Hongkeun Park

Impact of inocula and growth mode on the molecular microbial ecology of anaerobic ammonia oxidation (anammox) bioreactor communities.

The composition of distinctly inoculated granular anammox and biofilm-based completely autotrophic nitrogen removal over nitrite (CANON) bioreactors was investigated from start-up through continuous long-term operation via denaturing gradient gel electrophoresis (DGGE) and sequencing. The granular anammox reactor was seeded with sludge from an operational anammox reactor in Strass, Austria. The CANON reactor was seeded with activated sludge from a local wastewater treatment plant in New York City. The principal anammox bacteria (AMX) shifted from members related to Kuenenia stuttgartiensis present in the initial inoculum to members related to Candidatus Brocadia fulgida during pre-enrichment (before this study) and to members related to Candidatus Brocadia sp. 40 (during this study) in the granular reactor. AMX related to C. Brocadia sp. 40 were also enriched from activated sludge in the CANON reactor. The estimated doubling times of AMX in the granular and CANON reactors were 5.3 and 8.9 days, respectively, which are lower than the value of 11 days, reported previously. Both the granular anammox and CANON reactors also fostered significant amounts of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB). The fractions of AMX and two groups of NOB were generally similar in the granular anammox and CANON reactors. However, the diversity and fractions of AOB in the two reactors was markedly different. Therefore, it is suggested that the composition of the feed and extant substrate concentrations in the reactor likely select for the microbial community composition more than the inocula and reactor configuration. Further, such selection is not equivalent for all resident communities.

Impact of varying electron donors on the molecular microbial ecology and biokinetics of methylotrophic denitrifying bacteria

The goal of this study was to identify bacterial populations that assimilated methanol in a denitrifying sequencing batch reactor (SBR), using stable isotope probing (SIP) of 13C labeled DNA and quantitatively track changes in these populations upon changing the electron donor from methanol to ethanol in the SBR feed. Based on SIP derived 13C 16S rRNA gene clone libraries, dominant SBR methylotrophic bacteria were related to Methyloversatilis spp. and Hyphomicrobium spp. These methylotrophic populations were quantified via newly developed real‐time PCR assays. Upon switching the electron donor from methanol to ethanol, Hyphomicrobium spp. concentrations decreased significantly in accordance with their obligately methylotrophic nutritional mode. In contrast, Methyloversatilis spp. concentrations were relatively unchanged, in accordance with their ability to assimilate both methanol and ethanol. Direct assimilation of ethanol by Methyloversatilis spp. but not Hyphomicrobium spp. was also confirmed via SIP. The reduction in methylotrophic bacterial concentration upon switching to ethanol was paralleled by a significant decrease in the methanol supported denitrification biokinetics of the SBR on nitrate. In sum, the results of this study demonstrate that the metabolic capabilities (methanol assimilation and metabolism) and substrate specificity (obligately or facultatively methylotrophic) of two distinct methylotrophic bacterial populations contributed to their survival or washout in denitrifying bioreactors. Biotechnol. Bioeng. 2009;102: 1527–1536.

Propensity of activated sludge to amplify or attenuate tetracycline resistance genes and tetracycline resistant bacteria: a mathematical modeling approach.

The overall goal of this study was to quantify the propensity of the activated sludge (AS) process at three wastewater treatment plants (WWTP) to amplify or attenuate tetracycline resistant bacteria (TRB) and tetracycline resistance genes (TRG). Accordingly, the abundance and fraction of TRB and seven TRG in different unit operations of these WWTP were analytically measured and modeled using a mass balance approach widely used for AS design. Based on the model, the AS process of the different WWTP neither amplified nor attenuated the TRB and TRG fractions. Of the TRG tested, the ribosomal protection genes, tet(O) and tet(W) were the most abundant, along the treatment train of the WWTP, on all sampling dates and sampling locations. Significant amounts of TRB and TRG were discharged in the effluent streams. Notably, in selected samples, the fraction of TRB increased in response to ultraviolet disinfection of treated wastewater compared to chlorination. This study therefore implicates wastewater treatment processes as significant point sources of tetracycline resistance determinants to the environment, and provides a mathematical basis to compute the production capacity of these determinants in the AS process.

The effect of inorganic carbon on microbial interactions in a biofilm nitritation-anammox process.

The overarching goal of this study was to determine the role of inorganic carbon (IC) in influencing the microbial ecology, performance and nitrogen turnover by individual microbial communities of a biofilm based combined nitritation-anammox process. IC limitation was transiently imposed by reducing the IC input from 350% to 40% of the stoichiometric requirement for 40 days. The principal impact observed during IC limitation was the overgrowth of nitrite oxidizing bacteria (NOB) at the expense of anaerobic ammonia oxidizing bacteria (AMX). On the other hand, the concentrations of ammonia oxidizing bacteria (AOB) were relatively stable during the imposition of and recovery from IC limitation. The resulting dominance of NOB, in terms of their concentration and contribution to nitrite consumption over AMX, resulted, in turn, in a decrease in overall nitrogen removal from 78 ± 2.0% before IC limitation to 46 ± 2.9% during IC limitation. Upon recovery back to non-limiting IC input, it took an inordinately long time (about 57*HRT) for the N-removal to recover back to pre-limitation conditions. Even after recovery, NOB were still persistent in the biofilm and could not be washed out to pre-limitation concentrations. The emission of nitrous oxide (N₂O) and nitric oxide (NO), likely from AOB, transiently increased in concert with transient increases in ammonia and hydroxylamine concentrations during the period of IC limitation. Therefore, an unintended consequence of IC limitation in nitritation-anammox systems can be an increase in their greenhouse gas footprint, in addition to compromised process performance. Most emphasis to date on nitritation and anammox studies has been on the nitrogen cycle. The results of this study demonstrate that the differing strategies used by AOB, NOB and AMX to compete for their preferred assimilative carbon source can also significantly influence the microbial ecology, performance and carbon footprint of such processes.

Transfer of antibiotic resistance plasmids in pure and activated sludge cultures in the presence of environmentally representative micro-contaminant concentrations

The presence of antibiotics in the natural environment has been a growing issue. This presence could also account for the influence that affects microorganisms in such a way that they develop resistance against these antibiotics. The aim of this study was to evaluate whether the antibiotic resistant gene (ARG) plasmid transfer can be facilitated by the impact of 1) environmentally representative micro-contaminant concentrations in ppb (part per billion) levels and 2) donor-recipient microbial complexity (pure vs. mixed). For this purpose, the multidrug resistant plasmid, pB10, and Escherichia coli DH5α were used as a model plasmid and a model donor, respectively. Based on conjugation experiments with pure (Pseudomonas aeruginosa PAKexoT) and mixed (activated sludge) cultures as recipients, increased relative plasmid transfer frequencies were observed at ppb (μg/L) levels of tetracycline and sulfamethoxazole micro-contaminant exposure. When sludge, a more complex community, was used as a recipient, the increases of the plasmid transfer rate were always statistically significant but not always in P. aeruginosa. The low concentration (10 ppb) of tetracycline exposure led to the pB10 transfer to enteric bacteria, which are clinically important pathogens.

Molecular and biokinetic characterization of methylotrophic denitrification using nitrate and nitrite as terminal electron acceptors.

Although methanol is a widely employed carbon source for denitrification, relatively little is known on the abundance and diversity of methylotrophic bacteria in activated sludge. The primary aim of this study was to specifically identify bacteria that metabolized methanol in a sequencing batch denitrifying reactor (SBDR), using a novel technique, stable isotope probing (SIP) of 13C labeled DNA. A secondary aim was to quantitatively track dominant methylotrophic bacteria in the SBDR exposed to different terminal electron acceptors. SIP enabled 13C 16S rDNA clone libraries revealed that SBDR methylotrophic populations were related to Methyloversatilis spp. and Hyphomicrobium spp. Based on newly developed quantitative polymerase chain reaction (qPCR) assays, Hyphomicrobium spp. were more abundant than Methyloversatilis spp. throughout the period of SBDR operation. The relative population abundance was stable despite a shift in electron acceptor from nitrate to nitrite (keeping the same methanol dose). However, the shift to nitrite resulted in a significant decrease in denitrification biokinetics on both nitrate and nitrite.

Differentiation in the microbial ecology and activity of suspended and attached bacteria in a nitritation-anammox process.

A directed differentiation between the biofilm and suspension was observed in the molecular microbial ecology and gene expression of different bacteria in a biofilm nitritation‐anammox process operated at varying hydraulic residence times (HRT) and nitrogen loading rates (NLR). The highest degree of enrichment observed in the biofilm was of anaerobic ammonia‐oxidizing bacteria (AMX) followed by that of Nitrospira spp. related nitrite‐oxidizing bacteria (NOB). For AMX, a major shift from Candidatus “Brocadia fulgida” to Candidatus “Kuenenia stuttgartiensis” in both suspension and biofilm was observed with progressively shorter HRT, using discriminatory biomarkers targeting the hydrazine synthase (hzsA) gene. In parallel, expression of the hydrazine oxidoreductase gene (hzo), a functional biomarker for AMX energy metabolism, became progressively prominent in the biofilm. A marginal but statistically significant enrichment in the biofilm was observed for Nitrosomonas europaea related ammonia‐oxidizing bacteria (AOB). In direct contrast to AMX, the gene expression of ammonia monooxygenase subunit A (amoA), a functional biomarker for AOB energy metabolism, progressively increased in suspension. Using gene expression and biomass concentration measures in conjunction, it was determined that signatures of AOB metabolism were primarily present in the biofilm throughout the study. On the other hand, AMX metabolism gradually shifted from being uniformly distributed in both the biofilm and suspension to primarily the biofilm at shorter HRTs and higher NLRs. These results therefore highlight the complexity and key differences in the microbial ecology, gene expression and activity between the biofilm and suspension of a nitritation‐anammox process and the biokinetic and metabolic drivers for such niche segregation. Biotechnol. Bioeng. 2015;112: 272–279.

Long term assessment of factors affecting nitrifying bacteria communities and N-removal in a full-scale biological process treating high strength hazardous wastewater

Over a 3 year period, interactions between nitrifying bacterial communities and the operational parameters of a full-scale wastewater treatment plant were analyzed to assess their impact on nitrification performance. Throughout the study period, nitrification fluctuated while Nitrosomonas europaea and Nitrosomonas nitrosa, the two major ammonia oxidizing bacteria (AOB) communities, showed resistance to changes in operational and environmental conditions. Nitrobacter populations mostly exceeded those of Nitrospira within nitrite oxidizing bacteria (NOB). Meanwhile, principal component analysis (PCA) results revealed that a close association between Nitrobacter and nitrite concentration as well as a direct correlation between the quantity of AOB and influent SCN- concentration. The serial shifts of data points over time showed that the nitrification of a full-scale treatment plant has been gradually suppressed by the influence of influent COD and phenol concentrations.

Ammonia‐based intermittent aeration control optimized for efficient nitrogen removal

This work describes the development of an intermittently aerated pilot‐scale process (V = 0.45 m3) operated for optimized efficient nitrogen removal in terms of volume, supplemental carbon and alkalinity requirements. The intermittent aeration pattern was controlled using a strategy based on effluent ammonia concentration set‐points. The unique feature of the ammonia‐based aeration control was that a fixed dissolved oxygen (DO) set‐point was used and the length of the aerobic and anoxic time (anoxic time ≥25% of total cycle time) were changed based on the effluent ammonia concentration. Unlike continuously aerated ammonia‐based aeration control strategies, this approach offered control over the aerobic solids retention time (SRT) to deal with fluctuating ammonia loading without solely relying on changes to the total SRT. This approach allowed the system to be operated at a total SRT with a small safety factor. The benefits of operating at an aggressive SRT were reduced hydraulic retention time (HRT) for nitrogen removal. As a result of such an operation, nitrite oxidizing bacteria (NOB) out‐selection was also obtained (ammonia oxidizing bacteria [AOB] maximum activity: 400 ± 79 mgN/L/d, NOB maximum activity: 257 ± 133 mgN/L/d, P < 0.001) expanding opportunities for short‐cut nitrogen removal. The pilot demonstrated a total inorganic nitrogen (TIN) removal rate of 95 ± 30 mgN/L/d at an influent chemical oxygen demand: ammonia (COD/NH4+‐N) ratio of 10.2 ± 2.2 at 25°C within the hydraulic retention time (HRT) of 4 h and within a total SRT of 5–10 days. The TIN removal efficiency up to 91% was observed during the study, while effluent TIN was 9.6 ± 4.4 mgN/L. Therefore, this pilot‐scale study demonstrates that application of the proposed on‐line aeration control is capable of relatively high nitrogen removal without supplemental carbon and alkalinity addition at a low HRT. Biotechnol. Bioeng. 2015;112: 2060–2067.

Nitric oxide preferentially inhibits nitrite oxidizing communities with high affinity for nitrite

The prerequisite to the development success of the novel mainstream processes partial nitritation/anammox is the out-selection of nitrite oxidizing bacteria (NOB). A recent study suggested that this could be achieved through NO production by ammonium oxidizing bacteria under cyclic oxic-anoxic conditions. Indeed, it is known that among NOB, Nitrobacter species are reversibly inhibited by NO. However, the effect of NO on the activity of the NOB genus Nitrospira is not studied so far. Such an understanding is needed, since Nitrospira related NOB are mostly prevailing in sewage treatment plants. This study quantified the effect of NO on the nitratation activity of sludge types with different Nitrobacter/Nitrospira ratios. In an oxic bubbling column, a dosage of 4.4 mg NO L(-1) d(-1) (∼2 μg NO-N L(-1) in liquid phase) inhibited the Nitrobacter dominated sludge with 24%. For the Nitrospira dominated sludge types, the inhibition was strongly correlated with the nitrite half saturation constant (K(s)) ranging from 0% to 30-50% and 60-80% inhibition of the nitrite oxidation for K(s) of 0.72, 0.36 and 0.06 mg NO2(-)-N L(-1), respectively. This study showed that nitrifying communities with high affinity for nitrite and low specific nitrite oxidation rates (K-strategists) can be strongly inhibited by NO. The degree of inhibition could be confirmed in a set-up with NO dosage through an artificial alginate-based biofilm, ensuring a more direct contact between NO and the microorganisms.