Bioaugmentation as a Solution To Increase Methane Production from

Article pubs.acs.org/est

Bioaugmentation as a Solution To Increase Methane Production from an Ammonia-Rich Substrate Ioannis A. Fotidis,† Han Wang,† Nicolai R. Fiedel,† Gang Luo,‡ Dimitar B. Karakashev,† and Irini Angelidaki*,† †

Department of Environmental Engineering, Technical University of Denmark, Building 113, DK-2800 Kgs. Lyngby, Denmark Shanghai Key Laboratory of Atmospheric Particle Pollution and Prevention (LAP3), Department of Environmental Science and Engineering, Fudan University, 200433 Shanghai, China

‡

S Supporting Information *

ABSTRACT: Ammonia-rich substrates inhibit the anaerobic digestion (AD) process and constitute the main reason for low energy recovery in full-scale reactors. It is estimated that many fullscale AD reactors are operating in ammonia induced “inhibited steady-state” with significant losses of the potential biogas production yield. To date there are not any reliable methods to alleviate the ammonia toxicity effect or to efficiently digest ammoniarich waste. In the current study, bioaugmentation as a possible method to alleviate ammonia toxicity effect in a mesophilic continuously stirred-tank reactor (CSTR) operating under “inhibited steady state” was tested. A fast growing hydrogenotrophic methanogen (i.e., Methanoculleus bourgensis MS2T) was bioaugmented in the CSTR reactor at high ammonia levels (5 g NH4+-N L−1). A second CSTR reactor was used as control with no bioaugmentation. The results derived from this study clearly demonstrated a 31.3% increase in methane production yield in the CSTR reactor, at steady-state, after bioaugmentation. Additionally, high-throughput 16S rRNA gene sequencing analysis showed a 5-fold increase in relative abundance of Methanoculleus spp. after bioaugmentation. On the contrary to all methods used today to alleviate ammonia toxicity effect, the tested bioaugmentation process performed without interrupting the continuous operation of the reactor and without replacing the ammonia-rich feedstock.

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INTRODUCTION Ammonia-rich substrates are toxic to methanogens and are the main reason for imbalance and inefficient energy recovery in anaerobic digestion (AD) plants.1 Substrates such as pig and chicken manure, slaughterhouse residues, and food industries waste, etc., contain high ammonia (ammonium + free ammonia) levels that result in suboptimal bioconversion to methane in biogas reactors.2 Free ammonia has been reported as the ammonia form causing inhibition of the methanogenic archaea by increasing the maintenance energy requirement, affecting the intracellular pH, depleting the intracellular potassium, and inhibiting specific enzyme reactions.3 Free ammonia concentrations increase alongside with temperature and pH increase.1 AD is mediated by different groups of microorganisms (i.e., protozoa, fungi, bacteria, and archaea), to catabolize the different organic substrates to biogas.4 The methanogenic archaea participating in the AD process belong to the Methanosarcinales order (Methanosarcinaceae family: versatile acetoclastic methanogens; Methanosaetaceae family: strictly acetoclastic methanogens), and to Methanomicrobiales, Methanobacteriales, Methanococcales, Methanocellales, and Methanopyrales orders (strictly hydrogenotrophic).5 Hydrogenotrophic methanogens are in syntrophic association with syntrophic © 2014 American Chemical Society

acetate oxidizing bacteria (SAOB) which oxidize acetate to CO2 and H2 followed by hydrogenotrophic methanogenesis.6 Among the microorganisms mediating the AD process, methanogenic archaea are the most sensitive ones with respect to ammonia toxicity.7 Especially, acetoclastic methanogens seem to be more liable to ammonia toxicity compared to hydrogenotrophic methanogens.3 Many full-scale anaerobic digesters are operating in an ammonia-induced “inhibited steady-state” with up to 30% losses of potential methane production yield.7 This apparently stable but suboptimal process results in serious operational problems and economic losses for the biogas plants.8 Various solutions have been proposed to solve the ammonia toxicity problem. To date, the two most common methods to tackle the problem are (a) to lower the operating temperature of the reactor and (b) to dilute the reactor content with water. However, besides that these methods can alleviate ammonia toxicity only to a limited extent, had been proven to be costReceived: Revised: Accepted: Published: 7669

April 7, 2014 May 27, 2014 May 29, 2014 May 29, 2014 dx.doi.org/10.1021/es5017075 | Environ. Sci. Technol. 2014, 48, 7669−7676

Environmental Science & Technology

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expensive, and do not provide a long-term solution.8 Other methods, like the addition of ammonium binding ions and increasing the C/N ratio are still far away from any practical applicability.9,10 Eventually, to secure an optimal biogas production, biogas plant operators minimize or totally exclude the ammonia-rich substrates from their feedstocks. Nevertheless, the necessity to digest ammonia-rich substrates and/or alleviate the ammonia toxicity events in anaerobic reactors remains. A new approach to meet this challenge could be the bioaugmentation of ammonia tolerant methanogenic consortia. Bioaugmentation is a process where specific microorganisms are added to a biological system to improve the operating conditions.11 Bioaugmentation has been used to recover reactors from organic overload, to improve the lipids’ digestion rates, and to decrease the lag phase during the start-up period of the AD process.11,12 However, there have been doubts, whether the bioaugmented organisms and the introduced property would be established in the bioaugmented system (washout effect).13 Additionally, it has been claimed by many researchers that the specific microorganisms and consequently the desirable property, would eventually establish by themselves (acclimation), so the bioaugmentation is not necessary.1,3 Bioaugmentation of ammonia tolerant methanogenic consortia to fed-batch reactors with high ammonia content has been shown previously.1 However, it is doubtful whether suspended growth could support establishment of the new microorganisms in the fed-batch reactor system. Furthermore, previous researches have revealed that it is technically difficult (if possible) to bioaugment ammonia tolerant syntrophic methanogenic consortia (acetate oxidizing bacteria in syntrophic association with a hydrogenotrophic methanogen) in continuous reactors.14,15 Fotidis et al.14 have shown that hydrogenotrophic methanogens are the rate limiting organisms of ammonia tolerant syntrophic consortia. Accordingly, hydrogenotrophic methanogens, and not the SAOB partner(s), define the outcome of the bioaugmentation process in the complex environment of a manure-fed continuous reactor. To overcome this bottleneck the aforementioned researchers hypothesized that for successful bioaugmentation the use of a minimum amount (“critical biomass”) of fast growing-ammonia tolerant hydrogenotrophic methanogens, was of outmost importance for a successful bioaugmentation. The major challenge of introducing microorganisms in a continuously stirred-tank reactor (CSTR) is to ensure that the introduced microorganisms are going to thrive and not to washout from the reactor. Hence, the minimum amount of specific methanogen’s biomass required to avoid washout during bioaugmentation in a continuous reactor was defined as “critical biomass”. Up to our knowledge, successful bioaugmentation of ammonia tolerant methanogens in manurefed continuous methanogenic reactors has not been previously reported. Therefore, the aim of the current study was to develop a novel bioaugmentation method to alleviate ammonia inhibition in a mesophilic CSTR reactor operating under stable but at suboptimal state (ammonia induced “inhibited steady-state”). Methanoculleus bourgensis MS2T (MC culture) was chosen,16 as a mesophilic fast growing hydrogenotrophic methanogen that can produce methane at high ammonia levels (5 g NH4+-N L−1). The methane production efficiency was evaluated in comparison to one nonbioaugmented (control) CSTR reactor with equally high (5 g NH4+-N L−1) ammonia levels.

Article

EXPERIMENTAL SECTION

Inoculum and Feedstock. The inoculum used in this study was obtained from a full-scale mesophilic anaerobic reactor (Hashøj Biogas, Denmark), fed with pig and cattle manure (70 to 90%) and organic waste (10 to 30%). The feedstock used in the experiment was dairy manure derived from Hashøj municipality (Denmark). The dairy manure was sieved to remove coarse materials and kept at 4 °C before it was introduced to the reactors. The characteristics of the feedstock and the inoculum used in the experiment are presented in Table 1. Table 1. Characteristics of the Inoculum and the Cattle Manure Used as Feedstock parameter (unit) density (g·L−1) TS (g·L−1) VS (g·L−1) total Kjeldahl nitrogen (g N L −1 ) ammonia (g NH4+-N·L−1) pH total VFA (g L−1) a

inoculum value ± SDa 1002.39 29.88 18.53 4.04

± ± ± ±

0.8 0.2 0.1 0.22

3.18 ± 0.16 7.88 2.19 ± 0.33

feedstock value ± SD 1004.45 56.01 41.70 2.74

± ± ± ±

1.2 0.001 0.001 0.102

1.65 ± 0.045 7.15 10.97 ± 0.93

Standard deviation.

Methanogenic Culture Used for Bioaugmentation. Methanoculleus bourgensis MS2T (MC culture, DSM 3045) was obtained from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures. MC culture was grown by fed-batch cultivation in modified sterile Methanogenium bourgense (MB) medium (the accumulate methane production of the MC culture is shown in Supporting Information, Figure S1). The MB medium used contained per liter of Milli-Q water: 19.11 g of NH4Cl, 0.4 g of K2HPO4-3H2O, 0.1 g of MgCl2.6H2O, 0.5 g of L-cysteine hydrochloride, 5 g of sodium formate, 1 g of sodium acetate, 1 g of yeast extract, 1 g of trypticase peptone, 1 mg resazurin, 1.5 g Na2CO3 and Na2S.9H2O.16 The pH was adjusted at 6.7−7.0 under a H2/ CO2 (80%/20%) headspace pressurized at 2 bar. All the bottles had total and working volume of 118 and 40 mL, respectively, and were incubated at 37 ± 1 °C. Experimental Setup. The experiment was carried out in two identical lab-scale mesophilic (37 ± 1 °C) CSTR reactors (RControl: high ammonia loaded control reactor, abiotic augmentation; and RMC: high ammonia loaded reactor, bioaugmentation). The two reactors had 2.3 and 1.8 L total and working volume, respectively, hydraulic retention time (HRT) of 24 days and organic loading rate (OLR) of 1.74 g VS·L−1·d−1, throughout the experiment. Each reactor’s setup consisted of a feed vessel, a feeding peristaltic pump, an effluent bottle, two magnetic stirrers for the homogenization of substrate and mixing of the reactor, a water replacement gas meter and an electrical heating jacketed unit. The two CSTR reactors were started-up with an ammonia level of 1.65 g NH+4N L−1, whereafter the ammonia concentration was increased stepwise in the reactors to 3, 4, and 5 g NH4+-N·L−1, respectively (data not shown). The ammonia at each step was kept at the same level for a minimum time of 30 days. NH4Cl was used as ammonia source. The whole experiment was divided to three distinct experimental phases. One HRT after ammonia concentration 7670

dx.doi.org/10.1021/es5017075 | Environ. Sci. Technol. 2014, 48, 7669−7676


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Figure 1. Methane production yield of the two CSTR reactors. Phase-I, before bioaugmentation and abiotic augmentation; phase-II, bioaugmentation and abiotic augmentation; and phase-III, after bioaugmentation and abiotic augmentation. Error bars denote standard deviation from the mean of triplicate measurements (n = 3).

in the reactors was increased to 5 g NH4+-N·L−1, an ammonia induced “inhibited steady-state” was established (Phase-I, days 1−12) for both reactors. It was assumed that the process was at steady-state when a variation of the methane yield was less than 10% for at least ten consecutive days. The bioaugmentation process of the MC culture in the RMC reactor was performed during phase-II (days 13−15) in two distinct steps (days 13 and 15, respectively). In each step 100 mL of the MC culture (OD600 = 0.17−0.19, μmax = 0.022 ± 0.001, under exponential growth phase) was introduced to the reactor. At the same time, 100 mL of sterile MB medium with 5 g of NH4+-N·L−1, was also introduced in reactor RControl twice (days 13 and 15), in order to replicate the same hydraulic effect (abiotic augmentation) that the volume of the bioaugmented inoculum had on the RMC reactor. At the end of the bioaugmentation and abiotic augmentation processes 11% (200 mL) of the working volume of both the RMC and RControl reactors was replaced by inoculum and sterile media, respectively. The mixing in CSTR reactors was stopped 2 h before and resumed 2 h after each bioaugmentation step to avoid the loss of active biomass and allow the MC culture to settle in the reactors. During phase-II and phase-III (days 16−57), the reactors were operated continuously and the ammonia concentration in the feedstock was kept at 5 g NH4+-N L−1. Finally, on day 12 (Phase-I) and on day 39 (Phase-III), samples for high-throughput 16S rRNA gene sequencing analysis were taken from both reactors. Analyses. Methane content in the headspace of the two CSTR reactors was measured with a gas-chromatograph (GCTCD) fitted with a column of 1.1 m × 3/16 “Molsieve 137 and 0.7 m × 1/4” chromosorb 108 (MGC 82-12, Mikrolab A/S, Denmark).17 Volatile fatty acids (VFA) accumulation in the CSTR reactors was determined with a gas-chromatograph (HP 5890 series II) equipped with flame ionization detector and a FFAP fused silica capillary column, (30 m × 0.53 mm i.d., film thickness 1.5 μm), with nitrogen as carrier gas.18 Total solids (TS), volatile solids (VS), total Kjeldahl nitrogen (TKN), total ammonia, and pH were determined according to APHA’s Standard Methods.19 The pH fluctuation in the CSTR reactors was measured with PHM99 LAB pH meter. The optical density at 600 nm (OD600) was determined with a Spectronic 20D+ spectrophotometer (Thermoscientific, Soeborg, Denmark). Maximum growth rate (μmax) of the MC culture was calculated

as has been described before.9 All statistical analysis was made using the Graphpad PRISM program (Graphpad Software, Inc., San Diego, California). Analysis of variance (ANOVA) was used for statistically significant difference (p < 0.05). Archaea High-Throughput 16S rRNA Gene Sequencing and Analysis. On day 12 (Phase-I, before bioaugmentation) and on day 39 (Phase-III, after bioaugmentation) samples from both reactors were retrieved, for elucidating any possible changes in the relative abundance of archaea populations. The samples were treated using QIAamp DNA Stool Mini Kit (Qiagen Inc., Mississauga, Canada, Cat No. 51504) for total genomic DNA extraction from each sample according to the manufacturer’s instructions.20 Nested PCR was used with 20f (5′-TTCCGGTTGATCCYGCCRG-3′) and 958r (5′-YCCGGCGTTGAMTCCAATT-3′) as primers for the first amplification round, and 344f (5′-ACGGGGYGCAGCAGGCGCGA-3′) and 519r (5′-GWATTACCGCGGCKGCTG-3′) as primers for the second amplification round. Taq PCR Core Kit (QIAGEN) was used for all PCR amplifications with 1 uL template DNA and 20 pmol of each primer. The PCR conditions for the first and second amplification was 94 °C for 2 min, 30 cycles of three steps; 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1.5 min; followed by a final step at 72 °C for 10 min. QIAquick spin columns (QIAGEN) was used for purification and removal of the excess primer dimers and dNTPs form the PCR products. Afterward, the samples were sent for barcoded libraries preparation and sequencing on an Ion Torrent PGM apparatus with 316 chip using the Ion Sequencing 200 kit (all Life Technologies, Inc., Paisley, United Kingdom) according to the standard protocol (Ion XpressTM Plus gDNA and Amplicon Library Preparation, Life technologies).21 The sequencing results were deposited into the MGRAST metagenomic analysis server (http://metagenomics.anl. gov/) under the IDs: 4539366.3−4539369.3. The corrupted sequences, with length shorter than 100 bp, containing any ambiguous base calls and without exact matches to the forward and reverse primers, were removed from the raw sequencing data by RDP tools.22 The “Decipher Find Chimeras” web tool (http://decipher.cee.wisc.edu/FindChimeras.html) was used for chimeric sequences detection and exclusion.23 The numbers of high quality sequences and the average base pairs (bp) per sequence are presented in Supporting Information, Table S1. 7671



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This small improvement in the methan production of the RControl reactor could indicate that methanogenic populations were, to some extent, slowly adapting to the increased ammonia concentrations.1,27 RMC reactor demonstrated immediately after bioaugmentation (day 19), a significant improvement (p < 0.05) in methane production (16.7%) compared to the baseline steady state (Phase-I). This improvement led to the final steady-state (days 33−57) with the RMC operating continuously for more than one HRT with 31.3% higher methane production yield compared to the previous steady-state (days 1−12; Figure 2). Additionally, comparing directly the RMC and RControl during the final steady state (days 33−57), the RMC had an average of 31.8% higher methane production yield. Previous attempts to bioaugment ammonia tolerant methanogenic consortia in labscale continuous anaerobic reactors under high ammonia levels had no effect on methane production yield of the reactors.14,15 To our knowledge, this is the first time that bioaugmentation of an ammonia tolerant methanogen had such a fast and profound improvement in methane productivity of a CSTR reactor fed with ammonia-rich manure. Furthermore, the current bioaugmentation process was performed without interrupting the continuous operation of the reactor and without changing the ammonia-rich feedstock. This is an entirely new approach to solve the ammonia toxicity problem in anaerobic reactors compared to all the methods used today.8 The hypothesis that a “critical biomass” of ammonia tolerant methanogens is the key to successful bioaugmentation in continuous reactors14 seems to hold merit. However, since “critical biomass” is the minimum amount of ammonia tolerant inoculum needed to promote the desired microbial activity in a reactor, further experiments are necessary to determine whether smaller inoculum volumes can result in successful bioaugmentation in continuous reactor systems. Nevertheless, these results are very promising and could be potentially used for the development of an efficient continuous biomethanation process of ammonia-rich substrates in full-scale reactors. VFA Accumulation and pH Fluctuation. The significant difference (p < 0.05) in total VFA concentrations between the two reactors, during the final steady-state (days 33−57) at 5 g NH4+-N L−1, coincided with the methane production findings. Specifically, the VFA accumulation of the RMC reactor after bioaugmentation (Figure 3), was kept stable and within the limits for normal AD of dairy slurry in CSTR reactors.26 VFA accumulation in RControl reactor for the same period (after abiotic augmentation) evolved as expected for reactors subjected to ammonia inhibitory pressure.15 In the RControl reactor VFA levels were increased from 1.5 to 2.3 g HAc L−1 during phase-III and in combination with the reduced methane productivity, manifested a typical “inhibited steady state” (days 33−57). A VFA concentration threshold of 1.5 g HAc L−1 has been identified by previous researchers2,7,28 for a healthy AD process in both lab-scale and full-scale CSTR reactors. VFA levels in the RControl reactor remained over that threshold after abiotic augmentation addition of the cultivation medium, indicating a nonhealthy AD process. On the contrary, RMC reactor’s VFA levels after bioaugmentation stayed below 1.5 g HAc L−1 until the end of the experiment. Therefore, it became clear that bioaugmentation of the ammonia tolerant MC culture enhanced the yield of RMC reactor and led to lower VFA levels compared to RControl reactor. The pH levels in the two reactors was stabilized around 7.7 during the final steady-state. Contrary to expectations, VFA accumulation, which was the consequence

The numbers of archaea sequences were normalized to the same sequencing depth (52000 sequences) to simplify the comparison between the different samples by the MOTHUR program.24 “RDP Classifier” was used to phylogenetically assign the sequences for taxonomic classifications with a bootstrap cutoff of 50%.25 the MOTHUR program was used to cluster the sequences into operational taxonomic units (OTUs) by setting a 0.03 distance limit. Furthermore, the MOTHUR program was also used to generate rarefaction curves, species richness estimator of CHAO 1, Shannon diversity index, diversity coverage, Venn diagrams comparing the number of overlapping OTUs among the samples and dendrograms based on Bray−Curtis similarity matrix, as described before.21 Finally, high-throughput 16S rRNA gene sequencing was performed also for the bacteria in the CSTR reactors, but the data are not presented in this study since this research was focused on the changes in the relative abundance of the methanogens due to bioaugmentation.

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RESULTS AND DISCUSSION Reactors Performance. Phase-I was used as a baseline for the determination of any changes in methane production yield of the reactors RControl and RMC. Fang et al.26 have reported that the methane production yield of the continuous anaerobic digestion of dairy slurry derived from Hashøj municipality (as the substrate used in the current experiment) was 250 mL of CH4 g−1 VS under steady-state. Thus, throughout phase-I both reactors were at “inhibited steady-state” producing 23−29% less methane compared to the expected methane production yield assuming that the manure we used had the same characteristics as the one from the literature (Figure 1). During bioaugmentation (Phase-II) the methane production yield was not affected since the operational parameters of the reactors were kept stable (e.g., HRT, OLR, temperature). Thus, ammonia-rich manure was fed continuously into the reactors maintaining the same high ammonia levels (5 g NH4+-N L−1). This was in contrast to the conventional methods used to date (dilution, lower temperature, etc.) to alleviate ammonia toxicity in AD reactors affected by ammonia toxicity.8 In phase-III, the control reactor RControl presented an average of 4.5% increase (p > 0.05) in methane production compared to phase-I (Figure 2).

Figure 2. Average methane production yield comparison under different steady-states before and after abiotic augmentation and bioaugmentation for reactors RControl and RMC, respectively. 7672



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Figure 3. Total VFA accumulation in CSTR reactors. Phase-I, before bioaugmentation and abiotic augmentation; phase-II, bioaugmentation and abiotic augmentation; and phase-III, after bioaugmentation and abiotic augmentation. Error bars denote standard deviation from the mean of triplicate measurements (n = 3).

of the current experiment revealed that there was a limited amount of archaea species mediating methanogenesis in the two reactors before and after bioaugmentation and abiotic augmentation. Nevertheless, the Shannon diversity index also provides the evenness of the species among all the species in the community.21 Thus, the diversity was increased by 56.5% in the RMC(a) sample compared to the RMC(b) sample. On the contrary, diversity of the RControl reactor was slightly decreased by 14.8% after abiotic augmentation, compared to the RControl(b). Hence, from the samples’ diversity analysis, it can be concluded that bioaugmentation of the MC culture clearly and quickly (in only one HRT) enhanced the diversity in the RMC reactor. The rarefaction curves used to assess whether disparity differences between the four samples were robust to sample size differences. Rarefaction curves at 0.03 distances calculated from the observed OTUs were not reaching plateau phase (Supporting Information, Figure S1). These findings suggest that the archaea sequencing depth (52000) was not enough to cover the whole communities’ diversity and hence some minor groups were missed. However, the coverage values for archaea (≥99%) indicated that most common OTUs were detected in the current study. Overall, the results suggest that abundant taxonomic groups were well covered in the current sample, while a sufficient coverage of minor groups would require a considerable larger sample size, as has been suggested before for complex AD environments.35 Archaea Species Distribution and Relative Abundance. The evaluation of the Venn diagrams showed that only the 19% of the total 715 archaea species found mediating the AD process were common in the samples before (RMC(b)) and after (RMC(b)) bioaugmentation in the RMC reactor (Supporting Information, Figure S2a). The different species belonging to RMC(a) and not to RMC(b) might be related to the methane production yield improvement after bioaugmentation. At the same time, 23.9% of the 644 species were shared between groups RControl(b) and RControl(a) before and after abiotic augmentation, respectively (Supporting Information, Figure S2b). Furthermore, the total number of species was reduced by 16.5% in the sample after abiotic augmentation, compared to the sample prior. This reduction in the archaea species number mediating the AD process in the control reactor can be

of the ammonia toxicity effect, did not reduce the pH in RControl.29 That could be attributed to the strong buffer capacity of the manure used as feedstock in the experiment.26 Nevertheless, pH levels remained for both reactors inside the favorable range for the AD process throughout the experiment.30 Archaea High-Throughput 16S rRNA Gene Sequencing and Analysis-the Bioaugmentation Effect. The archaea species richness in the RMC reactor after bioaugmentation (RMC(a)) was 71% higher compared to the same reactor before bioaugmentation (RMC(b)) which was underlined by the big difference of OTUs and CHAO 1 numbers between the two samples (Table 2). Conversely, the species richness in the Table 2. Comparison of the Richness and Diversity of the 16S rRNA Gene Libraries Based on 0.03 Distance samples

OTUs

coverage (%)

CHAO1 richness estimation

Shannon diversity

RControl(a)a RControl(b)b RMC(a) RMC(b)

273 275 427 265

100 100 99 100

939 814 1497 874

0.23 0.27 0.36 0.23

a

After (a) bioaugmentation or abiotic augmentation samples. bBefore (b) bioaugmentation or abiotic augmentation samples.

RControl sample after abiotic augmentation (RControl(a)) was increased only by 15% (statistically significant, p < 0.05) compared to the sample before abiotic augmentation (RControl(b)) and the OTUs numbers remained similar (Table 2). As the two reactors RControl(a) and RMC(a) were operated identically, except for the bioaugmentation, we could conclude that the reason for the significant increase of species richness in the RMC reactor was caused by the addition of MC culture. The Shannon diversities of the four samples (Table 2), which is an indicator for the amount of information (entropy) in microbiological systems,31 were smaller compared with diversities reported in other studies testing different AD environments.32,33 Therefore, it seems that high ammonia levels (5 g NH4+-N L−1) were the major reason for the reduced archaea diversity in the current study versus the aforementioned investigations.34 The small diversities of the four samples 7673



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before and after bioaugmentation and abiotic augmentation. Specifically, Methanoculleus spp. increased its relative abundance among the archaea species mediating the AD process in the RMC reactor after bioaugmentation compared to the sample before bioaugmentation. Although, the relative abundance of Methanoculleus spp. was still smaller than the dominant Methanosarcina spp., it seems that it was sufficient to establish a change in the archaea community and consequently to increase the methane production yield of the reactor. It appears that the bioaugmentation of the MC culture, also triggered the growth of other ammonia tolerant hydrogenotrophic methanogens (i.e., Methanobrevibacter spp.).37 The most possible explanation is a “microbiological domino effect” triggered by the bioaugmentation of the MC culture. The MC culture reduced the hydrogen partial pressure in the reactor creating thermodynamically favorable conditions for the syntrophic acetate oxidation pathway which consequently enabled the growth of more hydrogenotrophic methanogens (i.e., Methanobrevibacter spp.).6 Hence, the hydrogenotrophic methanogenic pathway, which is more robust to ammonia toxicity effect,1 took over a fragment of the total methane production in the reactor. Finally, an additional factor contributing to the increased methane production in RMC appears to be the reduction of the ammonia−VFA synergistic effect7,38 caused by the VFA decrease (Figure 2) due to increased hydrogenotrophic (coupled with acetate oxidation) activity. Specifically, the reduced ammonia−VFA synergistic effect on the methanogens allowed the less ammonia-tolerant methanogens (e.g., Methanosarcina spp.) to produce methane more efficiently. Thus, the 31.3% increase in methane production after bioaugmentation was most probably derived from both the hydrogenotrophic methanogens and the less stressed aceticlastic methanogens. On the contrary to that in the RMC(a) sample, after abiotic augmentation in RControl the relative abundance of Methanoculleus spp. was stable and only Methanobrevibacter spp. increased its relative abundance (Figure 4b). It is known that the time-consuming process of acclimatization of methanogenic populations to high ammonia levels can occur in the anaerobic reactors.39 Therefore, the change in the relative abundance of the Methanobrevibacter spp. was due to slow ammonia acclimatization process which, according to the productivity of the reactor, was not enough to significantly alleviate the ammonia toxicity effect. Additionally, the deep sequencing analysis shown that the relative abundance of known SAO bacteria genera significantly increased in the RMC(a) compared to RMC(b) (data not shown). In conclusion, the high-throughput 16S rRNA gene sequencing results amply support the opinion that the MC culture was successfully bioaugmentated in the CSTR reactor. Thus, methane production increased immediately without changing the feedstock (ammonia-rich manure) or the operational parameters (i.e., temperature, OLR, HRT) of the CSTR reactor.

attributed to the ammonia toxicity effect on the nontolerant to ammonia methanogenic populations.27,34 The four samples were also assessed by generating a dendrogram from Bray−Curtis similarity matrix analysis taking into account the sequences abundance in each sample (Supporting Information, Figure S3).36 Bray−Curtis matrix showed that reactor RMC after bioaugmentation, displayed a different archaea community structure compared to the community profile of the same reactor before bioaugmentation and also compared to the profiles of the control reactor before and after bioaugmentation. From the remaining three samples, RMC(b) and RControl(a) were clustered together and RControl(b) was in close proximity with the other two. The similarity analysis indicated that bioaugmentation of the MC culture and not the high ammonia levels, was the decisive process parameter determining the archaea community structure in RMC. The sequences of archaea from the four samples were assigned to the order and genus level as shown in Figure 4. The

Figure 4. Taxonomic classification of the archaea communities (a) Order and (b) Genus. (b) Before bioaugmentation and abiotic augmentation samples and (a) after bioaugmentation and abiotic augmentation samples. Relative abundance was defined as the number of sequences affiliated with that taxon divided by the total number of sequences per sample. Individual genera making up less than 0.1% of total composition in all samples are summed and indicated as “Others”.

archaea mediating acetoclastic and hydrogenotrophic methanogenesis were found mainly within Methanosarcinales, Methanomicrobiales, and Methanobacteriales orders (Figure 4a). Acetoclastic Methanosarcina spp. was the dominant genus (>88%) in all the four samples (Figure 4b). Calli et al.34 also reported increased abundance of Methanosarcina spp. under high ammonia levels accompanied by great reduction in methane production. The strict acetoclastic methanogen Methanosaetaceae spp. was not found in any of the four samples which can be attributed to the high ammonia and/or acetate levels and the manure-based feedstock used in the reactors.7,27 In addition to the similarities, archaea relative abundance showed significant differences among the samples

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ASSOCIATED CONTENT

S Supporting Information *

Number of high quality sequences (Table S1), accumulate methane production MC culture (Figure S1), rarefaction curves (Figure S2), Venn diagrams (Figure S3), and dendrogram based on Bray−Curtis similarity matrix (Figure S4). This material is available free of charge via the Internet at http:// pubs.acs.org. 7674



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AUTHOR INFORMATION

Corresponding Author

*Phone: +45 45251429; fax: +45 45933850; e-mail: iria@env. dtu.dk. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

The current work was supported by Energinet.dk under the project framework ForskEL “Innovative process for digesting high ammonia wastes” (Program No. 2010-10537) and by the Bioref−Øresund project under EU INTERREG IVA.

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