bacteria were up to 48 times higher than background concentrations at the most impacted sites and populations of the dinoflagellate Prorocentrum texanum increased significantly as well. Laboratory microcosm experiments with a natural plankton community showed that P. texanum grew significantly faster under oiled conditions but monocultures of P. texanum did not. Additional laboratory experiments with natural communities from Tampa Bay, Florida showed similar results although a different species dominated, P. minimum. In both cases, tolerance to the presence of crude oil was enhanced by higher sensitivity of grazers led to a release from grazing pressure and allows Prorocentrum species to dominate after an oil spill. The results suggest careful monitoring for Vibrionales and Prorocentrum during future spills would be beneficial given the potential implications to human health. [ABSTRACT FROM AUTHOR]

Variations on hydrophobicity were monitored in two marine obligate hydrocarbonoclastic bacteria: Alcanivorax borkumensis SK2T and Thalassolituus oleivoras MIL-1T. These strains were inoculated, separately in ONR7a mineral medium with different concentration of sodium acetate. During 10 days measurements of cellular abundance and cellular hydrophobicity (capacity to adhere at polystyrene) were carried out. Data obtained revealed that the natures of carbon source and growth phase are important factors in the regulation of adhesion of bacteria to the surfaces. Moreover Alcanivorax showed a major capability to colonize polystyrene respect on Thalassolituus in the experimented conditions. The understanding of capacity to adhesion of these bacteria for utilization of hydrophobic compounds is fundamental for their potential use in the mitigation of oil spills phenomenon. [ABSTRACT FROM AUTHOR]

Background Synthetic microplastics (⩽5-mm fragments) are emerging environmental contaminants that have been found to accumulate within coastal marine sediments worldwide. The ecological impacts and fate of microplastic debris are only beginning to be revealed, with previous research into these topics having primarily focused on higher organisms and/or pelagic environments. Despite recent research into plastic-associated microorganisms in seawater, the microbial colonization of microplastics in benthic habitats has not been studied. Therefore, we employed a 14-day microcosm experiment to investigate bacterial colonization of lowdensity polyethylene (LDPE) microplastics within three types of coastal marine sediment from Spurn Point, Humber Estuary, U.K. Results Bacterial attachment onto LDPE within sediments was demonstrated by scanning electron microscopy and catalyzed reporter deposition fluorescence in situ hybridisation (CARDFISH). Log-fold increases in the abundance of 16S rRNA genes from LDPE-associated bacteria occurred within 7 days with 16S rRNA gene numbers on LDPE surfaces differing significantly across sediment types, as shown by quantitative PCR. Terminal-restriction fragment length polymorphism (T-RFLP) analysis demonstrated rapid selection of LDPEassociated bacterial assemblages whose structure and composition differed significantly from those in surrounding sediments. Additionally, T-RFLP analysis revealed successional convergence of the LDPE-associated communities from the different sediments over the 14- day experiment. Sequencing of cloned 16S rRNA genes demonstrated that these communities were dominated after 14 days by the genera Arcobacter and Colwellia (totalling 84-93% of sequences). Attachment by Colwellia spp. onto LDPE within sediments was confirmed by CARD-FISH. Conclusions These results demonstrate that bacteria within coastal marine sediments can rapidly colonize LDPE microplastics, with evidence for the successional formation of plastisphere-specific bacterial assemblages. Although the taxonomic compositions of these assemblages are likely to differ between marine sediments and the water column, both Arcobacter and Colwellia spp. have previously been affiliated with the degradation of hydrocarbon contaminants within low-temperature marine environments. Since hydrocarbon-degrading bacteria have also been discovered on plastic fragments in seawater, our data suggest that recruitment of hydrocarbonoclastic bacteria on microplastics is likely to represent a shared feature between both benthic and pelagic marine habitats. [ABSTRACT FROM AUTHOR]