Article pubs.acs.org/EF
Effects of Ammonia on Anaerobic Digestion of Food Waste: Process Performance and Microbial Community Hong Chen,† Wen Wang,*,† Lina Xue,† Chang Chen,† Guangqing Liu,† and Ruihong Zhang‡ †
Biomass Energy and Environmental Engineering Research Center, Beijing University of Chemical Technology, Beijing 100029, China ‡ Department of Biological & Agricultural Engineering, University of California, Davis, California95616, United States ABSTRACT: The effects of ammonia on semicontinuous anaerobic digestion (AD) of food waste were studied. Inhibition effects were observed when the total ammonium concentration in the AD reactor exceeded 2g/L. Ammonia strongly inhibited methanogenesis but minimally affected hydrolysis and acidification. Inhibition of AD by ammonia enhanced the accumulation of acetate and propionate and consequently decreased the pH, which worsened the inhibition effects. With increasing ammonium concentration, methane production from acetate shifted from acetoclastic methanogenesis (mostly by Methanosaeta) to syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis (mostly by Methanosarcina). The metabolism of hydrogenotrophic methanogens Methanobacterium and Methanospirillum were inhibited when the ammonium concentration further increased to about 6 g/L. Microorganisms irrelevant in the methane fermentation process were enriched with the increase of ammonium concentration. inhibit methanogens and reduce biogas production.8,9 Sung and Liu10 reported that methanogenic activity in soluble nonfat dry milk digestion was enhanced at TAN concentrations lower than 1500 mg/L, whereas methanogenesis was evidently inhibited at TAN concentrations higher than 4000 mg/L (thermophilic AD with HTR of 7d). Similarly, Angelidaki and Ahring11 found that the inhibition threshold concentration of ammonia ranged from 3000 mg/L to 4000 mg/L during thermophilic AD of livestock waste. However, Chen et al.6 reported that TAN concentrations ranging from 1700 mg/L to 14000 mg/L all decreased methane yield by more than 50%. Some other researchers found that the inhibition effect only occurred at ammonium concentrations higher than 6000 mg/ L.12,13 The discrepancies in ammonium threshold inhibition concentrations could be due to different substrates, inocula, operation parameters, and acclimation conditions employed in the studies. Microorganisms in AD are sensitive to environment conditions. The performance and efficiency of AD are directly affected by the structure of microorganisms. Leclerc et al.14 revealed that the development of microbial communities is strongly influenced by operating conditions, reactor designs, and substrate compositions. Methanogens are the most sensitive to ammonia among different microorganisms.15 Some researchers found that hydrogenotrophic methanogens are more robust than acetoclastic methanogens.16−19 However, several studies concluded that hydrogenotrophic methanogens are more sensitive to ammonia compared with acetoclastic methanogens.16,20 To date, the known mechanisms through which ammonia affects microorganisms in AD are complex and vary among different substrates and operational conditions.
1. INTRODUCTION Improper disposal and highly perishable characteristics of food waste (FW) have caused serious environment pollution in several countries; as such, economical and efficient FW management must be developed. In Beijing, China, a new regulation was issued stating that the costs of disposal of kitchen waste and other refuse should be charged separately. In this regulation, nonresidential buildings, including companies, office buildings, restaurants, and universities, need to pay 25 RMB for each ton of kitchen waste.1 Anaerobic digestion (AD) is a complex biological process that has been studied for more than 100 years; this process has attracted much interest in the last three decades for applications in organic substrate treatment to produce renewable energy in the form of biogas and control greenhouse gas emissions.2 FW contains abundant organic compounds and nutrients, which exhibit great potential to produce biogas with high methane content through AD. Economists confirmed that AD is an economically feasible technology for simultaneous waste treatment and energy recovery.3 Nitrogen has been identified as an important factor during AD.4,5 Biological degradation of N-rich organic compounds, such as proteins, nucleic acids, and urea, may result in accumulation of total ammonium nitrogen (NH3−N; TAN) in the form of free ammonium nitrogen (NH3; FAN) and ionized ammonium nitrogen (NH4+). The forms of ammonium nitrogen are affected by pH and temperature during organic waste treatment.6 Suitable concentrations of ammonium nitrogen can supply nitrogen as nutrient and ensure sufficient buffering capacity for AD systems. However, high concentrations of ammonia could lead to the failure of AD as a result of inhibited microbial activities.7 Researchers reported that AD could operate normally at TAN concentrations ranging from 600 mg/L to 800 mg/L (at pH 7.2−7.5 under mesophilic condition); conversely, higher TAN concentrations could © 2016 American Chemical Society
Received: March 28, 2016 Revised: June 5, 2016 Published: June 8, 2016 5749
DOI: 10.1021/acs.energyfuels.6b00715 Energy Fuels 2016, 30, 5749−5757
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NH4Cl to the corresponding ammonium concentrations. In the above experiments, the headspace of each bottle was purged with nitrogen gas to create an anaerobic environment, and then each bottle was sealed with a rubber stopper carrying a tube connected to a 2 L aluminum foil gas-collecting bag. The bottles were put in a reciprocating air bath shaker with a shaking speed of 90 rpm at 37 ± 1 °C. The volume of the biogas was measured with a syringe every day, and the composition of the biogas was analyzed as collected. Liquid samples were also collected and characterized periodically. 2.3. High-throughput 16S rRNA gene sequencing and analysis. Samples of the digestate in reactor R0, R3, and R6 with different ammonium concentrations were collected in phase II at the 78th day to explore the changes in the microbial community composition. Total genomic DNA was extracted using Omega D5625-01 soil DNA kits (Omega Bio-Tek, USA). The extracted DNA was qualitatively checked by agarose gel electrophoresis and then stored at −20 °C for subsequent use as a template in PCRamplifications. PCR was performed in a 50 μL (total volume) reaction mixture with 5 μL of each deoxynucleotide triphosphate (10 mM), 0.5 μL of Taq polymerase (5 U/μL), 50 pmol of each primer, and 0.5 μL of the extracted DNA. 341f (5′-CCTACGGGAGGCAGCAG-3′) and 805r (5′-GACTACCAGGGTATCTAATC-3′) were used as primers for bacteria. The PCR conditions for bacteria were: 94 °C for 2 min, 5 cycles of three steps (94 °C for 0.3 min, 45 °C for 0.3 min, and 65 °C for 1 min), 20 cycles of three steps (94 °C for 0.3 min, 60 °C for 0.3 min, and 72 °C for 0.3 min), followed by a final step at 72 °C for 5 min. Nested PCR was performed for the archaea, 109f (5′ACTGCTCAGTAACACGT-3′) and 1492r (5′-CGGCTACCTTGTTACGAC-3′) were used for the first round of amplification, and the same 109f and 519r (5′-GWATTACCGCGGCKGCTG-3′) were used for the second round. For the first round, the conditions were: 94 °C for 2 min, 35 cycles of three steps (94 °C for 1 min, 51 °C for 1 min, and 72 °C for 1 min), followed by a step at 72 °C for 10 min. For the second round, the conditions were: 94 °C for 3 min, 30 cycles of three steps (94 °C for 1 min, 53 °C for 1 min, and 72 °C for 2 min), followed by a final step at 72 °C for 10 min. The PCR products were further purified using the SAGON SK8131 kits (Sangon, China) to remove excess primer dimers and dNTPs, and after quantification the samples were sent for preparation of barcoded libraries. The samples were sequenced on an Illumina MiSeq platform according to standard protocols. The low-quality sequences without exact matches to the forward and reverse primers, with length shorter than 350 bp, and containing any ambiguous base calls, were removed from the raw sequencing data by Ribosomal Database Project (RDP) tools. Chimeras were removed from the data by using the Find Chimeras web tool. The sequencing yielded 30031 (R0), 30167 (R3), and 27568 (R6) high quality sequences for bacteria with an average length of 442 bp, and 24181 (R0), 26038 (R3), and 34085 (R6) high quality sequences for archaea with an average length of 416bp. In order to facilitate comparison between different samples, the numbers of the sequences were normalized to the same sequencing depth (Bacteria 27568 sequences, Archaea24181 sequences) with the MOTHUR program. The sequences were phylogenetically assigned to taxonomic classifications with the RDP Classifier with a confidence threshold of 50%. The sequences were clustered into operational taxonomic units (OTUs) by setting a 0.03 distance limit with the MOTHUR program. Rarefaction curves, the Shannon diversity index, the species richness estimator of Chao1, and diversity coverage were also generated by the MOTHUR program. Relative abundance was defined as the number of sequences affiliated with that taxon divided by the total number of sequences per sample. Phylum, order, and genus making up less than 1% of the total composition in all three samples were classified as “others”. 2.4. Analytical methods. After centrifugation at 10,000 rpm for 10 min, the supernatant of the samples was immediately analyzed as the soluble fraction. The pH value was read by a le438 pH electrode (Mettler Toledo, USA). Total solid (TS), volatile solid (VS), chemical oxygen demand (COD), total nitrogen, and TAN were determined according to standard methods.21 Elemental contents (C, H, N, S) were measured with an organic elemental analyzer (vario EL cube,
Although scholars investigated specific genera of bacteria and archaea affected by ammonia, only a few reports have focused on the diversity and structure of the entire microbial community. Therefore, the inhibition effects of ammonia on AD of FW and the underlying mechanisms must be elucidated. This study mainly aims to explore the mechanism of ammonium inhibition on AD of FW. The methane production and FW degradation performances were studied. Changes in the microbial community diversity and structure under different ammonium concentrations were examined through highthroughput sequencing of the 16S rRNA genes.
2. MATERIALS AND METHODS 2.1. Substrate and inoculum. FW was collected from a canteen of Beijing University of Chemical Technology. After sampling, inorganic components (such as plastics, bones, and toothpick) were removed manually. Then, after being shredded to smaller sizes and homogenized, the FW was stored at 4 °C before use to prevent biological decomposition. Inoculum used in this study was the mesophilic anaerobic digested sludge taken from a biogas station (with sewage sludge from a wastewater plant as substrate, HRT of 10 days, and temperature of 37 °C) in Shunyi District, Beijing, China. Before the inoculum was used, it was put in a 37 °C incubator for degassing. The characteristics of the substrate and inoculum are shown in Table 1.
Table 1. Characteristics of Food Waste as Substrate and of Sludge as Inoculuma
a
Parameters
Units
Food waste
Sludge
TS VS pH Alkalinity TCOD SCOD Total nitrogen Soluble protein Total ammonia nitrogen Element contents N C H S O
b
24.30 ± 2.11 22.50 ± 1.32 5.02 ± 0.03 2.25 ± 0.14 181.05 ± 0.24 103.53 ± 0.31 5.95 ± 0.13 33.75 ± 0.22 96 ± 3.5 2.31 ± 0.42 53.39 ± 1.22 6.93 ± 0.71 0.22 ± 0.01 29.75 ± 0.25
5.10 ± 0.23 2.78 ± 0.25 8.48 ± 0.02 ND ND ND ND ND 124 ± 15 2.85 ± 0.24 31.33 ± 1.25 4.23 ± 0.47 ND ND
% %b g/L g/L g/L g/L g/L mg/L %TS % TS % TS % TS % TS
ND: not determined. bAs total weight of sample.
2.2. Experimental design and procedures. The experiments were conducted using seven identical lab-scale anaerobic bottles as reactors with a working volume of 400 mL under mesophilic conditions. The operation mode was changed to semicontinuous after methane production was ceased in the first batch, and operated as a CSTR. The reactors were fed every 24 h with pretreated FW (20 mL of fresh mixing substrates, containing 2.47 g of pretreated FW and 17.53 g of distilled water), and an equivalent amount of the digestate was removed, which corresponded to an organic loading rate (OLR) of 1.5 g TS/L/d and a hydraulic retention time (HRT) of 20 days. The reactors were operated under the same conditions for about three HRTs with steady and similar biogas production performances (phase I), which reflected that similar microbial communities were established in all reactors. After 54 days, a sudden increase of TAN concentration was manipulated in each reactor, and different ammonium concentrations of 0, 0.5, 1, 2, 3, 4, and 5 g/L were adjusted by the addition of NH4Cl in different reactors labeled as R0, R1, R2, R3, R4, R5, and R6, respectively. At each concentration, the reactors were operated in triplicate. Afterward, the reactors were semicontinuously operated for 24 days (phase II), and the influent was also adjusted with 5750
DOI: 10.1021/acs.energyfuels.6b00715 Energy Fuels 2016, 30, 5749−5757
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Figure 1. Methane yields and methane contents during AD of FW with different ammonium concentrations. (Phase I: without NH4Cl addition; Phase II: with different concentrations of NH4Cl addition in R0−R6.) Germany). The element content of “O” was estimated by assuming C + H + O + N + S = 100% on a VS basis. The Lowry−Folin method was used to determine soluble protein concentrations using bovine serum albumin as the standard.22 Soluble carbohydrate concentrations were measured by the phenol sulfuric acid method using glucose as the standard.23 Alkalinity was measured by using a Hach digital titrator with 1.6 N sulfuric acid and bromcresol-green methyl-red indicator powder; the result was given in units of mg/L as CaCO3. Biogas (CH4 and CO2) compositions were determined with a gas chromatograph (Agilent, 7890B) equipped with a thermal conductivity detector and an analytical column of Agilent Hayesep Q. The operation temperature at the column oven and the detector were 60 and 220 °C, respectively. Helium was used as the carrier gas at a constant pressure of 5 psi. The concentrations of ethanol and VFA (acetate, propionate, isobutyrate, n-butyrate, isovalerate, n-valerate) were determined with a gas chromatograph (Agilent, 7890A) equipped with a flame ionization detector and a DB-wax capillary column (30 m × 530 μm × 1.0 μm). The temperature of the injector and the detector were 200 and 250 °C, respectively. Nitrogen was used as the carrier gas with a flow rate of 10 mL/min. The GC oven was programmed to begin at 55 °C for 1 min, increase at a rate of 30 °C/min to 110 °C, hold at 110 °C for 1 min, increase at a rate of 30 °C/min to 220 °C, and hold at 220 °C for 1 min. The injection volume was 1.0 μL. 2.5. Data analysis. The free ammonium concentrations were calculated based on eq 1.
⎛ ⎞−1 [NH3 (mg/L)] 10−pH = ⎜1 + 2729.92 ⎟ [NH3−N (mg/L)] ⎝ 10−(0.09018 + T(K) ) ⎠
An analysis of variance (ANOVA) was used to test the significance of the results, and p < 0.05 was considered to be statistically significant.
3. RESULTS AND DISCUSSION 3.1. Effects of ammonia on methane production. Methane yields (MYs) in the reactors are shown in Figure 1a. After the first 5 days, MY slowly increased and became stable for ∼2.5 HRTs before ammonium addition. On the 54th day, NH4Cl was fed to total concentrations of 0.5, 1, 2, 3, 4, and 5 g/L in reactors R0, R1, R2, R3, R4, R5, and R6, respectively. Methane production was intensely inhibited in R6 on the day when the TAN concentration was increased to 5 g/L. The MYs in R3, R4, and R5 with ammonium concentrations of 2 g/L to 4 g/L remained stable for 5−10 days of operation and gradually declined after 10 days. After 1 month of operation, AD in R3, R4, R5, and R6 was seriously affected by addition of ammonia and methane production was terminated. By contrast, the MYs in R0, R1, and R2 remained at approximately 400 mL/g VSadded. Therefore, with FW as the substrate, the ammonium concentrations in the AD reactors should be controlled below 2 g/L to achieve optimal methane fermentation performance. The biogas was mainly composed of methane and carbon dioxide, and the daily methane contents upon addition of different concentrations of NH4Cl are shown in Figure 1b. All the reactors exhibited similar methane contents of ∼50% prior to addition of NH4Cl. The methane content in R3 to R6 began to decrease at different rates after the addition of NH4Cl. The methane content in R6 started to decrease on the 56th day once NH4Cl was introduced into the reactor, whereas the methane contents in R5, R4, and R3 started to decrease on the 61st, 63rd, and 69th days, respectively. The profiles of methane content in the biogas were similar to the patterns of the MYs.
(1)
The methane production efficiency was evaluated using the methane content in the biogas (%) and the methane yield (MY) (the calculated methane production per gram VS, mL/g-VSadded). The gas volume reported in this study was calibrated to standard temperature (273 K) and pressure (1 atm). 5751
DOI: 10.1021/acs.energyfuels.6b00715 Energy Fuels 2016, 30, 5749−5757
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Energy & Fuels Table 2. Summary of the Reactor Performances in Phase I and Phase II Phase II (at the 78th day)
pH SCOD (mg/L) Soluble protein (mg/L) Soluble carbohydrate (mg/L) Total VFA/Ethanol (mg/L) Propionate/Acetate ratio Total ammonia (mg/L) Free ammonia (mg/L)
Phase I (R0−R6, steady state)
R0
R1
R2
R3
R4
R5
R6
7.20−7.25 800−1000 300−400
7.20 ± 0.02 798 ± 12 365 ± 6
7.15 ± 0.02 768 ± 10 328 ± 4
7.11 ± 0.03 825 ± 11 340 ± 7
5.77 ± 0.01 4200 ± 57 431 ± 6
5.46 ± 0.02 4900 ± 45 471 ± 5
5.36 ± 0.02 5689 ± 69 447 ± 4
4.69 ± 0.01 8500 ± 186 521 ± 6
80−100
77 ± 3
84 ± 2
89 ± 4
94 ± 4
103 ± 3
123 ± 3
212 ± 5
40% and dominated in R6. In this study, Phylum Firmicutes mainly consisted of the Order Clostridiales, which comprised Clostridium-related species. Some Clostridium-related species were previously reported to cleave acetate to CO2 and H2 through the SAO pathway. Clostridium IV and Clostridium sensustricto accounted for 0.02% and 3.79% in R0 and then increased to 1.62% and 5.46% in R3 as well as 4.31% and 10.04% in R6 (Figure 5). Both species may have the ability to transform acetate into H2 and CO2,40 thereby enriching the SAO-HM pathway. To date, only three mesophilic SAO bacteria have been reported; these species include Clostridium ultunense strain BST, Syntrophaceticusschinkii, and Tepidanaerobacteracetatoxydans.41 None of these species were observed in the present study. Nevertheless, some unclassified sequences could not be assigned to any known phylum, order, or genus; the percentage of these “other” species represented less than 1% of the total composition. Within these “other” species, some species may mediate the SAO pathway. The results obtained in this study by sequencing of the 16S rRNA genes can only provide a semiquantitative analysis of the microbial community structures due to PCR bias in this study.42 Therefore, future studies, such as direct metagenomic sequencing of the total genomic DNA extracted from the samples, could be performed to eliminate PCR bias; to obtain information about these low-percentage species and to identify the unknown bacterial species, the functional gene diversity of the microbial communities could be obtained simultaneously.43
bacterium (19.9%) and Methanospirillum (9.53%) were the two other main genera in R0; and their percentages increased to 42.73% and 15.54% in R3, which could be attributed to suppression of the acetoclastic pathway when the ammonium concentration was increased to 2 g/L. These phenomena implied that high ammonium concentrations suppressed the acetoclastic pathway and Methanosaeta activity but enhanced the SAO-HM pathway. These results are consistent with previous reports that the total ammonium concentration is the predominant inhibition factor for acetoclastic methanogens.32 Calli et al.27 found that acetoclastic methanogens are more sensitive to the presence of ammonia than hydrogenotrophic methanogens. Nevertheless, when the ammonium concentration was further increased to 5 g/L in R6, unlike Methanosarcina, the metabolism of Methanobacterium and Methanospirillum was inhibited, and their corresponding percentages decreased to 5.27% and 8.67%, respectively. The slight microdiversity variation of different archaea reflected the accumulation of VFAs, as illustrated in Figure 2. In AD, longer chain VFAs were converted into acetate before their conversion to methane; thus, the accumulation of acetate and propionate to high concentrations indicated that acetoclastic methanogens were inhibited. With VFA accumulation, the acetate consumption capacity of Methanosaeta would be further suppressed. Conklin et al. 33 reported that Methanosaeta was inhibited at high acetate levels and consequently promoted SAO-HM. Furthermore, low pH (5.6−6.5) would promote the shift of the methane generation pathway from acetoclastic methanogenesis to SAO-HM.34 In the present study, the pH in R3 and R6 decreased to below 6. The metabolism of acetoclastic methanogens was inhibited, whereas certain activities of hydrogenotrophic methanogens were retained. From the above-mentioned results, the combined inhibition effects of high ammonium concentrations, high VFA levels, and low pH suppressed the growth and substrate consumption capacity of Methanosaeta, whereas the translation of acetate to H2/CO2 and subsequent conversion of H2/CO2 into CH4 by SAO bacteria and hydrogenotrophic methanogens were partly enhanced. Nevertheless, methane productions were heavily affected and almost ceased. The percentages of Methanoculleus increased with increasing ammonium concentration. Methanoculleus is a mesophilic archaea that grows with H2/CO2 and formate; this genus may survive in a weakly acidic environment.35,36 Consequently, the Methanoculleus population increased when reactors R3, R4, R5, and R6 underwent acidification with ammonium inhibition. Thermogymnomonas was detected with a high percentage of 27.99% in R6. Thermogymnomonas is acidophilic, and most of its species are thermophilic. The increase in the percentage of Thermogymnomonas might be attributed to the acidification of high levels of ammonia concentrated in the reactor and the sharp decrease in pH, which resulted in a decrease in the populations of major common methanogens. The percentage of unclassified archaea also increased to 0.53% in R3 and 7.8% in R6, thereby indicating that the abundance of the microbiological population was enriched with increasing ammonium concentration. We could conclude that the dominant methanogenic pathway and the abundance of the dominant methanogens with ammonium concentrations greatly differed. As the ammonium concentration increased, the methane generation pathway shifted from strict acetoclastic methanogenesis to
4. CONCLUSIONS AD processes are sensitive to ammonia. This study showed that, with uncontrolled pH, ionized ammonium nitrogen is the key inhibitor during FW digestion. The inhibition effect would occur when the ammonium concentration exceeded 2 g/L. The disturbances of ammonia nitrogen on AD processes were mainly attributed to the inhibition of methanogenesis, whereas hydrolysis and acidification were less affected. The inhibition effects of high ammonium concentrations on AD would lead to 5755
DOI: 10.1021/acs.energyfuels.6b00715 Energy Fuels 2016, 30, 5749−5757
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VFA accumulation and pH decrease. These factors would suppress the acetoclastic pathway and Methanosaeta activity but on some level enhance the SAO-HM pathway. When the ammonium concentration further increased to about 6g/L, the metabolism of the hydrogenotrophic methanogens Methanobacterium and Methanospirillum was inhibited. With the increase of ammonium concentration, microorganisms irrelevant to the methane fermentation process were enriched.
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AUTHOR INFORMATION
Corresponding Author
*Tel.:+861064429591. E-mail:
[email protected]. Address: A507 Zonghe Building, Beijing University of Chemical Technology, 15 North Third Ring East Road, Beijing 100029, China. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by Science and Technology Commission of Beijing Municipality (Z151100001115006), National Science Foundation of China (51508015), and Fundamental Research Funds for the Central Universities (buctrc201505, ZY1510).
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NOMENCLATURE AD = anaerobic digestion ANOVA = an analysis of variance CSTR = continuously stirred tank reactor FAN = free ammonia nitrogen FW = food waste HRT = hydraulic retention time MY = methane yield MYs = methane yields OLR = organic loading rate OUT = operational taxonomic units RDP = ribosomal database project SAO = syntrophic acetate oxidation SCOD = soluble chemical oxygen demand TAN = total ammonia nitrogen TS = total solids TVFA = total volatile fatty acids VFA = volatile fatty acids VS = volatile solids SAO-HM = syntrophic acetate oxidation−hydrogenotrophic methanogenesis
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