Syntroph diversity and abundance in anaerobic digestion revealed through a comparative core microbiome approach.

Anaerobic digestion is an important biotechnology treatment process for conversion of waste to energy. In this study, a comparative core microbiome approach, i.e., determining taxa that are shared in functioning digesters but not shared in non-functioning digesters, was used to determine microbial taxa that could play key roles for effective anaerobic digestion. Anaerobic digester functions were impaired by adding the broad-spectrum antimicrobial triclosan (TCS) or triclocarban (TCC) at different concentrations, and the core microbiomes in both functioning and non-functioning anaerobic digesters were compared. Digesters treated with high (2500 mg/kg) or medium (450 mg/kg) TCS and high (850 mg/kg) TCC concentrations lost their function, i.e., methane production decreased, effluent volatile fatty acid concentrations increased, and pH decreased. Changes in microbial community diversity and compositions were assessed using 16S rRNA gene amplicon sequencing. Microbial richness decreased significantly in non-functioning digesters (p < 0.001). Microbial community compositions in non-functioning digesters significantly differed from those in functioning digesters (p = 0.001, ANOSIM). Microbes identified as potentially key taxa included previously known fatty acid-degrading syntrophs and amino acid-degrading syntrophs. A diverse group of syntrophs detected in this study had low relative abundance in functioning digesters, suggesting the importance of rare microbes in anaerobic digester operation. The comparative microbiome approach used in this study can be applied to other microbial systems where a community-driven biological phenomena can be observed directly.

Metagenomics reveal triclosan-induced changes in the antibiotic resistome of anaerobic digesters.

Triclosan (TCS) is a broad-spectrum antimicrobial used in a variety of consumer products. While it was recently banned from hand soaps in the US, it is still a key ingredient in a top-selling toothpaste. TCS is a hydrophobic micropollutant that is recalcitrant under anaerobic digestion thereby resulting in high TCS concentrations in biosolids. The objective of this study was to determine the impact of TCS on the antibiotic resistome and potential cross-protection in lab-scale anaerobic digesters using shotgun metagenomics. It was hypothesized that metagenomics would reveal selection for antibiotic resistance genes (ARGs) not previously found in pure culture studies or mixed-culture studies using targeted qPCR. In this study, four different levels of TCS were continuously fed to triplicate lab-scale anaerobic digesters to assess the effect of TCS levels on the antibiotic resistance gene profiles (resistome). Blasting metagenomic reads against antibiotic/metal resistance gene database (BacMet) revealed that ARG diversity and abundance changed along the TCS concentration gradient. While loss of bacterial diversity and digester function were observed in the digester treated with the highest TCS concentration, FabV, which is a known TCS resistance gene, increased in this extremely high TCS environment. The abundance of several other known ARG or metal resistance genes (MRGs), including corA and arsB, also increased as the concentrations of TCS increased. Analysis of other functional genes using SEED database revealed the increase of potentially key genes for resistance including different types of transporters and transposons. These results indicate that antimicrobials can alter the abundance of multiple resistance genes in anaerobic digesters even when function (i.e. methane production) is maintained. This study also suggests that enriched ARGs could be released into environments with biosolids land application.

Altered antibiotic tolerance in anaerobic digesters acclimated to triclosan or triclocarban.

Bench-scale anaerobic digesters were amended to elevated steady-state concentrations of triclosan (850 mg/kg) and triclocarban (150 mg/kg) using a synthetic feed. After more than 9 solids retention time (SRT) values of acclimatization, biomass from each digester (and a control digester that received no antimicrobials) was used to assess the toxicity of three antibiotics. Methane production rate was measured as a surrogate for activity in microcosms that received doses of antibiotics ranging from no-antibiotic to inhibitory concentrations. Biomass amended with triclocarban was more sensitive to tetracycline compared to the control indicating synergistic inhibitory effects between this antibiotic and triclocarban. In contrast, biomass amended with triclosan was able to tolerate statistically higher levels of ciprofloxacin indicating that triclosan can induce functional resistance to ciprofloxacin in an anaerobic digester community.

Chronic exposure to triclosan sustains microbial community shifts and alters antibiotic resistance gene levels in anaerobic digesters.

Triclosan, an antimicrobial chemical found in consumer personal care products, has been shown to stimulate antibiotic resistance in pathogenic bacteria. Although many studies focus on antibiotic resistance pertinent to medical scenarios, resistance developed in natural and engineered environments is less studied and has become an emerging concern for human health. In this study, the impacts of chronic triclosan (TCS) exposure on antibiotic resistance genes (ARGs) and microbial community structure were assessed in lab-scale anaerobic digesters. TCS concentrations from below detection to 2500 mg kg(-1) dry solids were amended into anaerobic digesters over 110 days and acclimated for >3 solid retention time values. Four steady state TCS concentrations were chosen (30-2500 mg kg(-1)). Relative abundance of mexB, a gene coding for a component of a multidrug efflux pump, was significantly higher in all TCS-amended digesters (30 mg kg(-1) or higher) relative to the control. TCS selected for bacteria carrying tet(L) and against those carrying erm(F) at concentrations which inhibited digester function; the pH decrease associated with digester failure was suspected to cause this selection. Little to no impact of TCS was observed on intI1 relative abundance. Microbial communities were also surveyed by high-throughput 16S rRNA gene sequencing. Compared to the control digesters, significant shifts in community structure towards clades containing commensal and pathogenic bacteria were observed in digesters containing TCS. Based on these results, TCS should be included in studies and risk assessments that attempt to elucidate relationships between chemical stressors (e.g. antibiotics), antibiotic resistance genes, and public health.

Recovery of agricultural nutrients from biorefineries.

This review lays the foundation for why nutrient recovery must be a key consideration in design and operation of biorefineries and comprehensively reviews technologies that can be used to recover an array of nitrogen, phosphorus, and/or potassium-rich products of relevance to agricultural applications. Recovery of these products using combinations of physical, chemical, and biological operations will promote sustainability at biorefineries by converting low-value biomass (particularly waste material) into a portfolio of higher-value products. These products can include a natural partnering of traditional biorefinery outputs such as biofuels and chemicals together with nutrient-rich fertilizers. Nutrient recovery not only adds an additional marketable biorefinery product, but also avoids the negative consequences of eutrophication, and helps to close anthropogenic nutrient cycles, thereby providing an alternative to current unsustainable approaches to fertilizer production, which are energy-intensive and reliant on nonrenewable natural resource extraction.

Triclocarban Influences Antibiotic Resistance and Alters Anaerobic Digester Microbial Community Structure.

Triclocarban (TCC) is one of the most abundant organic micropollutants detected in biosolids. Lab-scale anaerobic digesters were amended with TCC at concentrations ranging from the background concentration of seed biosolids (30 mg/kg) to toxic concentrations of 850 mg/kg to determine the effect on methane production, relative abundance of antibiotic resistance genes, and microbial community structure. Additionally, the TCC addition rate was varied to determine the impacts of acclimation time. At environmentally relevant TCC concentrations (max detect = 440 mg/kg), digesters maintained function. Digesters receiving 450 mg/kg of TCC maintained function under gradual TCC addition, but volatile fatty acid concentrations increased, pH decreased, and methane production ceased when immediately fed this concentration. The concentrations of the mexB gene (encoding for a multidrug efflux pump) were higher with all concentrations of TCC compared to a control, but higher TCC concentrations did not correlate with increased mexB abundance. The relative abundance of the gene tet(L) was greater in the digesters that no longer produced methane, and no effect on the relative abundance of the class 1 integron integrase encoding gene (intI1) was observed. Illumina sequencing revealed substantial community shifts in digesters that functionally failed from increased levels of TCC. More subtle, yet significant, community shifts were observed in digesters amended with TCC levels that did not inhibit function. This research demonstrates that TCC can select for a multidrug resistance encoding gene in mixed community anaerobic environments, and this selection occurs at concentrations (30 mg/kg) that can be found in full-scale anaerobic digesters (U.S. median concentration = 22 mg/kg, mean = 39 mg/kg).

The impact of triclosan on the spread of antibiotic resistance in the environment.

Triclosan (TCS) is a commonly used antimicrobial agent that enters wastewater treatment plants (WWTPs) and the environment. An estimated 1.1 × 10(5) to 4.2 × 10(5) kg of TCS are discharged from these WWTPs per year in the United States. The abundance of TCS along with its antimicrobial properties have given rise to concern regarding its impact on antibiotic resistance in the environment. The objective of this review is to assess the state of knowledge regarding the impact of TCS on multidrug resistance in environmental settings, including engineered environments such as anaerobic digesters. Pure culture studies are reviewed in this paper to gain insight into the substantially smaller body of research surrounding the impacts of TCS on environmental microbial communities. Pure culture studies, mainly on pathogenic strains of bacteria, demonstrate that TCS is often associated with multidrug resistance. Research is lacking to quantify the current impacts of TCS discharge to the environment, but it is known that resistance to TCS and multidrug resistance can increase in environmental microbial communities exposed to TCS. Research plans are proposed to quantitatively define the conditions under which TCS selects for multidrug resistance in the environment.