mcrA Gene abundance correlates with hydrogenotrophic methane production rates in full-scale anaerobic waste treatment systems.

UNLABELLED: Anaerobic treatment is a sustainable and economical technology for waste stabilization and production of methane as a renewable energy. However, the process is under-utilized due to operational challenges. Organic overload or toxicants can stress the microbial community that performs waste degradation, resulting in system failure. In addition, not all methanogenic microbial communities are equally capable of consistent, maximum biogas production. Opinion varies as to which parameters should be used to monitor the fitness of digester biomass. No standard molecular tools are currently in use to monitor and compare full-scale operations. It was hypothesized that determining the number of gene copies of mcrA, a methanogen-specific gene, would positively correlate with specific methanogenic activity (SMA) rates from biomass samples from six full-scale anaerobic digester systems. Positive correlations were observed between mcrA gene copy numbers and methane production rates against H2  : CO2 and propionate (R(2)  = 0·67-0·70, P < 0·05) but not acetate (R(2)  = 0·49, P > 0·05). Results from this study indicate that mcrA gene targeted qPCR can be used as an alternate tool to monitor and compare certain methanogen communities in anaerobic digesters. SIGNIFICANCE AND IMPACT OF THE STUDY: Using quantitative PCR (qPCR), we demonstrate that the abundance of mcrA, a gene specific to methane producing archaea, correlated with specific methanogenic activity (SMA) measurements when H2 and CO2 , or propionate were provided as substrates. However, mcrA abundance did not correlate with SMA with acetate. SMA values are often used as a fitness indicator of anaerobic biomass. Results from qPCR can be obtained within a day while SMA analysis requires days to weeks to complete. Therefore, qPCR for mcrA abundance is a sensitive and fast method to compare and monitor the fitness of certain anaerobic biomass. As a monitoring tool, qPCR of mcrA will help anaerobic digester operators optimize treatment and encourage more widespread use of this valuable technology.

Biochar from Pyrolysis of Biosolids for Nutrient Adsorption and Turfgrass Cultivation.

At water resource recovery facilities, nutrient removal is often required and energy recovery is an ever-increasing goal. Pyrolysis may be a sustainable process for handling wastewater biosolids because energy can be recovered in the py-gas and py-oil. Additionally, the biochar produced has value as a soil conditioner. The objective of this work was to determine if biochar could be used to adsorb ammonia from biosolids filtrate and subsequently be applied as a soil conditioner to improve grass growth. The maximum carrying capacity of base modified biochar for NH3-N was 5.3 mg/g. Biochar containing adsorbed ammonium and potassium was applied to laboratory planters simulating golf course putting greens to cultivate Kentucky bluegrass. Planters that contained nutrient-laden biochar proliferated at a statistically higher rate than planters that contained biosolids, unmodified biochar, peat, or no additive. Nutrient-laden biochar performed as well as commercial inorganic fertilizer with no statistical difference in growth rates. Biochar from digested biosolids successfully immobilized NH3-N from wastewater and served as a beneficial soil amendment. This process offers a means to recover and recycle nutrients from water resource recovery facilities.

Correlating methane production to microbiota in anaerobic digesters fed synthetic wastewater.

A quantitative structure activity relationship (QSAR) between relative abundance values and digester methane production rate was developed. For this, 50 triplicate anaerobic digester sets (150 total digesters) were each seeded with different methanogenic biomass samples obtained from full-scale, engineered methanogenic systems. Although all digesters were operated identically for at least 5 solids retention times (SRTs), their quasi steady-state function varied significantly, with average daily methane production rates ranging from 0.09 ± 0.004 to 1 ± 0.05 L-CH4/LR-day (LR = Liter of reactor volume) (average ± standard deviation). Digester microbial community structure was analyzed using more than 4.1 million partial 16S rRNA gene sequences of Archaea and Bacteria. At the genus level, 1300 operational taxonomic units (OTUs) were observed across all digesters, whereas each digester contained 158 ± 27 OTUs. Digester function did not correlate with typical biomass descriptors such as volatile suspended solids (VSS) concentration, microbial richness, diversity or evenness indices. However, methane production rate did correlate notably with relative abundances of one Archaeal and nine Bacterial OTUs. These relative abundances were used as descriptors to develop a multiple linear regression (MLR) QSAR equation to predict methane production rates solely based on microbial community data. The model explained over 66% of the variance in the experimental data set based on 149 anaerobic digesters with a standard error of 0.12 L-CH4/LR-day. This study provides a framework to relate engineered process function and microbial community composition which can be further expanded to include different feed stocks and digester operating conditions in order to develop a more robust QSAR model.

Anaerobic digester bioaugmentation influences quasi steady state performance and microbial community.

Nine anaerobic digesters, each seeded with biomass from a different source, were operated identically and their quasi steady state function was compared. Subsequently, digesters were bioaugmented with a methanogenic culture previously shown to increase specific methanogenic activity. Before bioaugmentation, different seed biomass resulted in different quasi steady state function, with digesters clustering into three groups distinguished by methane (CH4) production. Digesters with similar functional performance contained similar archaeal communities based on clustering of Illumina sequence data of the V4V5 region of the 16S rRNA gene. High CH4 production correlated with neutral pH and high Methanosarcina abundance, whereas low CH4 production correlated to low pH as well as high Methanobacterium and DHVEG 6 family abundance. After bioaugmentation, CH4 production from the high CH4 producing digesters transiently increased by 11 ± 3% relative to non-bioaugmented controls (p < 0.05, n = 3), whereas no functional changes were observed for medium and low CH4 producing digesters that all had pH higher than 6.7. The CH4 production increase after bioaugmentation was correlated to increased relative abundance of Methanosaeta and Methaospirillum originating from the bioaugment culture. In conclusion, different anaerobic digester seed biomass can result in different quasi steady state CH4 production, SCOD removal, pH and effluent VFA concentration in the timeframe studied. The bioaugmentation employed can result in a period of increased methane production. Future research should address extending the period of increased CH4 production by employing pH and VFA control concomitant with bioaugmentation, developing improved bioaugments, or employing a membrane bioreactor to retain the bioaugment.

Influent wastewater microbiota and temperature influence anaerobic membrane bioreactor microbial community.

Sustainable municipal wastewater recovery scenarios highlight benefits of anaerobic membrane bioreactors (AnMBRs). However, influences of continuous seeding by influent wastewater and temperature on attached-growth AnMBRs are not well understood. In this study, four bench-scale AnMBR operated at 10 and 25°C were fed synthetic (SPE) and then real (PE) primary effluent municipal wastewater. Illumina sequencing revealed different bacterial communities in each AnMBR in response to temperature and bioreactor configuration, whereas differences were not observed in archaeal communities. Activity assays revealed hydrogenotrophic methanogenesis was the dominant methanogenic pathway at 10°C. The significant relative abundance of Methanosaeta at 10°C concomitant with low acetoclastic methanogenic activity may indicate possible Methanosaeta-Geobacter direct interspecies electron transfer. When AnMBR feed was changed to PE, continual seeding with wastewater microbiota caused AnMBR microbial communities to shift, becoming more similar to PE microbiota. Therefore, influent wastewater microbiota, temperature and reactor configuration influenced the AnMBR microbial community.

Pyrolysis of wastewater biosolids significantly reduces estrogenicity.

Most wastewater treatment processes are not specifically designed to remove micropollutants. Many micropollutants are hydrophobic so they remain in the biosolids and are discharged to the environment through land-application of biosolids. Micropollutants encompass a broad range of organic chemicals, including estrogenic compounds (natural and synthetic) that reside in the environment, a.k.a. environmental estrogens. Public concern over land application of biosolids stemming from the occurrence of micropollutants hampers the value of biosolids which are important to wastewater treatment plants as a valuable by-product. This research evaluated pyrolysis, the partial decomposition of organic material in an oxygen-deprived system under high temperatures, as a biosolids treatment process that could remove estrogenic compounds from solids while producing a less hormonally active biochar for soil amendment. The estrogenicity, measured in estradiol equivalents (EEQ) by the yeast estrogen screen (YES) assay, of pyrolyzed biosolids was compared to primary and anaerobically digested biosolids. The estrogenic responses from primary solids and anaerobically digested solids were not statistically significantly different, but pyrolysis of anaerobically digested solids resulted in a significant reduction in EEQ; increasing pyrolysis temperature from 100°C to 500°C increased the removal of EEQ with greater than 95% removal occurring at or above 400°C. This research demonstrates that biosolids treatment with pyrolysis would substantially decrease (removal>95%) the estrogens associated with this biosolids product. Thus, pyrolysis of biosolids can be used to produce a valuable soil amendment product, biochar, that minimizes discharge of estrogens to the environment.

Thermophilic anaerobic digester with ultrafilter for solids stabilization.

A thermophilic anaerobic digester with ultrafilter (TADU) for solids separation offers potential advantages of higher VS destruction, biomass retention, and pathogen removal. However, potential disadvantages include ultrafilter fouling, decreasing flux, and high VFA concentrations. In this study, a thermophilic anaerobic digester coupled to a sintered titanium, cross-flow ultrafilter was operated for over five months. Dairy manure was digested (HRT of 23 days). The filtrate VFA concentration was low (220 mg/L as HAc), average VS destruction was 49%, and a low average effluent fecal coliform concentration of 10(2) MPN/100 mL was observed. The low coliform value may be beneficial if dewatered biosolids are used for livestock bedding since low pathogen counts help prevent mastitis. Ultrafilter fluxes of 40-80 L/m2-hr were maintained by cleaning using caustic (3.5% NaOH) followed by water and acid (3% phosphoric acid). Sand from livestock bedding was found to damage the pump and ultrafilter. If TADU were implemented at full scale, then replacing sand bedding with dewatered biosolids should be considered.

Bioaugmentation of overloaded anaerobic digesters restores function and archaeal community.

Adding beneficial microorganisms to anaerobic digesters for improved performance (i.e. bioaugmentation) has been shown to decrease recovery time after organic overload or toxicity upset. Compared to strictly anaerobic cultures, adding aerotolerant methanogenic cultures may be more practical since they exhibit higher methanogenic activity and can be easily dried and stored in ambient air for future shipping and use. In this study, anaerobic digesters were bioaugmented with both anaerobic and aerated, methanogenic propionate enrichment cultures after a transient organic overload. Digesters bioaugmented with anaerobic and moderately aerated cultures recovered 25 and 100 days before non-bioaugmented digesters, respectively. Increased methane production due to bioaugmentation continued a long time, with 50-120% increases 6 to 12 SRTs (60-120 days) after overload. In contrast to the anaerobic enrichment, the aerated enrichments were more effective as bioaugmentation cultures, resulting in faster recovery of upset digester methane and COD removal rates. Sixty days after overload, the bioaugmented digester archaeal community was not shifted, but was restored to one similar to the pre-overload community. In contrast, non-bioaugmented digester archaeal communities before and after overload were significantly different. Organisms most similar to Methanospirillum hungatei had higher relative abundance in well-operating, undisturbed and bioaugmented digesters, whereas organisms similar to Methanolinea tarda were more abundant in upset, non-bioaugmented digesters. Bioaugmentation is a beneficial approach to increase digester recovery rate after transient organic overload events. Moderately aerated, methanogenic propionate enrichment cultures were more beneficial augments than a strictly anaerobic enrichment.

Relating methanogen community structure and anaerobic digester function.

Much remains unknown about the relationships between microbial community structure and anaerobic digester function. However, knowledge of links between community structure and function, such as specific methanogenic activity (SMA) and COD removal rate, are valuable to improve anaerobic bioprocesses. In this work, quantitative structure-activity relationships (QSARs) were developed using multiple linear regression (MLR) to predict SMA using methanogen community structure descriptors for 49 cultures. Community descriptors were DGGE demeaned standardized band intensities for amplicons of a methanogen functional gene (mcrA). First, predictive accuracy of MLR QSARs was assessed using cross validation with training (n = 30) and test sets (n = 19) for glucose and propionate SMA data. MLR equations correlating band intensities and SMA demonstrated good predictability for glucose (q(2) = 0.54) and propionate (q(2) = 0.53). Subsequently, data from all 49 cultures were used to develop QSARs to predict SMA values. Higher intensities of two bands were correlated with higher SMA values; high abundance of methanogens associated with these two bands should be encouraged to attain high SMA values. QSARs are helpful tools to identify key microorganisms or to study and improve many bioprocesses. Development of new, more robust QSARs is encouraged for anaerobic digestion or other bioprocesses, including nitrification, nitritation, denitrification, anaerobic ammonium oxidation, and enhanced biological phosphorus removal.

Methanogen community structure-activity relationship and bioaugmentation of overloaded anaerobic digesters.

Accumulation of acids in anaerobic digesters after organic overload can inhibit or stop CH4 production. Therefore, methods to reduce acid concentrations would be helpful. One potential method to improve recovery involves bioaugmentation, addition of specific microorganisms to improve performance. In this study, transiently overloaded digesters were bioaugmented with a propionate-degrading enrichment culture in an effort to decrease recovery time. Biomass samples from 14 different, full-scale anaerobic digesters were screened for specific methanogenic activity (SMA) against propionate; the microbial communities were also compared. SMA values spanned two orders of magnitude. Principal component analysis of denaturing gradient gel electrophoresis (DGGE) banding patterns for a functional gene (mcrA) suggested an underlying community structure-activity relationship; the presence of hydrogenotrophic methanogens closely related to Methanospirillum hungatei and Methanobacterium beijingense was associated with high propionate SMA values. The biomass sample demonstrating the highest SMA was enriched for propionate degrading activity and then used to bioaugment overloaded digesters. Bioaugmented digesters recovered more rapidly following the organic overload, requiring approximately 25 days (2.5 solids retention times (SRTs)) less to recover compared to non-bioaugmented digesters. Benefits of bioaugmentation continued for more than 12 SRTs after organic overload. Bioaugmentation is a promising approach to decrease recovery time after organic overload.