Dead in the water - Role of relic DNA and primer choice for targeted sequencing surveys of anaerobic sewage sludge intended for biological monitoring.

DNA-based monitoring of microbial communities that are responsible for the performance of anaerobic digestion of sewage wastes has the potential to improve resource recoveries for wastewater treatment facilities. By treating sludge with propidium monoazide (PMA) prior to amplicon sequencing, this study explored how the presence of DNA from dead microbial biomass carried over with feed sludge may mislead process-relevant biomarkers, and whether primer choice impacts such assessments. Four common primers were selected for amplicon preparation, also to determine if universal primers have sufficient taxonomic or functional coverage for monitoring ecological performance; or whether two domain-specific primers for Bacteria and Archaea are necessary. Anaerobic sludges of three municipal continuously stirred-tank reactors in Victoria, Australia, were sampled at one time-point. A total of 240 amplicon libraries were sequenced on a Miseq using two universal and two domain-specific primer pairs. Untargeted metabolomics was chosen to complement biological interpretation of amplicon gene-based functional predictions. Diversity, taxonomy, phylogeny and functional potentials were systematically assessed using PICRUSt2, which can predict community wide pathway abundance. The two chosen universal primers provided similar diversity profiles of abundant Bacteria and Archaea, compared to the domain-specific primers. About 16 % of all detected prokaryotic genera covering 30 % of total abundances and 6 % of PICRUSt2-estimated pathway abundances were affected by PMA. This showed that dead biomass in the anaerobic digesters impacted DNA-based assessments, with implications for predicting active processes, such as methanogenesis, denitrification or the identification of organisms associated with biological foams. Hence, instead of running two sequencing runs with two different domain-specific primers, we propose conducting PMA-seq with universal primer pairs for routine performance monitoring. However, dead sludge biomass may have some predictive value. In principal component analysis the compositional variation of 239 sludge metabolites resembled that of 'dead-plus-alive' biomass, suggesting that dead organisms contributed to the potentially process-relevant sludge metabolome.

Wood biochar enhances methanogenesis in the anaerobic digestion of chicken manure under ammonia inhibition conditions.

The process of breaking down chicken manure through anaerobic digestion is an effective waste management technology. However, chicken manure can be a challenging feedstock, causing ammonia stress and digester instability. This study examined the impacts of adding wood biochar and acid-alkali-treated wood biochar to anaerobically digest chicken manure under conditions of ammonia inhibition. The results highlighted that only the addition of 5 % acid-alkali-treated wood biochar by volume can achieve cumulative methane production close to the typical methane potential range of chicken manure. The treated wood biochar also exhibited highest total ammonia nitrogen removal compared to the Control treatment. Scanning Electron Microscope revealed growing interactions between biochar and methanogens over time. Real-time polymerase chain reaction showed that treated wood biochar produced the highest number of bacterial biomass. In addition, 16S amplicon-based sequencing identified a more robust archaeal community from treated biochar addition. Overall, the acid-alkali treatment of biochar represents an effective method of modifying biochar to improve its performance in anaerobic digestion.

Remediation of groundwater contaminated with dye using carbon dots technology: Ecotoxicological and microbial community responses.

Groundwater pollution poses a serious threat to the main source of clean water globally. Nanoparticles have the potential for remediation of polluted aquifers; however, environmental safety concerns associated with in situ deployments of such technology include potential detrimental effects on microorganisms in terms of toxicity and functional disruptions. In this work, we evaluated a new and ecofriendly approach using carbon dots (CDs) as Fenton-like catalysts to catalyse the degradation of dye-containing groundwater samples. This investigation aimed at evaluating the efficacy of a novel remediation technology in terms of dye degradation and toxicity reduction while assessing its impacts on aquatic microorganisms. Uncontaminated Australian groundwater samples were spiked with methylene blue and incubated in the dark, at 18 °C, under slow agitation, using CDs at 0.5 mg mL-1 and H2O2 at 73.5 mM for 25 h. The dye degradation rate was determined as well as the toxicity of the treated solutions using the Microtox® bioassay. Further, to determine the changes in the groundwater microbial community, 16 S rRNA sequencing was used and evenness and diversity indices were analysed using Pielou's evenness and Simpson index, respectively. This study revealed that dye-containing groundwater were effectively treated by CDs showing a degradation rate of 78-82% and a significant 4-fold reduction in the toxicity. Characterisation of the groundwater microbiota revealed a predominance of at least 60% Proteobacteria phylum in all samples where diversity and evenness were maintained throughout the remediation process. The results showed that CDs could be an efficient approach to treat polluted groundwater and potentially have minimum impact on the environmental microbiome.

The effects of biochar aging on rhizosphere microbial communities in cadmium-contaminated acid soil.

Biochars are widely used in the remediation of Cd-contaminated soils. However, changes in the bacterial communities in the rhizosphere contaminated with Cd in response to biochar aging are poorly studied. Addressing this gap in knowledge is important to improving micro-ecological services on healthy growth of plants with mitigation strategies against Cd contamination. An aging experiment (270 days) was conducted with biochars derived from poultry litter and sugar-gum wood added to a Cd-contaminated acid soil. Bacterial communities in the rhizosphere of Brassica rapa and bulk soils were investigated after 1, 90 and 270 days of biochar aging. There was no significant difference (P > 0.05) in bacterial Shannon and Simpson indices between the control and biochar treatments. However, compared to the no-Cd control, the addition of Cd decreased the relative abundances of Firmicutes, Chloroflexi and Acidobacteriota but increased those of Actinobacteriota and Proteobacteria. Poultry-litter biochar had the largest effect on bacterial community composition, especially in the rhizosphere. Aging of poultry-litter biochar increased the abundance of Armatimonadota over time more than the sugar-gum-wood biochar, which was attributed to a lower pH and higher bioavailability of Cd in the sugar-gum-wood biochar treatment. The addition of poultry-litter biochar to the contaminated soil mitigated the bioaccumulation of Cd by increasing soil pH and restoring soil bacterial ecology in contaminated acid soils over time.

Elevated atmospheric CO2 alters the microbial community composition and metabolic potential to mineralize organic phosphorus in the rhizosphere of wheat.

BACKGROUND: Understanding how elevated atmospheric CO2 (eCO2) impacts on phosphorus (P) transformation in plant rhizosphere is critical for maintaining ecological sustainability in response to climate change, especially in agricultural systems where soil P availability is low. METHODS: This study used rhizoboxes to physically separate rhizosphere regions (plant root-soil interface) into 1.5-mm segments. Wheat plants were grown in rhizoboxes under eCO2 (800 ppm) and ambient CO2 (400 ppm) in two farming soils, Chromosol and Vertosol, supplemented with phytate (organic P). Photosynthetic carbon flow in the plant-soil continuum was traced with 13CO2 labeling. Amplicon sequencing was performed on the rhizosphere-associated microbial community in the root-growth zone, and 1.5 mm and 3 mm away from the root. RESULTS: Elevated CO2 accelerated the mineralization of phytate in the rhizosphere zones, which corresponded with increases in plant-derived 13C enrichment and the relative abundances of discreet phylogenetic clades containing Bacteroidetes and Gemmatimonadetes in the bacterial community, and Funneliformis affiliated to arbuscular mycorrhizas in the fungal community. Although the amplicon sequence variants (ASVs) associated the stimulation of phytate mineralization under eCO2 differed between the two soils, these ASVs belonged to the same phyla associated with phytase and phosphatase production. The symbiotic mycorrhizas in the rhizosphere of wheat under eCO2 benefited from increased plant C supply and increased P access from soil. Further supportive evidence was the eCO2-induced increase in the genetic pool expressing the pentose phosphate pathway, which is the central pathway for biosynthesis of RNA/DNA precursors. CONCLUSIONS: The results suggested that an increased belowground carbon flow under eCO2 stimulated bacterial growth, changing community composition in favor of phylotypes capable of degrading aromatic P compounds. It is proposed that energy investments by bacteria into anabolic processes increase under eCO2 to level microbial P-use efficiencies and that synergies with symbiotic mycorrhizas further enhance the competition for and mineralization of organic P. Video Abstract.

Biochar reduced extractable dieldrin concentrations and promoted oligotrophic growth including microbial degraders of chlorinated pollutants.

The role of organic amendments for natural degradation of aged persistent organic pollutants (POPs) in agricultural soils remains controversial. We hypothesised that organic amendments enhance bacterial activity and function at the community level, facilitating the degradation of aged POPs. An incubation study was conducted in a closed chamber over 12 months to assess the effects of selected organic amendments on extractable residues of aged dieldrin. The role of bacterial diversity and changes in community function was explored through sequenced marker genes. Linear mixed effect models indicated that, independent of amendment type, cumulative CO2 release was negatively associated with decreases in dieldrin concentration, by up to 7% per µmol CO2-C respired by microorganisms. The addition of poultry litter led to the highest daily carbon mineralisation, which was associated with low dieldrin dissipation after 9 months. In comparison, biochar resulted in significant decreases in extractable dieldrin residues over time, which coincided with shifts towards aerobic, oligotrophic, gram-negative bacteria, some with dehalogenation metabolism, and with increased potentials for biosynthesis of membrane components such as fatty acids and high redox quinones. The results supported an alternative theory that labile carbon promoted blooms of copiotrophic growth, which suppressed the required community-level traits and oligotrophic diversity to degrade chlorinated pollutants.

The role of microbial ecology in improving the performance of anaerobic digestion of sewage sludge.

The use of next-generation diagnostic tools to optimise the anaerobic digestion of municipal sewage sludge has the potential to increase renewable natural gas recovery, improve the reuse of biosolid fertilisers and help operators expand circular economies globally. This review aims to provide perspectives on the role of microbial ecology in improving digester performance in wastewater treatment plants, highlighting that a systems biology approach is fundamental for monitoring mesophilic anaerobic sewage sludge in continuously stirred reactor tanks. We further highlight the potential applications arising from investigations into sludge ecology. The principal limitation for improvements in methane recoveries or in process stability of anaerobic digestion, especially after pre-treatment or during co-digestion, are ecological knowledge gaps related to the front-end metabolism (hydrolysis and fermentation). Operational problems such as stable biological foaming are a key problem, for which ecological markers are a suitable approach. However, no biomarkers exist yet to assist in monitoring and management of clade-specific foaming potentials along with other risks, such as pollutants and pathogens. Fundamental ecological principles apply to anaerobic digestion, which presents opportunities to predict and manipulate reactor functions. The path ahead for mapping ecological markers on process endpoints and risk factors of anaerobic digestion will involve numerical ecology, an expanding field that employs metrics derived from alpha, beta, phylogenetic, taxonomic, and functional diversity, as well as from phenotypes or life strategies derived from genetic potentials. In contrast to addressing operational issues (as noted above), which are effectively addressed by whole population or individual biomarkers, broad improvement and optimisation of function will require enhancement of hydrolysis and acidogenic processes. This will require a discovery-based approach, which will involve integrative research involving the proteome and metabolome. This will utilise, but overcome current limitations of DNA-centric approaches, and likely have broad application outside the specific field of anaerobic digestion.

An Insight Into the Effect of Organic Amendments on the Transpiration Efficiency of Wheat Plant in a Sodic Duplex Soil.

Transpiration efficiency, the shoot biomass produced per unit of transpired water, is generally considered to be a constant property for a given crop in a given environment. To determine whether deep-banded organic amendments affect the transpiration efficiency (TE) of wheat plants and to provide a possible explanation for any changes in the TE, two-column experiments were carried out under controlled environment conditions. A Sodosol soil with physically constrained subsoils and a well-structured Vertosol were subjected to treatments including a control, fertilizer nutrients alone, and fertilizer-enriched organic amendments. The addition of fertilizer-enriched organic amendments in Sodosol consistently increased the canopy TE compared to the control and inorganic fertilizer treatments. The instantaneous TE, at the leaf level, was also increased by the organic-based amendments due to greater reductions in stomatal conductance and transpiration rates during periods of moderate water-deficit stress and the subsequent recovery from this stress. Shoot nitrogen (N) status could not explain the increases in TE following the addition of organic amendments relative to inorganic amendments. The increases in canopy TE were directly associated with increases in the absolute abundance of indigenous Bacillus (R 2 = 0.92, p <0), a well-known genus comprising many strains of plant beneficial rhizobacteria, in subsoil below the amendment band. In contrast, there were no differences in the canopy TE and instantaneous leaf TE between the organic and fertilizer amendments in the Vertosol with a well-structured subsoil. The positive effect of organic amendments on TE in the Sodosol should be attributed to their direct or indirect effect on improving the physical structure or biological properties of the subsoil.

Highly decomposed organic carbon mediates the assembly of soil communities with traits for the biodegradation of chlorinated pollutants.

To improve biodegradation strategies for chlorinated pollutants, the roles of soil organic matter and microbial function need to be clarified. It was hypothesised that microbial degradation of specific organic fractions in soils enhance community metabolic capability to degrade chlorinated pollutants. This field study used historic records of dieldrin concentrations since 1988 and established relationships between dieldrin dissipation and soil carbon fractions together with bacterial and fungal diversity in surface soils of Kurosol and Chromosol. Sparse partial least squares analysis linked dieldrin dissipation to metabolic activities associated with the highly decomposed carbon fraction. Dieldrin dissipation, after three decades of natural attenuation, was associated with increased bacterial species fitness for the decomposition of recalcitrant carbon substrates including synthetic chlorinated pollutants. These metabolic capabilities were linked to the decomposed carbon fraction, an important driver for the microbial community and function. Common bacterial traits among taxonomic groups enriched in samples with high dieldrin dissipation included their slow growth, large genome and complex metabolism which supported the notion that metabolic strategies for dieldrin degradation evolved in an energy-low soil environment. The findings provide new perspectives for bioremediation strategies and suggest that soil management should aim at stimulating metabolism at the decomposed, fine carbon fraction.

Composition of soil organic matter drives total loss of dieldrin and dichlorodiphenyltrichloroethane in high-value pastures over thirty years.

The residues of dieldrin and dichlorodiphenyltrichloroethane (DDT), internationally-banned agricultural insecticides, continue to exceed government guidelines in some surface soils 30 years after use. Little is known regarding the soil factors and microbial community dynamics associated with the in-situ biodegradation of these organochlorine chemicals. We hypothesised that soil organic matter, a key factor affecting microbial biomass and diversity, affects the biodegradation and total loss of the pollutants 30 years after use. We sampled 12 contaminated paddocks with residue concentrations monitoring data since 1988 that represent two different agricultural surface-soils. The total loss and current concentrations of the residues was correlated with soil physicochemical properties, microbial biomass carbon, microbial community diversity indices and microbial community abundance. Current dieldrin and DDT residue concentrations were positively correlated with soil organic matter and clay contents. However, key indicators for loss of residues after 23-30 years were low carbon-to­nitrogen ratios, high microbial-C-to-total-C ratios and high fungal community evenness. The results support the composition of soil organic matter as an important factor affecting degradation of organochlorines and that co-metabolism of dieldrin and DDT could be enhanced by manipulating the composition of soil organic matter to cater for a broad diversity of microbial function.