Article pubs.acs.org/est
Investigation of Intertidal Wetland Sediment as a Novel Inoculation Source for Anaerobic Saline Wastewater Treatment Xueqing Shi,† Kok Kwang Ng,† Xiao-Ran Li,‡ and How Yong Ng*,† †
Centre for Water Research, Department of Civil and Environmental Engineering, National University of Singapore, 1 Engineering Drive 2, Singapore 117576 ‡ Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China S Supporting Information *
ABSTRACT: Biological treatment of saline wastewater is considered unfavorable due to salinity inhibition on microbial activity. In this study, intertidal wetland sediment (IWS) collected from a high saline environment was investigated as a novel inoculation source for anaerobic treatment of saline pharmaceutical wastewater. Two parallel lab-scale anaerobic sequencing batch reactors (AnSBR) were set up to compare the organic removal potential of IWS with conventional anaerobic digested sludge (ADS). Under steady-state condition, IWS reactor (Ri) showed organic reduction performance significantly superior to that of ADS reactor (Ra), achieving COD removal efficiency of 71.4 ± 3.7 and 32.3 ± 6.1%, respectively. In addition, as revealed by fluorescent in situ hybridization (FISH) analysis, a higher relative abundance of methanogenic populations was detected in Ri. A further 16S rRNA gene pyrosequencing test was conducted to understand both the bacterial and archaeal community populations in the two AnSBRs. A predominance of halophilic/tolerant microorganisms (class Clostridia of bacteria, genera Methanosarcina, and Methanohalophilus of archaea) in Ri enhanced its organic removal efficiency. Moreover, several microbial groups related with degradation of hardly biodegradable compounds (PAHs, n-alkenes, aliphatic hydrocarbons, and alkanes, etc.) were detected in the IWS. All these findings indicated that IWS is a promising inoculation source for anaerobic treatment of saline wastewater.
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INTRODUCTION Huge amounts of salt (NaCl) are consumed by industries, mainly for the purpose of chemical synthesis, food processing, tanning and refining processes, generating enormous quantities of saline wastewaters that are rich in both salt and organic matter.1 With ever-tightening regulations for wastewater discharge, interest in saline wastewater treatment processes has been rapidly increased over the past two decades. Nowadays, biological means have been intensively applied to both municipal and industrial wastewater treatments, while saline effluents are still primarily treated by physicochemical approaches, because the microbial activity was found to be strongly inhibited by salinity.2 However, due to the high energy consumption and operational cost of physicochemical technologies, alternative solutions such as biological-based processes are still being attempted, with most of the focus on the adaptation of conventional microorganisms to a highsalinity environment.3−5 In addition, considering the high organic strength of saline wastewaters, anaerobic treatment process is preferred over aerobic treatment process. This is because anaerobic treatment process could be operated at higher organic loading, and methane gas could be recovered for energy production. On the other hand, aerobic treatment © 2015 American Chemical Society
process requires intensive energy input due to the strong aeration required to maintain proper dissolved oxygen level in the system and to meet the carbonaceous oxygen demand; consequently, a foaming issue may lead to process instability or even system failure.6,7 The salinity inhibition of microbial activity is mainly caused by high osmotic pressure, which would result in water loss from cell membranes and reduce enzyme activities, leading to plasmolysis or even cell death. Unlike freshwater microbial species, halophilic/tolerant microorganisms have their own strategies to survive in saline environments. These include (1) a specific enzyme structure allowing them to remain active and stable under high-saline conditions,8 (2) the ability to actively extrude Na+ out of their cells through Na+/H+ antiporters to maintain the internal ion concentration,9 and (3) the ability (possessed by most halophilic/tolerant species) to synthesize and accumulate compatible solutes such as sugars, amino acids, glycine betaine, trehalose, and ectoine10 to maintain an osmotic Received: Revised: Accepted: Published: 6231
January 30, 2015 April 17, 2015 April 17, 2015 April 17, 2015 DOI: 10.1021/acs.est.5b00546 Environ. Sci. Technol. 2015, 49, 6231−6239
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Environmental Science & Technology
for investigation and comparison of their organic degradation efficiencies in the saline environment. The ADS was obtained from an anaerobic digester of a wastewater reclamation plant in Singapore, with an initial mixed liquor suspended solids (MLSS) of 5142 ± 337 mg/L and pH of 7.4; the IWS was collected from an intertidal mangrove zone at Lim Chu Kang, Singapore (Supporting Information (SI) Figure S1). The selection of this specific sediment was mainly based on two considerations: (1) the salinity level of the IWS (i.e., salinity of ∼3%) is similar to that of the wastewater used in this study, which can probably minimize the microbial shock and reduce the duration of process startup, and (2) as a major biological component that contributes to the remarkable productivity of intertidal mangrove ecosystem,15 IWS has been reported to have higher numbers and activities of microbial degraders involved in the carbon and nitrogen cycles than many other ecosystems.16 The IWS (subsurface sediment of between 5 and 20 cm deep) was uniformly dark gray in color (the surface sediment was a light brown color), reflecting an anoxic/ anaerobic condition, and contained mainly inorganic fine particles with a low VSS/TSS ratio of 0.19 ± 0.01. The TDS and pH for in situ seawater were 27 130 ± 1880 mg/L and 6.8, respectively. Prior to the experiment, the IWS was sieved through a 0.2-mm screen, and the initial MLSS was diluted to ∼10 000 mg/L by the addition of seawater. System Setup and Continuous Operation. In this study, two anaerobic sequencing batch reactors (AnSBRs) with an effective volume of 5 L (SI Figure S2) each were set up in parallel for the pharmaceutical wastewater treatment. The reactors were operated at ambient temperature (27 ± 1 °C) with a mixing rate of 150 rpm. The anaerobic condition was provided by an initial N2 purging of the reactors. The AnSBRs were operated with 12-h cycles throughout the experiment, and the details of each cycle were as follows: 15 min feeding, 11 h reaction, 30 min settling, and 15 min decanting. Initially, the two AnSBRs, denoted as Ra and Ri, were seeded with concentrated ADS (Ra) and diluted IWS (Ri), respectively (with an initial MLSS of ∼10 000 mg/L for both the reactors), and the wastewater was gradually fed into the systems during continuous operation. The volumetric exchange rate for each batch cycle was fixed at 20%, resulting in a hydraulic retention time (HRT) of 60 h and an organic loading rate (OLR) of 7.3 ± 1.1 g COD/L/d. A long SRT of 80 d was selected for both the reactors in this study because, based on preliminary tests (data not shown), a SRT shorter than 80 d could result in complete biomass washout in the Ra. Notably, during system startup (especially for the initial 20 d), the washout of biomass in both the reactors was significant, resulting in the actual SRTs of both reactors being shorter than 80 d with considerable variations. When the MLSS concentration in the systems became stabilized, daily sludge withdrawal was applied. The calculation of SRT was based on the MLSS concentration in the reactor, amount of daily sludge withdrawal, TSS concentration in the effluent, and amount of mixed liquor taken for weekly analysis. The amount of daily sludge withdrawal was adjusted on a weekly basis to ensure the SRTs of both the reactors were maintained at 80 d. The biogas production, influent and effluent characteristics, and biomass concentrations in the AnSBRs were monitored and measured regularly in order to analyze the treatment performance of the two systems. Microbial Community Diversity Analyses by Pyrosequencing. Four sets of biomass samples were collected from
balance by increasing the internal osmotic pressure. Owing to their dual characteristics of being halo-resistant and able to degrade organic pollutants, these microorganisms possess a huge application potential in saline wastewater treatment.11 However, to date only a few studies have been reported on applying halophilic biomass for the treatment of industrial saline effluents, and most of them used halophilic strains (pure culture) isolated from marine environments.12,13 Nevertheless, compared with those widely applied mixed-culture treatment processes, the pure-culture systems are vulnerable to environmental variation and foreign species contamination, and thus are nonviable for full-scale application. Intertidal wetlands, in particular temperate-zone salt marshes, tropical mangroves, and mudflats, are environments with primary productivity that provide many essential ecosystem services.14 Given its high salinity nature, it is believed that intertidal wetland sediment (IWS) contains considerable amounts of halophilic/tolerant microorganisms that could be used as an inoculation source for a biological process to treat saline wastewater. However, current studies of IWS merely cover its ecological and microbiological roles, and there has been no application of IWS in the saline wastewater treatment field. Therefore, the objective of this study was to investigate the potential of IWS as a novel inoculation source for anaerobic treatment of saline wastewater. Several treatment parameters, including organic removal efficiency, biomass settling property, and microbial community composition were assessed and compared with those of a conventional anaerobic sludge system, to offer a comprehensive understanding of this potential alternative for the biological treatment of saline wastewater.
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MATERIALS AND METHODS Wastewater Characterization and Inoculant Preparation. Pharmaceutical wastewater is one typical saline wastewater that contains a high concentration of salt and organic matter. The pharmaceutical wastewater used for this study was collected from the equalization tank of a pharmaceutical factory located in Singapore. It was generated during the manufacturing of antibiotics (from the penicillin family), and therefore contained a variety of organic and inorganic constituents, such as intermediates, catalysts, and spent solvents, etc. The wastewater was stored at 4 °C to prevent microbial degradation, and prior to treatment the wastewater was brought to ambient temperature (27 ± 1 °C) and its pH was adjusted to 7 by the addition of concentrated phosphoric acid (15.8 M). The main characteristics of the pharmaceutical wastewater were chemical oxygen demand (COD) of 18 320 ± 2846 mg/L, total organic carbon (TOC) of 8988 ± 1126 mg/ L, total nitrogen (TN) of 1212 ± 189 mg/L, ammonia nitrogen (NH3−N) of 89 ± 67 mg/L, total suspended solids (TSS) of 173 ± 108 mg/L, and total dissolved solids (TDS) of 28 046 ± 3012 mg/L. Because of its high salinity and organic strength, the on-site aerobic treatment plant (i.e., a trickling filtration process followed by an activated sludge process) in the pharmaceutical factory encountered a serious foaming issue. Consequently, significant biomass loss was observed, which led to low organic removal performance (less than 40%) and system instability. To address this problem, anaerobic treatment combined with halophilic biomass (IWS) was therefore investigated in this study. Two types of biomass inoculaanaerobic digested sludge (ADS) and intertidal wetland sediment (IWS)were prepared 6232
DOI: 10.1021/acs.est.5b00546 Environ. Sci. Technol. 2015, 49, 6231−6239
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Figure 1. Biomass growth during system startup.
bacterial or archaeal groups were expressed as percentage of the whole bacterial or archaeal community DNA sequences, respectively. Analytical Methods. The biogas composition (i.e., N2, CH4, and CO2) was measured using a gas chromatograph (GC17A, Shimadzu, Japan). The Standard Methods22 were used for the measurement of the COD, TDS, TSS, and VSS. Particularly, chloride ion concentration exceeding 100 mg/L in the sample is known to interfere with COD measurement (Closed Reflux Titrimetric Method, APHA). In this study, considering the high chloride concentration and COD content of the wastewater, the samples were diluted at 150−200 times prior to COD analysis to eliminate the chloride interference, while maintaining the COD concentration in the diluted samples to be within the detection range. The obtained COD results were regularly compared with those obtained by the HACH Closed Reflux Colorimetric Method (0−150 mg/L) to ensure the accuracy of the results. The TOC and TN were analyzed with a TOC/TN analyzer (TOC-VCSH, Shimadzu, Japan) and the ammonia nitrogen (NH3−N) was determined according to the Salicylate method (Nitrogen, Ammonia, High Range, Test ‘N tube) with a detection range of 0−50 mg/L measured at 425 nm wavelength. Fluorescence in situ hybridization (FISH) analysis was conducted for the detection of methanogenic populations in the two AnSMBRs (on day 262) using a FITC-labeled oligonucleotide probe arc915 (5′FITC-GTG CTC CCC CGC CAA TTC CT-3′). The methodology was adopted from a previous study.23 Hybridization was conducted in a humidified chamber at 46 °C for 3.5 h, and subsequently subjected to DAPI counter-staining to visualize all microorganisms. Quantification of the FISH results was accomplished with the computer program COMSTAT.24
Ra and Ri at the initial phase (day 1) and steady-state condition (day 262) with triplicates. On day 1, triplicate samples were collected from both the inoculants before the startup of the AnSBRs; on day 262, mixed liquor samples were taken from both the AnSBRs during the reaction stage (i.e., 0.5, 5.5, and 10.5 h) of the batch cycle. The DNA from all these samples was extracted from equal amounts (0.5 g) of biomass (whereby mixed liquor samples were first centrifuged at 10 000 rpm for 10 min to remove the supernatant) with an UltraClean DNA extraction kit (Mobio Laboratories, USA) according to the manufacturer’s instructions. To ensure the representativeness of the final DNA sequencing results, the exacted DNA from replicates of each biomass sample set was pooled with equal amount (DNA concentration was determined by a NanoDrop system). Further, the 16S rRNA gene fragments from the pooled DNA of each sample set were amplified by polymerase chain reaction (PCR) using DreamTaq Green PCR Master Mix (Thermo Scientific, USA), with two commonly applied primer sets: 343F (5′-TACGGRAGGCAGCAG-3′) 17/926R (5′CCGTCAATTYYTTTRAGTTT-3′)18 for bacterial populations and 341F (5′-CCT ACG GGR SGC AGC AG-3′)19/ 958R (5′-YCC GGC GTT GAM TCC AAT T-3′)20 for archaeal populations. To conduct pyrosequencing, barcodes were incorporated between the 454 adaptor and forward primers. The PCR amplification program consisted of an initial denaturing at 95 °C for 5 min, followed by 35 cycles of denaturing at 95 °C for 30 s, annealing at 55 °C for 1 min, and extension at 72 °C for 45 s, and finally an extension at 72 °C for 10 min. The amplicons with different barcodes were then mixed in equal concentration and sequenced by a Roche 454 GS-FLX Titanium sequencer (Roche, USA). Raw sequences from pyrosequencing were screened, the adaptors, barcodes, and primers were trimmed, and those sequences less than 200 bp or containing ambiguous bases were excluded. The taxonomic identities of the sequences were then assigned using the Classifier program of the RDP-II, with a minimum confidence level of 80%. The rarefaction curves were generated, and the Chao 1 and abundance-based coverage estimator (ACE) indices were calculated to compare the microbial diversity and richness between the inocula and adapted biomasses according to the literature.21 The relative abundance values of
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RESULTS AND DISCUSSION Biomass Adaptation during System Startup and Treatment Efficiencies at Steady-State Condition. Microbial responses to chemical stress are important for engineered systems, especially during the startup period. Biomass concentrations were monitored as mixed liquor volatile suspended solids (MLVSS) for both the AnSBRs to enable an understanding of the microbial growth and adaptation ability 6233
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was achieved by Ri, compared with 32.3 ± 6.1% by Ra. This outstanding organic removal potential of the IWS for the saline wastewater treatment was validated by the results of a previous study,25 in which a COD removal efficiency of 41.3 ± 2.2% was achieved when an upflow anaerobic sludge blanket (UASB) with anaerobic granular sludge was applied for the treatment of the same pharmaceutical wastewater. UASB is a widely applied anaerobic technology designed especially for high-strength wastewater treatment, and granular-form sludge can benefit microorganisms to survive and resist harsh environments,26,27 hence the combination makes this wastewater treatment technology popular in full-scale industrial applications. Nevertheless, low COD removal efficiencies (less than 50%) were observed in both the UASB25 and Ra, even though the former showed a slight enhancement. Therefore, the significantly higher performance of Ri implied that halophilic biomass could be one of the key solutions for biological saline wastewater treatment. In addition, a stable CH4 yield of 241 ± 53 mL CH 4 /g COD consumed was observed for R i , which was approximately two times higher than that of Ra (122 ± 75 mL CH4/g CODconsumed), reflecting the excellent functioning of the methanogenic populations in the IWS than in the ADS under the saline environment. According to the literature, similar CH4 yields of 220−270 CH4/g CODconsumed were also observed in anaerobic treatment of different industrial wastewaters (i.e., cheese whey wastewater, landfill leachate, and bioethanol industrial wastewater, etc.) at varying salinity conditions, with excellent COD removal efficiencies of 82− 99%.28−30 Solid content is another important parameter of effluent qualities. The TSS concentration in the effluent of Ri (166 ± 103 mg/L) was found to be significantly lower than that of Ra (574 ± 74 mg/L). This finding could be attributed to the superior sludge-settling properties of the IWS, compared with those of the ADS (SI Figure S3). The better settling of IWS would also facilitate higher biomass retention in Ri, as shown in Figure 1, which could further explain its higher organic
of different inocula during the initial 65 d. As shown in Figure 1, because of the high inorganic content of the IWS, a significantly lower initial MLVSS concentration was observed in Ri when compared with Ra. During system operation, a gradual increase of the biomass concentration in Ri from 1965 to 3486 mg/L was recorded, while that of Ra was dramatically decreased from 7755 to 3024 mg/L in the initial few days (from day 1 to 21). These changes in MLVSS could be attributed to faster microbial growth and adaptation of the IWS than ADS to the saline wastewater environment. On the other hand, the VSS/ TSS ratio in Ri was considerably increased to 0.73 by day 56, similar to that of conventional sludge (between 0.6 and 0.8), which suggests a rapid establishment of functional microbial populations. Subsequently, the AnSBRs were operated for another 220 d to achieve steady-state condition, and the stable treatment efficiencies and effluent qualities are summarized in Table 1. Table 1. Process Efficiencies and Effluent Qualities at Steady-State Condition parameter COD reduction (%) TOC reduction (%) methane yield (mL CH4/g CODconsumed) COD (mg/L) TOC (mg/L) TN (mg/L) NH3−N (mg/L) TDS (mg/L) TSS (mg/L) pH
effluent Ri
effluent Ra
71.4 ± 3.7 74.6 ± 5.4 241 ± 53
32.3 ± 6.1 36.9 ± 8.1 122 ± 75
5,240 ± 678 2,283 ± 485 1,017 ± 27 661 ± 44 28,312 ± 3,422 166 ± 103 7.4 ± 0.1
12,403 ± 1,118 5,672 ± 810 1,006 ± 43 729 ± 26 27,988 ± 3,117 537 ± 74 7.2 ± 0.1
With regard to the reduction of organic pollutants, a considerably higher COD removal efficiency of 71.4 ± 3.7%
Figure 2. FISH images of adapted biomass samples in the AnSBRs collected under steady-state condition: (a) Ri (DAPI staining for all microorganisms, blue); (b) Ri (FITC-labeled probe detecting methanogenic populations, green); (c) Ra (DAPI staining for all microorganisms, blue); (d) Ra (FITC-labeled probe detecting methanogenic populations, green). Bar, 10 μm. 6234
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Environmental Science & Technology Table 2. Summary of Pyrosequencing Data for the Two Inocula and Adapted Biomass Samples bacterial community Number of Sequences OTUsa ACEa Chao1a a
archaeal community
IWS inoculum
ADS inoculum
IWS adapted
ADS adapted
IWS inoculum
ADS inoculum
IWS adapted
ADS adapted
1,510 1,032 5,469 3,263
998 522 3,307 1,571
1,305 481 3,132 1,478
828 97 312 203
2,650 1,253 12,462 5,202
1,678 274 1,900 1,024
2,339 125 481 269
1,865 234 877 550
Generated at 97% similarity threshold.
each sample (SI Figure S4), the IWS inoculum for both the bacterial and archaeal communities had the greatest steepness, suggesting that it had the highest evenness of community composition among different samples.35 Bacterial Community Composition. Various groups of bacteria are involved in substrate hydrolysis and acidification during the anaerobic digestion processes. Figure 3A demonstrates the phylum level distributions of bacterial populations among different biomass samples. It can be observed that for all the samples, Bacteroidetes, Firmicutes, and Proteobacteria were the dominant phyla, comprising between 70.0 and 90.2% of the total bacterial sequences. On the other hand, another popular phylum present in conventional anaerobic digesters, Actinobacteria,21,36 was found in very low abundance, accounting for only 0.1−2.7% of the total bacterial sequences. Among the three dominant phyla, Bacteroidetes showed the highest average abundance (41.9%) in both the inocula and adapted biomasses, which revealed its high richness in the natural sediment environment,37,38 as well as its remarkable adaptation potential in the engineered saline wastewater treatment system.39,40 In addition, it has been reported that members of Bacteroidetes are important heterotrophs involved in cycling organic carbon and proteinaceous substances,41,42 and hence Bacteroidetes should play a vital role in the anaerobic degradation. Proteobacteria is another key bacterial group in conventional biological wastewater treatment processes. However, its abundance decreased remarkably in both the AnSBRs under steady-state condition, which suggested that this specific wastewater environment was not favorable for the growth of Proteobacteria populations. For investigation of more specific taxa with this phylum, the classlevel distribution within Proteobacteria is illustrated in Figure 3B. It can be observed that in the IWS inoculum, similar to in other sediment samples, γ-proteobacteria (16.4%) and δproteobacteria (24.2%) were the dominant classes,43 while for conventional anaerobic sludge (ADS inoculum), α-proteobacteria, β-proteobacteria, γ-proteobacteria, and δ-proteobacteria were prevalent,44 accounting for 3.2, 21.4, 3.2, and 9.9% of the whole bacteria populations, respectively. Notably, in the adapted biomasses from the two AnSBRs, only γ-proteobacteria and δproteobacteria were detected, which indicates that within this bacterial phylum, these two classes could have relatively higher resistance to the unfavorable saline environment. On the other hand, the quantity of phylum Firmicutes, members of which have been reported to withstand harsh environmental conditions because of their spore-forming capability,45 was found to be significantly increased during system operation. Moreover, as illustrated in Figure 3C, the increasing abundance of Firmicutes was mostly resulted by the thriving of class Clostridia, whose quantity in both Ri and Ra rose sharply during the operation, from 3.8 to 45.7% and from 3.1 to 18.1%, respectively. Clostridia is a very important bacterial taxa in anaerobic fermentation, given its versatile metabolism for organic substrate hydrolysis and acidification, and spore-
treatment capacity. It has been reported that decantation problems have occurred in biological plants when saline wastewater was treated, as cell plasmolysis caused by high osmotic pressure could result in a reduction of particle size and density.31 Therefore, the presence of halophilic microorganisms in the IWS can maintain floc structure in a saline environment, and further be attributed to the enhanced sludge settling in Ri. Nevertheless, considering the incomplete oxidation as one main drawback of anaerobic processes, a subsequent aerobic posttreatment would be recommended for further removal of residual COD and NH3−N from the anaerobic effluent. Relative Abundance of Methanogenic Populations Revealed by FISH. Methanogenic populations play crucial roles in the mineralization of organic carbon. Therefore, to understand the different treatment efficiency of the two AnSBRs, relative abundance of methanogens in the biomasses was quantified from FISH images as the proportion of the fluorescent area of a methanogen-specific probe versus the area of all microorganisms (Figure 2). As a result, a significantly greater abundance of methanogenic populations was observed in Ri (32%) than in Ra (5%), which was in accordance with its preferred treatment efficiency, indicating that the higher quantity of adapted methanogens in Ri could be one main reason for the satisfactory organic reduction. On the other hand, aggregation of cells was noted in both samples (especially that from Ri), suggesting this growth strategy could benefit the methanogens to resist the saline and probably toxic environmental conditions. In addition, filamentous populations were not observed in either of the AnSBRs; this observation is supported by literature which reported that a high salinity environment could inhibit the growth of filamentous bacteria.1 Overall Analysis of Pyrosequencing Results. To study the underlying mechanism of organic degradation, the bacterial and archaeal communities from the inocula and adapted biomasses of both the AnSBRs were investigated and compared using 454-pyrosequencing technology. As demonstrated in Table 2, a total of 4641 and 8532 effective sequences were obtained after filtering from 6004 and 10 692 raw sequences for bacterial and archaeal communities, with average sequence lengths of 544 and 394 bp, respectively. With regard to the inocula samples (both bacteria and archaea), higher operational taxonomic units (OTUs) and community richness estimator values (Ace and Chao 1) were observed for the IWS than the ADS, indicating a more diverse microbial community in the IWS; the high diversity correlated positively with great metabolic versatility,32,33 which could enable the community to survive under various environments by utilization of different organic sources. Whereas for the adapted biomass samples collected at steady-state condition, the microbial community diversity was remarkably decreased compared with the inocula samples, which could be the result of the re-establishment of dominant microbial groups under specific environmental stress.34 Moreover, as illustrated by the rarefraction curves of 6235
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Considering its great potential for the anaerobic treatment of saline wastewater, the bacterial genus-level distribution in the IWS (inoculum) was further assessed, and the populations related to wastewater decontamination are listed in Table 3. It Table 3. Summary of Wastewater Decontamination-Related Genera Detected in the IWS Inoculum genus name
relative abundance (%)
contaminant
ref.
Desulfococcus Desulfosarcina Desulfobulbus Desulfatiferula Acidithiobacillus Desulfobacterium Desulfonema Novosphingobium
1.9 1.4 1.0 0.7 0.7 0.5 0.2 0.1
PAHs PAHs PAHs n-alkenes heavy metals aliphatic hydrocarbons alkanes PPCPs (triclosan)
48 48 49 50 51 52 53 54
was noted that a large number of sulfate-reducing bacteria (SRB) populations (genera Desulfatiferula, Desulfobacterium, Desulfobulbus, Desulfococcus, Desulfonema, and Desulfosarcina) were detected, which were capable of breaking down several groups of hardly biodegradable organics, such as polycyclic aromatic hydrocarbons (PAHs), n-alkenes, aliphatic hydrocarbons, and alkanes, etc.48,50,52,53 In addition, the contribution of genus Acidithiobacillus to precious metal extraction during the bioleaching process has been reported,51 and genus Novosphingobium has been found to have the ability to reduce pharmaceutical and personal care products (PPCPs).54 Although the relative abundance of these bacterial groups was considered low in the IWS inoculum (less than 2%), which could be resulted by the limited substrate concentration in the natural sediment environment, these specific microbial populations in the sediments had been successfully enriched and became dominant in the engineered systems.53,55 Thus, in general, the presence of halophilic microorganisms and the aforementioned valuable bacterial groups suggests that IWS could be an ideal inoculation source for saline wastewater treatment plants, and could also be applied as a bioaugmentation source to improve treatment efficiencies for in-operation anaerobic processes. Archaeal Community Composition. Methanogenic archaea plays an indispensable role in anaerobic processes, removing excess hydrogen and fermentation metabolites generated during acidogenisis, and hence ensuring that the organic pollutants are finally degraded and removed in the form of methane and carbon dioxide. Figure 4A illustrates the classlevel distributions of archaeal populations among the four biomass samples. On the basis of classification, there are three methanogenic classes in total,56 two of which (classes Methanobacteria and Methanomicrobia) were detected in all the samples. For the IWS inoculum, a most even distribution was observed, consistent with its highest diversity degree as discussed in the Pyrosequencing Results section. However, in the ADS inoculum, the methanogenic population (class Methanomicrobia) was found to be the only predominant group, indicating its active condition for anaerobic degradation purposes. In addition, the relative abundance of methanogenic populations was increased in Ri (from 18.6 to 70.0%) but decreased in Ra (from 79.8 to 37.2%). This finding suggested that the methanogens from the IWS could possess a greater ability to adapt to the saline and possibly toxic wastewater environment. Moreover, it was noted that for all the biomass
Figure 3. Bacterial community composition between inocula and adapted biomasses. (A) Phylum-level distributions. Some phyla were assigned to “other” if detected abundance was fewer than 2%. (B) Class-level distributions within phylum Proteobacteria. (C) Class-level distributions within phylum Firmicutes. The relative abundance of a given bacterial group is expressed as a percentage of the whole bacterial community.
forming ability to survive under unfavorable environments.46 Hence, the higher abundance of Clostridia in Ri than in Ra under the steady-state condition could markedly contribute to the superior COD reduction efficiency of Ri. The above findings for the bacterial population distributions are supported by a previous study by Li et al.,47 in which classes δproteobacteria, ε-proteobacteria, Clostridia, and Bacilli were found to be associated with antibiotic-containing environments, which shared similar characteristics with the pharmaceutical wastewater used in this study. 6236
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distribution was observed, with seven major methanogenic genera detected: Methanococcoides (0.8%), Methanogenium (2.0%), Methanohalophilus (0.1%), Methanoplanus (1.5%), Methanosaeta (5.5%), Methanosalsum (1.4%), and Methanosarcina (0.7%); while in the ADS inoculum, Methanosaeta (63.5%) was found to be the only abundant genus. In addition, the archaeal community composition was shifted significantly in the adapted biomasses. The abundance of Methanosaeta was reduced drastically in both the AnSBRs during operation: under steady-state condition, in Ri, only 0.1% of the archaeal sequences were affiliated to Methanosaeta, as Methanosarcina and Methanohalophilus became the dominant groups, accounting for 23.6 and 41.0% respectively; while in Ra, the abundance of Methanosaeta dropped from 63.5 to 16.6%. According to previous literature, Methanosaeta and Methanosarcina are two abundant genera always reported from anaerobic treatment plants,57,58 where the Methanosaeta species are often observed to be the most predominant, especially in the seed sludge. However, it has been agreed that Methanosarcina could outcompete Methanosaeta in high ammonia or acetate environments.56 To understand the decreased abundance of Methanosaeta, the acetate concentrations were measured in both the AnSBRs under steady-state condition, and it was found that the acetate levels were within 130 mg/L, which was considered suitable for the growth of methanogens. Therefore, the high ammonia level (as shown in Table 1) could be responsible for the inhibition of Methanosaeta species. The resilience of genera Methanosarcina and Methanohalophilus could be one important reason for the superior treatment efficiency of Ri. Several studies have reported the domination of Methanosarcina in unfavorable wastewater environments, owing to its metabolic versatility in substrate utilization and its relatively high toxicity resistance.59,60 It has been documented that members of this genus can anaerobically degrade triethylamine (TEA),61 which is known as the major COD contributor in the pharmaceutical wastewater.25 In addition, S layer protein, which is responsible for enhanced chemical resistance, has been found in the cell wall of Methanosarcina other than Methanosaeta.62 On the other hand, members of Methanohalophilus have been found to be moderately halophilic, with optimum growth conditions in a NaCl solution with salinity of 0.5−2.5 M,63 which is similar to the salinity level of the pharmaceutical wastewater used in this study. In general, the valuable archaeal populations detected in the IWS in this study suggest the potential of IWS as an inoculation source, especially for saline industrial wastewater treatment.
Figure 4. Archaeal community composition between inocula and adapted biomasses. (A) Class-level distributions. Some classes were assigned to “other” if detected abundance was fewer than 2%. (B) Genus-level distributions of methanogenic populations. Some genera were assigned to “other” if detected abundance was fewer than 0.1%. The relative abundance of a given archaeal group is expressed as a percentage of the whole archaeal community.
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ASSOCIATED CONTENT
S Supporting Information *
Figures S1−S4. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.est.5b00546.
samples, class Thermoplasmata was found to be the subdominant group, which accounted for 7.7−29.7% of the total archaeal sequences. This observation illustrates the wide distribution and strong adaptation capability of this archaeal class, as well as its potential role in the anaerobic processes. An investigation of more specific taxa could offer a deeper understanding of microbial community functions. Genus-level distributions among methanogenic populations are further illustrated in Figure 4B. In the IWS inoculum, a highly diverse
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AUTHOR INFORMATION
Corresponding Author
*Phone: +65-6516 4777; fax: +65-6774 4202; e-mail:
[email protected]. Notes
The authors declare no competing financial interest. 6237
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DOI: 10.1021/acs.est.5b00546 Environ. Sci. Technol. 2015, 49, 6231−6239
