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A rat model was used to determine resistant starch (RS) in vivo and three different methods to analyze it in vitro. The first method involves a Termamyl/gelatinization step, the next utilizes pancreatic amylase and the last method uses Termamyl and amyloglucosidase. It was shown that the first method is not suitable for the assay of indigestible starch fractions in raw materials. In addition, a fraction of heat-processed starch was observed to survive prolonged incubation with pancreatin but was susceptible to Termamyl at high temperatures. In vivo studies with mice proves to be efficient in measuring undigested starch in the small intestines.

Upon entering the marine environment, plastics are colonized by a plethora of microorganisms to form a plastisphere, influencing the fate and transport of the plastic debris and the health of marine ecosystems. The assembly of marine plastisphere is generally believed to be dominated by stochastic processes. However, it remains elusive whether microbial interaction in the assembly of plastisphere microbial communities is conserved or not. We analyzed the plastisphere microbiomes of 137 plastic debris samples from intertidal zones at different geographical locations and habitats (seagrass, coral, mangrove, beach, and open ocean) and compared them with the surrounding sediment and seawater microbiomes. Microbial community structures of the plastisphere from different locations were more similar to each other but differed substantially from the surrounding sediment and water microbiomes, implying a common mechanism of plastisphere assembly. We used different machine learning algorithms (Multinomial Logistic Regression, Support Vector Machine, Decision Trees, Random Forest, and Artificial Neural Networks) to classify plastic debris samples with high sensitivity based on the microbiome composition. Eukaryotic and prokaryotic phototrophic organisms such as green algae, diatoms, and cyanobacteria, were found to be enriched on the plastic surfaces. Network analysis revealed the central role of the phototrophic organisms in the formation and sustenance of the plastispheres. We found that phototrophs served as core members interacting strongly with heterotrophic organisms in marine plastisphere, irrespective of the sampling location, habitats, and polymer types. This would explain the stochastic assembly of the plastisphere along with conserved properties driven by the phototrophs in the surrounding environment. Our results highlight the importance of phototrophic organisms in shaping the marine plastisphere microbial communities., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Gut microbial communities of plastic-munching worms provide novel insights for the development of plastic-processing biotechnologies. Considering the complexity of worm maintenance and the gut microbial communities, it is challenging to apply the worms directly in plastic processing. Harnessing the power of microbial communities derived from the worm gut microbiomes in vitro may enable a promising bioprocess for plastic degradation. Here, we established stable and reproducible plastic-associated biofilm communities derived from the gut microbiome of a superworm, Zophobas atratus, through a two-stage enrichment process: feeding with plastics (HDPE, PP, and PS) and in vitro incubation of gut microbiomes obtained from the plastic-fed worms. Plastic feeding exhibited marginal influence on bacterial diversity but substantially changed the relative abundance of different bacterial groups, and intriguingly, enriched potential plastic degraders. More prominent shifts of microbial communities were observed during the in vitro incubation of the gut microbiomes. Taxa containing plastic-degrading strains were further enriched, while other taxa represented by lactic acid bacteria were depleted. Additionally, the plastic characterization confirmed the degradation of the incubated plastics by the plastic-associated microbial communities. Community functional inference for both gene abundance and community phenotype suggested that the in vitro incubation enhanced plastic-degrading potential. Deterministic ecological effects, in particular, selection processes, were identified as the main driving force of the observed community shifts. Our findings provide novel insights into plastic-munching-worm-inspired bioprocessing of plastic wastes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Coastal habitats have been suggested to serve as a sink for unaccounted plastic debris, i.e., "missing plastic" in the sea, and hence, a hotspot of plastic pollution in the marine and coastal environments. Although the accumulation of plastic debris may pose significant threats to coastal ecosystems, we know little about the fate of these plastic debris and their ecological impacts due to the lack of studies on plastic-microbe interactions in coastal habitats, especially for the tropical marine and coastal environments. In this study, we collected plastic debris from 14 sites consisting of various coastal ecosystems (seagrass meadows, mangrove forests, and beaches), and marine ecosystem (coral reef) around Singapore and characterized the prokaryotic and eukaryotic microbial communities colonized on them. Our results showed that the composition of plastisphere communities in these intertidal ecosystems was predominantly influenced by the sediment than by the plastic materials. Compared with surrounding sediment and seawater, the plastic debris enriched potential plastic degraders, such as Muricauda, Halomonas, and Brevundimonas. The plastic debris was also found to host taxa that play significant roles in biogeochemical cycles (e.g., cyanobacteria, Erythrobacter), hygienically relevant bacteria (e.g., Chryseobacterium, Brevundimonas), and potential pathogens that may negatively impact the health of coastal ecosystems (e.g., Thraustochytriaceae, Labyrinthulaceae, Flavobacterium). Taken together, our study provides valuable insights into the plastic-microbe interactions in tropical coastal and marine ecosystems, highlighting the urgent need for plastisphere studies to understand the fate and ecological impacts of plastic debris accumulated in coastal habitats., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Approximately 9 million metric tons of plastics enters the ocean annually, and once in the marine environment, plastic surfaces can be quickly colonised by marine microorganisms, forming a biofilm. Studies on plastic debris-biofilm associations, known as plastisphere, have increased exponentially within the last few years. In this review, we first briefly summarise methods and techniques used in exploring plastic-microbe interactions. Then we highlight research gaps and provide future research opportunities for marine plastisphere studies, especially, on plastic characterisation and standardised biodegradation tests, the fate of "environmentally friendly" plastics, and plastisphere of coastal habitats. Located in the tropics, Southeast Asian (SEA) countries are significant contributors to marine plastic debris. However, plastisphere studies in this region are lacking and therefore, we discuss how the unique environmental conditions in the SEA seas may affect plastic-microbe interaction and why there is an imperative need to conduct plastisphere studies in SEA marine environments. Finally, we also highlight the lack of understanding of the pathogenicity and ecotoxicological effects of plastisphere on marine ecosystems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Monochloramine (MCA) is a widely used secondary disinfectant to suppress microbial growth in drinking water distribution systems. In monochloraminated drinking water, a significant amount of extracellular DNA (eDNA) has been reported, which has many implications ranging from obscuring DNA-based drinking water microbiome analyses to posing potential health concerns. To address this, it is imperative for us to know the origin of the eDNA in drinking water. Using Pseudomonas aeruginosa as a model organism, we report for the first time that MCA induces the release of nucleic acids from both biofilms and planktonic cells. Upon exposure to 2 mg/L MCA, massive release of DNA from suspended cells in both MilliQ water and 0.9% NaCl was directly visualized using live cell imaging in a CellASIC ONIX2 microfluidic system. Exposing established biofilms to MCA also resulted in DNA release from the biofilms, which was confirmed by increased detection of eDNA in the effluent. Intriguingly, massive release of RNA was also observed, and the extracellular RNA (eRNA) was also found to persist in water for days. Sequencing analyses of the eDNA revealed that it could be used to assemble the whole genome of the model organism, while in the water, certain fragments of the genome were more persistent than others. RNA sequencing showed that the eRNA contains non-coding RNA and mRNA, implying its role as a possible signaling molecule in environmental systems and a snapshot of the past metabolic state of the bacterial cells.

In densely populated cities with limited land, storage of surface water in underground spaces is a potential solution to meet the rising demand of clean water. In addition, due to the imperative need of renewable solar energy and limited land resources, the deployment of floating solar photovoltaic (PV) systems over water has risen exponentially. In both scenarios, microbial communities in the water do not have access to sunlight. How the absence of sunlight influences microbial community function and the water quality is largely unknown. The objective of this study was to reveal microbial processes in surface water stored in the dark and water quality dynamics. Water from a freshwater reservoir was stored in the dark or light (control) for 6 months. Water quality was monitored at regular intervals. RNA sequencing was performed on the Illumina MiSeq platform and qPCR was used to substantiate the findings arising from the sequencing data. Our results showed that storage of surface water in the dark resulted in the accumulation of nitrate in the water. Storage in the dark promoted the decay of algal cells, increasing the amount of free nitrogen in the water. Most of the free nitrogen was eventually transformed into nitrate through microbial processes. RNA sequencing-based microbial community analyses and pure culture experiments using nitrifying bacteria Nitrosomonas europaea and Nitrobacter sp. revealed that the accumulation of nitrate in the dark was likely due to an increase in nitrification rate and a decrease in the assimilation rate of nitrate back into the biomass. IMPORTANCE Microbial communities play an essential role in maintaining a healthy aquatic ecosystem. For example, in surface water reservoirs, microorganisms produce oxygen, break down toxic contaminants and remove excess nitrogen. In densely populated cities with limited land, storing surface water in underground spaces and deploying floating solar photovoltaic (PV) systems over water are potential solutions to address water and energy sustainability challenges. In both scenarios, surface water is kept in the dark. In this work, we revealed how the absence of sunlight influences microbial community function and water quality. We showed that storage of surface water in the dark affected bacterial activities responsible for nitrogen transformation, resulting in the accumulation of nitrate in the water. Our findings highlight the importance of monitoring nitrate closely if raw surface water is to be stored in the dark and the potential need of downstream treatment to remove nitrate.

Environmental DNA, i.e., DNA extracted directly from environmental samples, has been used to understand microbial communities in the environment and to monitor contemporary biodiversity in the conservation context. Environmental DNA often contains both intracellular DNA (iDNA) and extracellular DNA (eDNA). eDNA can persist in the environment and complicate environmental DNA sequencing-based analyses of microbial communities and biodiversity. Although several studies acknowledged the impact of eDNA on DNA-based profiling of environmental communities, eDNA is still being neglected or ignored in most studies dealing with environmental samples. In this article, we summarize key findings on eDNA in environmental samples and discuss the methods used to extract and quantify eDNA as well as the importance of eDNA on the interpretation of experimental results. We then suggested several factors to consider when designing experiments and analyzing data to negate or determine the contribution of eDNA to environmental DNA-based community analyses. This field of research will be driven forward by (i) carefully designing environmental DNA extraction pipelines by taking into consideration technical details in methods for eDNA extraction/removal and membrane-based filtration and concentration; (ii) quantifying eDNA in extracted environmental DNA using multiple methods, including qPCR and fluorescent DNA binding dyes; (iii) carefully interpreting the effect of eDNA on DNA-based community analyses at different taxonomic levels; and (iv) when possible, removing eDNA from environmental samples for DNA-based community analyses.

The current study was aimed to explore the differential effects on Gram-positive and Gram-negative freshwater sediment bacterial isolates upon exposure to nano-particles and bulk particles of Al2O3 at low concentrations (0.25, 0.5, and 1 mg/L). The Gram-negative Pseudomonas aeruginosa was more susceptible to both the nano-forms and bulk forms than the Gram-positive Bacillus altitudinis. The generation of reactive oxygen species (ROS) and release of lipopolysaccharide due to membrane damage were dependent on the dose of nano-Al2O3. The Fourier transform infrared spectroscopy (FT-IR) studies confirmed the attachment of nano-Al2O3 on bacterial cells, which may lead to subsequent changes in the cell membrane composition and integrity. Internalization of nano-Al2O3 was estimated to be more for P. aeruginosa than for B. altitudinis cells. As a role of defense mechanism, the biofilm formation and production of extracellular polymeric substances (EPSs; polysaccharide and protein) were increased with respect to the concentration of toxicant. Nano-Al2O3 was estimated to cause more DNA damage than the bulk particles in both Gram-positive and Gram-negative bacterial strains.