Rethinking growth and decay kinetics in activated sludge - towards a new adaptive kinetics approach.

Growth kinetics in activated sludge modelling (ASM) are typically assumed to be the result of intrinsic growth and decay properties and thus process parameters are deemed to be constant. The activity change in a microbial population is expressed in terms of variance of the active biomass fraction and not actual shifts in bacterial cellular activities. This approach is limited, in that it does not recognise the reality that active biomass is highly physiologically adaptive. Here, a strong correlation between maximum specific growth rate (µmax) and decay rate (be) of ordinary heterotrophic organisms was revealed in both low solids retention times (SRT) and high SRT activated sludge systems. This relationship is indicative of physiological adaptation either for growth (high µmax and be) or survival optimization (low µmax and be). Further, the nitrifier decay process was investigated using molecular techniques to measure decay rates of ammonia oxidizing bacteria and nitrite oxidizing bacteria over a range of temperatures. This approach revealed decay rates 10-12% lower than values previously accepted and used in ASM. These findings highlight potential benefits of incorporating physiological adaptation of heterotrophic and nitrifying populations in future ASM.

Effects of electron acceptors on removal of antibiotic resistant Escherichia coli, resistance genes and class 1 integrons under anaerobic conditions.

Anaerobic biotechnologies can effectively remove antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs), but there is a need to better understand the mechanisms. Here we employ bioelectrochemical systems (BES) as a platform to investigate the fate of a native tetracycline and sulfonamide-resistant Escherichia coli strain and its ARGs. The E. coli strain carrying intI1, sulI and tet(E) was isolated from domestic wastewater and dosed into a tubular BES. The BES was first operated as a microbial fuel cell (MFC), with aeration in the cathode, which resulted in enhanced removal of E. coli and ARGs by ~2 log (i.e., order of magnitude) when switched from high current to open circuit operation mode. The BES was then operated as a microbial electrolysis cell (MEC) to exclude the effects of oxygen diffusion, and the removal of E. coli and ARGs during the open circuit configuration was again 1-2 log higher than that at high current mode. Significant correlations of E. coli vs. current (R(2)=0.73) and ARGs vs. E. coli (R(2) ranged from 0.54 to 0.87), and the fact that the BES substrate contained no electron acceptors, implied that the persistence of the E. coli and its ARGs was determined by the availability of indigenous electron acceptors in the BES, i.e., the anode electrode or the electron shuttles generated by the exoelectrogens. Subsequent experiments with pure-culture tetracycline and sulfonamide-resistant E. coli being incubated in a two-chamber MEC and serum bottles demonstrated that the E. coli could survive by respiring anode electrode and/or electron shuttles released by exoelectrogens, and ARGs persisted with their host E. coli.

Survival of Antibiotic Resistant Bacteria and Horizontal Gene Transfer Control Antibiotic Resistance Gene Content in Anaerobic Digesters.

Understanding fate of antibiotic resistant bacteria (ARB) vs. their antibiotic resistance genes (ARGs) during wastewater sludge treatment is critical in order to reduce the spread of antibiotic resistance through process optimization. Here, we spiked high concentrations of tetracycline-resistant bacteria, isolated from mesophilic (Iso M1-1-a Pseudomonas sp.) and thermophilic (Iso T10-a Bacillus sp.) anaerobic digested sludge, into batch digesters and monitored their fate by plate counts and quantitative polymerase chain reaction (QPCR) of their corresponding tetracycline ARGs. In batch studies, spiked ARB plate counts returned to baseline (thermophilic) or 1-log above baseline (mesophilic) while levels of the ARG present in the spiked isolate [tet(G)] remained high in mesophilic batch reactors. To compare results under semi-continuous flow conditions with natural influent variation, tet(O), tet(W), and sul1 ARGs, along with the intI1 integrase gene, were monitored over a 9-month period in the raw feed sludge and effluent sludge of lab-scale thermophilic and mesophilic anaerobic digesters. sul1 and intI1 in mesophilic and thermophilic digesters correlated positively (Spearman rho = 0.457-0.829, P < 0.05) with the raw feed sludge. There was no correlation in tet(O) or tet(W) ratios in raw sludge and mesophilic digested sludge or thermophilic digested sludge (Spearman rho = 0.130-0.486, P = 0.075-0.612). However, in the thermophilic digester, the tet(O) and tet(W) ratios remained consistently low over the entire monitoring period. We conclude that the influent sludge microbial composition can influence the ARG content of a digester, apparently as a result of differential survival or death of ARBs or horizontal gene transfer of genes between raw sludge ARBs and the digester microbial community. Notably, mesophilic digestion was more susceptible to ARG intrusion than thermophilic digestion, which may be attributed to a higher rate of ARB survival and/or horizontal gene transfer between raw sludge bacteria and the digester microbial community.

Effect of silver nanoparticles and antibiotics on antibiotic resistance genes in anaerobic digestion.

Water resource recovery facilities have been described as creating breeding ground conditions for the selection, transfer, and dissemination of antibiotic resistance genes (ARGs) among various bacteria. The objective of this study was to determine the effect of direct addition of antibiotic and silver nanoparticles (Ag NPs, or nanosilver) on the occurrence of ARGs in thermophilic anaerobic digesters. Test thermophilic digesters were amended with environmentally-relevant concentrations of Ag NP (0.01, 0.1, and 1.0 mg-Ag/L; corresponding to approximately 0.7, 7.0, and 70 mg-Ag/kg total solids) and sulfamethoxazole (SMX) that span susceptible to resistant classifications (1, 5, and 50 mg/L) as potential selection pressures for ARGs. Tetracycline (tet(O), tet(W)) and sulfonamide (sulI, sulII) ARGs and the integrase enzyme gene (intI1) associated with Class 1 integrons were measured in raw sludge, test thermophilic digesters, a control thermophilic digester, and a control mesophilic digester. There was no apparent effect of Ag NPs on thermophilic anaerobic digester performance. The maximum SMX addition (50 mg/L) resulted in accumulation of volatile fatty acids and low pH, alkalinity, and volatile solids reduction. There was no significant difference between ARG gene copy numbers (absolute or normalized to 16S rRNA genes) in amended thermophilic digesters and the control thermophilic digester. Antibiotic resistance gene copy numbers in digested sludge ranged from 10(3) to 10(6) copies per microL (approximately 8 x10(1) to 8 x 10(4) copies per microg) of sludge as result of a 1-log reduction of ARGs (2-log reduction for intI1). Quantities of the five ARGs in raw sludge ranged from 10(4) to 10(8) copies per microL (approximately 4 x 10(2) to 4 x 10(6) per microg) of sludge. Test and control thermophilic digesters (53 degrees C, 12-day solids retention time [SRT]) consistently reduced but did not eliminate levels of all analyzed genes. The mesophilic digester (37 degrees C, 20-day SRT) also reduced levels of sulI, sulII, and intI1 genes, but levels of tet(O) and tet(W) were the same or higher than in raw sludge. Antibiotic resistance gene reductions remained constant despite the application of selection pressures, which suggests that digester operating conditions are a strong governing factor of the bacterial community composition and thus the prevalence of ARGs.