Shotgun metagenomics of soil invertebrate communities reflects taxonomy, biomass and reference genome properties

Metagenomics - shotgun sequencing of all DNA fragments from a community DNA extract - is routinely used to describe the composition, structure and function of microorganism communities. Advances in DNA sequencing and the availability of genome databases increasingly allow the use of shotgun metagenomics on eukaryotic communities. Metagenomics offers major advances in the recovery of biomass relationships, in comparison to taxonomic marker gene based approaches (metabarcoding). However, little is known about the factors that influence metagenomics data from eukaryotic communities, such as differences among organism groups, properties of reference genomes and genome assemblies. We evaluated how shotgun metagenomics records composition and biomass in artificial soil invertebrate communities. We generated mock communities of controlled biomass ratios from 28 species from all major soil mesofauna groups: mites, springtails, nematodes, tardigrades and potworms. We shotgun-sequenced these communities and taxonomically assigned them with a database of over 270 soil invertebrate genomes. We recovered 90% of the species, and observed relatively high false positive detection rates. We found strong differences in reads assigned to different taxa, with some groups consistently attracting more hits than others. Biomass could be predicted from read counts after considering taxon-specific differences. Larger genomes more complete assemblies consistently attracted more reads than genomes. The GC content of the genome assemblies had no effect on the biomass-read relationships. The results show considerable differences in taxon recovery and taxon specificity of biomass recovery from metagenomic sequence data. Properties of reference genomes and genome assemblies also influence biomass recovery, and they should be considered in metagenomic studies of eukaryotes. We provide a roadmap for investigating factors which influence metagenomics-based eukaryotic community reconstructions. Understanding these factors is timely as accessibility of DNA sequencing, and momentum for reference genomes projects show a future where the taxonomic assignment of DNA from any community sample becomes a reality.

Shotgun metagenomics of soil invertebrate communities reflects taxonomy, biomass and reference genome properties

Metagenomics - shotgun sequencing of all DNA fragments from a community DNA extract - is routinely used to describe the composition, structure and function of microorganism communities. Advances in DNA sequencing and the availability of genome databases increasingly allow the use of shotgun metagenomics on eukaryotic communities. Metagenomics offers major advances in the recovery of biomass relationships in a sample, in comparison to taxonomic marker gene based approaches (metabarcoding). However, little is known about the factors which influence metagenomics data from eukaryotic communities, such as differences among organism groups, the properties of reference genomes and genome assemblies. We evaluated how shotgun metagenomics records composition and biomass in artificial soil invertebrate communities. We generated mock communities of controlled biomass ratios from 28 species from all major soil mesofauna groups: mites, springtails, nematodes, tardigrades and potworms. We shotgun-sequenced these communities and taxonomically assigned them with a database of over 270 soil invertebrate genomes. We recovered 90% of the species, and observed relatively high false positive detection rates. We found strong differences in reads assigned to different taxa, with some groups (e.g. springtails) consistently attracting more hits than others (e.g. enchytraeids). Original biomass could be predicted from read counts after considering these taxon-specific differences. Species with larger genomes, and with more complete assemblies consistently attracted more reads than species with smaller genomes. The GC content of the genome assemblies had no effect on the biomass-read relationships. The results show considerable differences in taxon recovery and taxon specificity of biomass recovery from metagenomic sequence data. The properties of reference genomes and genome assemblies also influence biomass recovery, and they should be considered in metagenomic studies of eukaryotes. We provide a roadmap for investigating factors which influence metagenomics-based eukaryotic community reconstructions. Understanding these factors is timely as accessibility of DNA sequencing, and momentum for reference genomes projects show a future where the taxonomic assignment of DNA from any community sample becomes a reality.

Microalgal-bacterial flocs and extracellular polymeric substances for optimum function of integrated algal pond systems

Despite the dire state of sanitation infrastructures, water scarcity, and the dwindling reserve of natural resources due to ever-increasing population growth, implementation of a suitable technology that can provide a solution to all these issues continues to be ignored. The integrated algal pond system (IAPS) is a wastewater treatment technology that combines the processes of anaerobic digestion and photosynthetic oxygenation to achieve wastewater treatment and facilitate the recovery of treated water and resources in the form of biogas and microalgal-bacterial biomass. The natural process of bioflocculation through microalgal-bacterial mutualism and production of extracellular polymeric substances (EPS) in high rate algal oxidation ponds (HRAOPs) of an IAPS increases efficiency of wastewater treatment and potentially enhances harvestability and biomass recovery, which could contribute significantly to the successful establishment of a biorefinery. Using a 500 PE pilot-scale IAPS supplied domestic sewage coupled with laboratory experiments, this study investigated the importance and function of in situ EPS production and MaB-floc formation in HRAOP. A metagenomic study revealed the biological components of the biomass or mixed liquor suspended solids (MLSS) produced in HRAOP and showed that the suspended biomass is composed largely of eukaryotes that were dominated by the colonial microalgae Pseudopediastrum sp. and Desmodesmus sp., and a diverse range of prokaryotes including bacteria and cyanobacteria. Dominance, within the bacterial population, by a sulphur-oxidizing bacterium, Thiothrix which comprised up to 80% of the prokaryotes, coincided with a period of poor flocculation and was therefore rationalized to have contributed to bulking and poor biomass settleability. Otherwise, good flocs were formed in the MLSS with settleability up to 95% and, within 1 h. The formation of MaB-flocs appeared to be dependent on EPS concentration of the mixed liquor due to the observed positive correlation between soluble EPS (S-EPS), biomass concentration, and settleability. The contribution and role of MLSS components towards the formation and sustenance of MaB-flocs were further demonstrated in laboratory experiments using pure strains of microalgae, cyanobacteria, and bacteria. Results showed that pure cultures of dominant microalgae in MLSS, Pseudopediastrum sp. and Desmodesmus sp. achieved a rapid 92 and 75% settleability within 3 h. A self-flocculating filamentous cyanobacterium, Leptolyngbya strain ECCN 20BG was isolated, characterized, and shown to achieve 99% settleability within 5 min by forming large tightly aggregated flocs. In further experiments, this strain was found to improve the settleability of MLSS by an average of 20%. Bacterial strains identified as Bacillus strain ECCN 40b, Bacillus strain ECCN 41b, Planococcus strain ECCN 45b, and Exiguobacterium strain ECCN 46b were also observed to produce sticky EPS-like materials in pure cultures that could also contribute to the aggregation of cells in a mixed environment. Given these results, various factors and/or mechanisms that might enhance microbial aggregation and biomass recovery from HRAOP MLSS were identified in this study and include; (1) dominance by larger colonial microalgae prevents disintegration of MaB-flocs and enhances recovery of biomass from MLSS by gravity sedimentation, (2) presence of filamentous cyanobacteria species that can self-flocculate to form an interwoven network of filaments may play an important role in the structural stability and settleability of MaB-flocs in MLSS, and (3) production of EPS to form the matrix or scaffold whereon all microbial components aggregate to develop a microenvironment. Indeed, all forms of EPS, except for that produced by Bacillus strain ECCN 41b, showed bioflocculating property and were able to serve as flocculants for the recovery of Chlorella, an alga known for its poor settleability. A combination of biochemical analyses and FTIR spectroscopy revealed the importance of carbohydrate enrichment of these biopolymers. Carbohydrate concentration in all forms of EPS was between 12 and 41% suggesting that production of these compounds by microbes within the MLSS contributed to MaB-floc formation. EPS extracted from bulk MLSS and EPS produced by Bacillus strains possessed some surface-active properties that were comparable to Triton X-100, indicating potential application in bioremediation and recovery of oil from contaminated soil and water. In particular, EPS generated from Bacillus strain ECCN 41b displayed relatively distinct properties including the quantity produced (> 500 mg/L), increased viscosity, inability to flocculate microalgal cells, a rhamnolipid content of 32%, and a higher surface-activity. Based on these results, Bacillus strain ECCN 41b was rationalized to produce anionic EPS with potential application in metal or oil recovery. In addition to EPS production, the bacteria Planococcus strain ECCN 45b and Exiguobacterium strain ECCN 46b appeared pigmented. Based on partial characterization using UV/Vis spectrophotometry, thin-layer chromatography, FTIR, and NMR, the pigments produced by these two strains appeared to be identical and were tentatively identified as ketocarotenoids. This study successfully demonstrated the importance of EPS production and formation of MaB-flocs in the MLSS from HRAOP of an IAPS treating domestic sewage. It is evident that increased settleability of the biomass does contribute to the reported efficiency of wastewater treatment by IAPS and would reduce both total suspended solids (TSS) and chemical oxygen demand (COD). In addition, demonstration that this biomass contains products of value such as carotenoids and EPS with potential for commercial use strengthens the idea of using IAPS as a platform technology for innovation of the wastewater treatment process to a biorefinery.

Analysing the Impact of Harvesting Methods on the Quantity of Harvesting Residues: An Australian Case Study

Many parameters can influence the weight of harvesting residues per hectare that remain on plantation sites after extracting sawlogs and pulpwoods. This study aimed at quantifying the impact of the cut-to-length and whole-tree harvesting methods on the weight of harvesting residues using 26 case studies in Australian plantations. A database was created using case studies conducted in different plantations, to measure the weight of harvesting residues per hectare and the components of harvesting residues. An analysis of variance was applied to test the impact made by the harvesting methods. The results confirmed that the cut-to-length harvesting method produced a larger weight of residues (104.0 tonnes of wet matter per hectare (tWM/ha) without additional biomass recovery and 64.7 tWM/ha with additional biomass recovery after sawlog/pulpwood extraction) than the whole-tree harvesting method (12.5 tWM/ha). The fraction test showed that stem wood formed the largest proportion of the harvesting residues in cut-to-length sites and needles were the largest component of the pine harvesting residues in sites cleared by the whole-tree harvesting method. The outcomes of this study could assist plantation managers to set proper strategies for harvesting residues management. Future research could study the impact of product type, silvicultural regime, stand quality, age, equipment, etc., on the weight of harvesting residues.

Light And Oxygen Impelling Rubrivivax Gelatinosus Directly Used Food Processing Wastewater To Accumulate Poly-β-Hydroxybutyrate, 5-Aminolevulinic Acid, Pigment

Background: Rubrivivax gelatinosus have the advantage of using wastewater to realize biomass recovery. However, they still cannot be large-scale applied because they cannot directly treat the wastewater containing macromolecular organics. Thus, this paper investigated the effect of light-oxygen condition on Rubrivivax gelatinosus (R. gelatinosus) directly recycling wastewater containing macromolecular organics to produce biomass, poly-β-hydroxybutyrate (PHB), 5-Aminolevulinic acid (5-ALA), pigment.Results: R. gelatinosus directly treated the macromolecules organic (soybean protein and starch) wastewaters and achieved biomass recovery under light-anaerobic and light-micro oxygen in six conditions. COD, protein, starch removals for two wastewaters all reached above 70%. Renewable bio-resource such as biomass, PHB, 5-ALA, pigment productions were 10 times of initial content. Theoretical analysis indicated that light activated two component signal pathway by stimulating its first hk gene expression, which regulated synthesis of protease and amylase. However, oxygen concentration decided the level of gene expression and the amount of enzymes. When oxygen was at micro-oxygen or anaerobic, above these expression and synthesis were conducted. Conclusion: In summary, this study expanded the view point ignored by traditional theory. It was realized that PNSB directly treated wastewater and accumulated nutrients (biomass, PHB, pigment and 5-ALA) for recycling, which reduced the secondary pollution of excess sludge to the environment.

Identification of a Paraquat Resistant Goosegrass (Eleusine indica) Population from a Central Alabama Vegetable Production Field

A goosegrass (Eleusine indica (L.) Gaertn.) population uncontrolled by paraquat (R) in a vegetable production field in St. Clair County, Alabama was collected in Summer 2019. Research was conducted to assess the level of resistance of the suspected resistant population compared to three population with no suspected paraquat resistance (S1, S2, and S3). Visual injury at all rating dates and biomass reduction at 28 days after treatment (DAT) of S populations occurred exponentially to increasing paraquat rates. S biotypes were injured greater than R at 3 DAT with biomass recovery at 28 DAT only occurring at rates < 0.28 kg ha−1. Plant death or biomass reduction did not occur for any rate at any date for R. Paraquat rates that induced 50% or 90% injury or reduced biomass 50% or 90% compared to the non-treated (I50 or I90, respectively) ranged from 10 to 124X higher I50 for R compared to S and 54 to 116X higher I90 for R compared to S biotypes. These data confirm a paraquat resistant E. indica biotype in Alabama providing additional germplasm for study of Resistance to photosystem I-electron diverting (PSI-ED) resistance mechanisms.

Mangrove selective logging sustains biomass carbon recovery, soil carbon, and sediment

West Papua’s Bintuni Bay is Indonesia’s largest contiguous mangrove block, only second to the world’s largest mangrove in the Sundarbans, Bangladesh. As almost 40% of these mangroves are designated production forest, we assessed the effects of commercial logging on forest structure, biomass recovery, and soil carbon stocks and burial in five-year intervals, up to 25 years post-harvest. Through remote sensing and field surveys, we found that canopy structure and species diversity were gradually enhanced following biomass recovery. Carbon pools preserved in soil were supported by similar rates of carbon burial before and after logging. Our results show that mangrove forest management maintained between 70 and 75% of the total ecosystem carbon stocks, and 15–20% returned to the ecosystem after 15–25 years. This analysis suggests that mangroves managed through selective logging provide an opportunity for coastal nature-based climate solutions, while provisioning other ecosystem services, including wood and wood products.