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Microbial community distribution and extracellular enzyme activities in leach bed reactor treating food waste: Effect of different leachate recirculation practices
Accepted Manuscript Microbial community distribution and extracellular enzyme activities in leach bed reactor treating food waste: effect of different leachate recirculation practices Su Yun Xu, Obuli.P. Karthikeyan, Ammaiyappan Selvam, Jonathan W.C. Wong PII: DOI: Reference:
S0960-8524(14)00668-3 http://dx.doi.org/10.1016/j.biortech.2014.05.009 BITE 13417
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
21 January 2014 30 April 2014 3 May 2014
Please cite this article as: Xu, S.Y., Karthikeyan, Obuli.P., Selvam, A., Wong, J.W.C., Microbial community distribution and extracellular enzyme activities in leach bed reactor treating food waste: effect of different leachate recirculation practices, Bioresource Technology (2014), doi: http://dx.doi.org/10.1016/j.biortech.2014.05.009
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Microbial community distribution and extracellular enzyme activities in leach bed reactor treating food waste: effect of different leachate recirculation practices Su Yun Xu1,2,4, Obuli. P. Karthikeyan,2,3,4, Ammaiyappan Selvam2 and Jonathan W.C. Wong2,*
1
Department of Environmental & Low-Carbon Science, School of Environment and Architecture,
University of Shanghai for Science and Technology, Shanghai, China 2
Sino-Forest Applied Research Centre for Pearl River Delta Environment and Department of Biology,
Hong Kong Baptist University, Hong Kong SAR 3
School of Marine and Tropical Biology, Faculty of Engineering, James Cook University, Townsville,
Queensland, Australia 4
Contributed equally
*Corresponding author.
Prof. Jonathan W.C. Wong Ph.D., MH Sino-Forest Applied Research Centre for Pearl River Delta Environment and Department of Biology Hong Kong Baptist University Hong Kong SAR Tel.: +852 3411 7056 Fax: +852 3411 2355 E-mail: [email protected]
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Abstract
This study aimed at understanding the relationship between microbial community and extracellular enzyme activities of leach bed reactor (LBR) treating food waste under different leachate recirculation practices (once per day and continuous) and liquid to solids (L/S) ratios (1:1 and 0.5:1). Microbial community analysis using PCR-DGGE revealed that Lactobacillus sp., Bifidobacter sp., and Proteobacteria were the most abundant species. Number of phylotypes were higher in LBRs with intermittent recirculation; whereas, a lower number of phylotypes dominated by the key players of degradation was observed with continuous recirculation. The L/S ratio of 1:1 significantly enhanced the volatile solids removal compared with 0.5:1; however, this effect was insignificant under once a day leachate recirculation. Continuous leachate recirculation with 1:1 L/S ratio significantly improved the organic leaching (240 g COD/kg volatile solid) and showed distinct extracellular enzyme activities suitable for food waste acidogenesis.
Key words: Food waste; leach bed reactor; hydrolysis; extracellular enzymes; microbial community; DGGE
1. Introduction In Hong Kong, food waste (FW) represented about 36% of the municipal solid waste stream disposed in landfills during 2012 (HKEPD, 2014). The FW are generally characterized with high moisture (> 70%), volatile solids (85-92% of total solids) contents and carbon to nitrogen (C/N) ratio of 14.5-20.0% (Browne et al., 2013; Zhang et al., 2007a). Therefore, anaerobic digestion of
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FW is considered a more feasible option due to its energy recovery benefits and associated greenhouse gas mitigation. Due to technical simplicity and high organic loading/conversion rates, dry anaerobic digestion technology (> 20% of total solids) is considered to be more advantageous over the wet technology (<10% of total solids) to treat the highly biodegradable waste components like FW (Karthikeyan and Visvanathan, 2013). The leach led reactor (LBR) is conceptualized by Ghosh (1981) for treating high-solid organic substrate and more commonly used for FW treatment under single or two-phase configurations in recent studies (Browne et al., 2013; Selvam et al., 2010; Stabnikova et al., 2008; Wang et al., 2005; Xu et al., 2012; Zhu et al., 2009). Advantages of two-phase anaerobic digestion of FW using LBR and UASB (up-flow anaerobic sludge blanket) reactors were well documented in earlier publications (Browne et al., 2013; Lü et al 2008; Wang et al., 2005; Xu et al., 2011). However, hydrolysis of FW is a critically rate limiting step depending on the solids retention time (SRT) in LBRs and subsequent biomethanation of organics in UASB, which is essential to be well understood to improve the process rate. Leachate recirculation is the most commonly used approach (as detailed in Table 1) to improve the rate of hydrolysis/acidogenesis in LBRs that redistributes the available nutrient contents and buffer the system, leading to a more effective microbial activity. In many cases, combination of leachate recirculation with other process controls namely, particle size reduction (Kim et al., 2008), pH adjustment (Selvam et al., 2010, Xu et al., 2011), micro-aeration (Xu et al., 2014; Zhu et al., 2009), enzyme addition (Romano et al., 2009), inoculum addition (Charles et al., 2009) and temperature control (Lee et al., 2008), were also considered but most of these studies did not have a clear understanding of metabolic complexity during hydrolysis/acidogenesis of FW in LBRs.
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Each of the above mentioned process variables are expected to positively influence the distribution of microbial communities and associated extracellular enzyme activities within the LBRs under various stages of operation/waste stabilization (Cirne et al., 2007; Dearman et al., 2006; Lü et al., 2009; Wang et al., 2010). Therefore, a clear understanding is thus required for further control of the reactors positively. Lü et al. (2009) found divergence in microbial community and metabolites in anaerobic batch reactors due to the effect of pH. Dearman et al. (2006) found that the methane production rate is significantly correlated with the bacterial community distribution structure within the old and new LBRs operated in a sequential mode. The development and distribution of microorganisms in LBRs treating grass silage with continuous leachate recirculation revealed that bacteria belonging to Bacteroidetes, Betaproteobacteria, Alphaproteobacteria, and Gammaproteobacteria were the dominant ones (Wang et al., 2010). In addition, they found Archaea (hydrogenotrophic genus Methanobacterium) in the 10th and 17th day of leachate samples. Members of the phylum Firmicutes, Actinobacteria, Chloroflexi and Flavobacterium were reported from 1st stage LBRs while treating energy crops (Cirne et al., 2007). Thus, it is very clear that the favorable physical and chemical conditions namely pH, buffering capacity and metabolite re-distribution through leachate recirculation, and feedstock characteristics were probably of equal importance for microbial distribution and effective enzyme production in LBRs. However, the available literature on the distribution of microbial diversity and enzyme activities associated with solid organic substrates under various liquid recirculation regimes are inadequate. Thus, the main challenge for maturation of two phase technology for FW treatment is the inadequate information on the microbial dynamics under optimized acidogenic LBR.
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Particularly, the microbial metabolic complexity i.e., microbial composition, enzymes and metabolites distribution pattern in LBRs, as this governs the degradation and gas production rates, needs to be investigated. Therefore, this study aimed to investigate the metabolic complexity of hydrolytic/acidogenic LBRs treating FW under various leachate recirculation conditions (buffering) mainly to establish the correlation between microbial community and enzyme distribution patterns to address the existing knowledge gaps.
2.
Methods
2.1
Food waste and inoculum FW was prepared using bread, boiled rice, cabbage and cooked meat at 35, 25, 25 and 15 %
(on wet weight basis), respectively. Particle size of the FW was reduced to less than 10 mm before feeding into the reactor. The total solids (TS) and volatile solids (VS) contents of the FW were 39.5±1.3% and 97.1±0.8% of TS, respectively; and total organic carbon and total nitrogen contents were 56.4% and 4.5%, respectively. The active inoculum used as the seed in LBRs was collected from the anaerobic sludge digester at Shek Wu Hui wastewater treatment plant, Hong Kong and stored at 4oC before use.
2.2
Reactor design, loading and operational sequences Four identical LBRs, as reported previously (Xu et al., 2011), were used in this study. Each
reactor was initially loaded with 1 kg of FW and 0.2 kg of anaerobic sludge as inoculum. About 75 g of wood chips, as a bulking agent, was mixed with the FW in all the LBRs to avoid the substrate compaction and channeling of leachate. Bulk density of the substrate mixture was 0.65
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kg/L. Organic acids were found to be effectively leached from the FW with wood chips in the LBRs (Demirer and Chen, 2008, Xu et al., 2011). The LBRs were loaded with food waste at two different liquid to solid (L/S) ratios of 1:1 (LBR-A and LBR-C) and 0.5:1 (LBR-B and LBR-D) and received intermittent (LBR-A and LBR-B) and continuous (LBR-C and LBR-D) leachate recirculation (Fig. 1). Initially, LBR-A and LBR-C were added 1.0 L tap water, while LBR-B and LBR-D were added 0.5 L to achieve the determined L/S ratio. In LBR-A and LBR-B, the percolated leachate was collected at the bottom and manually recycled back into the LBRs once a day. Whereas, leachate recirculation was practiced continuously at a flow rate of 5 mL/min using a peristaltic pump in LBR-C and LBR-D. Exactly 50% of the total leachate volume was replaced with a buffer solution (0.5 N NaHCO3) to adjust the pH to 6.0 (once in a day) in all the LBRs. The LBRs were continuously operated and monitored for 16 days at 35oC. Leachate samples were collected at regular intervals and characterized for pH, COD, total Kjeldahl nitrogen (TKN) and ammonium nitrogen following Standard methods (APHA, 2005). The residual solid materials after digestion were analyzed at the end of the operation to determine VS reduction.
2.3
Enzyme activities Qualitative and quantitative enzyme assays were performed to understand the
hydrolysis/acidogenesis of FW in the LBRs operated under various conditions and also to interpret with the microbial community distributions. Three replicate samples for each treatment were collected during 1st, 9th and 16th day of LBRs operation, and API ZYMTM strips (BioMerieux, Marcy l' Etoile, France) were used for qualitative enzyme assay. Each API ZYMTM strip had 20 micro-well containing dehydrated chromogenic substrates of phosphatases (3),
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esterases (3), aminopeptidases (3), proteases (2), glycosyl-hydrolases (8) and 1 well without any substrates for control measurement. About 65 µL of leachate samples were pipetted out into each micro-well and strips were incubated at 37 oC for 4 h. After that, one drop each of ZYM A and ZYM B reagents was added into well and illuminated under light for 30-60 sec for color development. A numerical value of 1-5 (from low, 5 nmol, to high, >20, nmol) was assigned to each micro-well according to the color chart provided by the manufacturer. This kit has been successfully used for monitoring extracellular enzyme activities in anaerobic digestion study (Charles et al., 2009). Three major enzymes, namely α-amylase, β-galactosidase and protease were also assayed quantitatively. Samples collected on day 1, 2, 3, 5, 9, 13 and 16 of LBRs operations were assayed for these enzyme activities. All the chemicals and reagents used for enzyme assays were obtained from Sigma (St. Louis, USA). Amylase activity was assayed by determining the amount of reducing sugar released from the starch substrate according to the method of Bernfeld (1955). The β-galactosidase activity in leachate samples were determined by reducing lactose into glucose (Bergmeyer and Bernt, 1974) while protease was quantified from L-tyrosine release from casein during peptide bond cleavage (Folin and Ciocalteu, 1927). Enzyme unit (IU/mL) in each case was defined as the amount of enzyme, which releases 1 µmol of end products under the assay conditions per minute.
2.4
Microbial community distribution analysis Leachate samples collected on day 1, 5, 9 and 16 of LBR operations were used for
microbial community analysis. Leachate samples, 2 mL, were centrifuged at 10,000 g for 3 minutes and the biomass pellet was used for the extraction of DNA.
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2.4.1 DNA extraction and PCR amplification Genomic DNA was extracted from the pellet samples using the Soil MasterTM DNA extraction kit (Epicenter® Biotech., WI, USA) as per the manufacturer’s instructions. The quality of the DNA was checked with 0.8% (w/v) agarose gel electrophoresis and further quantified using Nanodrop® ND-1000 spectrophotometer. Besides ratio of 260/280 and 260/230 wavelengths were used to assess the contamination of protein and humic substances, respectively. RedsafeTM nucleic acid staining solution (iNtRON biotechnology Inc., Korea) was used for staining the agarose gel. The extracted DNA was used as a template for polymerase chain reaction (PCR) amplifications. Each PCR reaction mixture contained 25 µL of 2× PCR master mix (Promega, USA), 2 µL each of forward (BAC-338F: 5’- GC clampACTCCTACGGGAGGCAG-3’) and reverse (BAC-805R: 5’GACTACCAGGGTATCTAATCC-3’) primers for bacteria (Yu et al., 2005), 2 µL of genomic DNA and made up to 50 µl with nuclease free water. The 5’ end of the forward primer was capped with 40 bp GC-clamp to stabilize the melting behavior of the DNA. The PCR reaction mixtures were amplified using DNA Engine Gradient thermal cycler (Bio-Rad, USA) under the programmed conditions of: initial denaturation at 94oC for 3 min; followed by 35 cycles of denaturation at 94 oC for 30 s, annealing at 55 oC for 20 s and extension at 72 oC for 45 s and a final extension at 72 oC for 7 min.
2.4.2 Denaturing gradient gel electrophoresis (DGGE), Sequencing and BLAST analysis For DGGE analysis, the PCR products were separated using DCodeTM Universal Mutation Detection System (Bio-Rad Laboratories, Inc., California, USA) as follows: 50 µL of the PCR
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product, mixed 6× loading dye (Promega, USA), was loaded onto 6.5% polyacrylamide gel (acrylamide-bisacrylamide, 37.5:1) containing a linear gradient between 40% and 60%, where 100% denaturants is a mixture of 7 M urea and 40% (v/v) formamide. Electrophoresis was run at a constant voltage of 150 for 7 h in 1× TBE buffer (at 60 oC) and the gel was stained with RedsafeTM Nucleic Acid staining solution for 60 min. Gel image was captured using a Bio-Rad gel documentation system (ChemiDoc XRS, USA) and analyzed using the Quantity One 4.5.0 program (Bio-Rad image) for qualitative analysis. Bands that were present in relatively high abundance in different samples, appeared in most of the samples or only appeared in distinct samples were selected for excision and cloning. Selected DGGE bands were excised from the gel and the DNA were eluted using 200 µL DI water under overnight incubation at 4 oC. Eluted DNA samples were further PCR-amplified using corresponding forward and reverse primer without GC-clamp. The PCR-products were cloned in Escherichia coli (JM109) using pGEM-T Easy Vector system (Promega, WI, USA) according to the manufacturer’s instructions. Positive clones were isolated randomly from the LB agar plate (with 100 µg/mL of Ampicilin, 100 mM IPTG, 50 mg/mL of X-gal) and cultured in LB broth with ampicillin. Plasmid DNA were extracted from the clones using Wizard® plus mini preparation kit (DNA purification systems, Promega) and used as templates for sequencing (Tech Dragon Ltd., Hong Kong). The BLASTn tool was then used to search for closely related sequences in the GenBank at the National Center for Biotechnology Information (NCBI) Database (http://www.ncbi.nlm.nih.gov/BLAST).
3.
Results and Discussion
3.1
Hydrolysis/acidogenesis of FW under various recirculation regimes in the LBRs
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Hydrolysis/acidogenesis of FW in the LBRs under different recirculation regimes were monitored over 16 days and the changes in the leachate characteristics are depicted in Fig. 2, while the data are summarized in Table 2. The cumulative leachate productions were 8.3 and 8.5 L from LBR-A and LBR-C, respectively; whereas, LBR-B and LBR-D produced 4.2 and 4.1 L of leachate, respectively. Further, the leachate production was almost equal to that of buffer (pH 6.0) replacement in all the four reactors, indicating that the LBRs were performed efficiently without any clogging problems. Even though the acidic leachate from LBRs were replaced (50% of leachate volume) with pH 6.0 buffer solution from day 1, the average pH values were 5.04±0.42, 4.71±1.62, 5.14±1.41 and 4.71±0.58 in LBR-A, LBR-B, LBR-C and LBR-D, respectively. Leachate pH was too acidic (3.94 to 4.17) during initial periods and increased slightly during the later stages in all the LBRs that could be linked to the rapid acidification rate of FW. After 16 days, the leachate pH values were between 5.20 and 5.40, except LBR-C, in which a near neutral pH of 6.54 was observed (Fig. 2). The possible reason could be that the continuous leachate recirculation with the L/S ratio of 1:1 in LBR-C may have continuously flushed out the organic acids (from hydrolysis/acidogenesis of FW) and buffered the system efficiently than the intermittent recirculation practices and lower L/S ratio (0.5:1) practiced in other LBRs. The total VFA production was significantly high in LBR-C, followed by LBR-A and LBR-D with almost similar quantities, and LBR-B showed the lowest leaching quantities (Table 2). It is also evident that pH is one of the major factors governing the product spectrum, especially VFA (data not shown), of hydrolysis/acidogenesis in LBRs along with the leachate recirculation regimes. Kim et al. (2008) reported that the major end product of acidogenesis of FW was acetate at the pH 4.5-5.5 and lactate at the pH 3.3-3.4. Similar results were observed in the present study (data not
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shown), in which lactate dominated during the initial periods and acetate dominated during the later stage of LBRs operations with respect to pH changes. Furthermore, continuous leachate recirculation (in LBR C and D) was superior than daily leachate recirculation practices (in LBRA and LBR-B) in accelerating the hydrolysis/acidogenesis through; (i) maintaining FW completely wet in LBRs, (ii) uniform buffering of the system, (iii) improving mass transportation, and (iv) minimizing nutrient deficiency for microbial activity as reported earlier (Browne et al., 2013; Kusch et al., 2012; Lü et al. 2008). Similar to total VFA production, cumulative COD production was maximum (240 g/kg FW) in LBR-C and minimum in LBR-B (159 g/kg FW). The peak COD was observed during 2nd, 12th and 13th day in all the four LBRs and initial peak (2nd day) may be due to simple washout of organics (Fig. 2). Wang et al. (2005) observed maximum COD production on 8th day of pilotscale LBR operation with FW as a substrate. Lai et al. (2001) reported that the initial high COD values in LBRs were mainly contributed by the readily available organic particulates and partially decomposed organics from MSW. Further, they stated that the generation of COD from approximately day 10 onwards can be taken as a true indicator for the particulate solubilization in LBRs due to enzymatic activities. Overall rates of hydrolysis/acidogenesis in LBRs under different leachate recirculation regimes were calculated and compared in Table 2. The initial FW and residual digested solids from LBRs were also analyzed for VS, TOC and TKN contents and differences were accounted as removal. The results showed that the continuous leachate recirculation in LBR-C and LBR- D showed slightly higher VS, TOC and TKN removal than LBR-A and LBR-B. Lü et al. (2008) observed that the partial recirculation of hydrolytic/acidogenic leachate was favorable for the hydrolysis of recalcitrant biomass and achieved maximum TS (60.7 %), VS (62.9 %) and C (58.4
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%) removal efficiencies with 1:3 of hydrolytic: methanogenic leachate mix. While, the present study showed better hydrolysis/acidogenesis in LBR-C (69.4% VS reduction) with continuous leachate recirculation with 50% replacement of pH 6.0 buffer solution. The specific yields of COD and VFA were the highest and significantly higher than other treatments. Therefore, understanding the metabolic network, i.e., enzyme activity and microbial community distribution, of LBR-C in comparison with the other LBRs appears important for further process improvements.
3.2
Enzyme activities in LBRs Enzyme profiles at different time intervals of the LBR operations were determined
qualitatively using API ZYMTM strips and the results are shown in Fig. 3. Activities of aminopeptidases (leucine, valine and cystein arylamidases), esterases (lipase, esterase-lipase and esterase) and proteases (trypsin and chymotrypsin) were in low levels throughout the study in all the LBRs, while, the phosphatases (acid and alkaline phosphatases) and glycosyl-hydrolases (βgalactosidase, α-glucosidase and glucosaminidase) activities were higher. Especially, LBR-B and LBR-D with the lower L/S ratio of 0.5 showed higher activities than those with an L/S ratio of 1.0 (LBR-A and LBR-C). The maximum enzyme activities were recorded on day 9 and 16 in all LBRs, except LBR-D (Fig. 3), in which the enzyme activity on day 1 was higher for phosphatases and glycosyl-hydrolases. It was observed that the distribution of iso-valeric and iso-butyric acids in LBR-D was 3-4 times higher than the other LBRs on day 1, while caproic acid was comparatively higher in LBR-B on days 9 and 16 than the other treatments (data was not shown). Enzyme activities mainly depends on the substrate availability, in the other words few of the above mentioned enzyme activities are substrate-inducible (Tiquia, 2002). The
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enzyme profile of LBR-C showed higher activities of α-mannosidase, α–fucosidase, lipase and βgalactosidase. Therefore, it is very clear from the results that the enzyme activities are depend mainly on individual substrate distribution and LBR operational sequences. Further, the quantitative analysis of major hydrolytic enzymes, namely α-amylase, βgalactosidase and protease at different time intervals of the LBR operations are presented in Fig. 4. During hydrolysis, complex insoluble organic materials are hydrolyzed by extracellular enzymes secreted by hydrolytic microorganisms. In general, these cell-free extracellular enzyme activities were found to be relatively higher than the cell-bound extracellular enzyme activities in LBRs (Parawira et al., 2005; Zhang et al., 2007b). Therefore, in the present study only cell-free enzyme activities were considered. Initial enzyme activities in the LBRs were higher and then decreased to lower levels with respect to time, except for protease, which showed the peak value on day 5. The α-amylase, β-galactosidase and protease activities for the L/S of 1.0 was comparatively lower than the L/S of 0.5 and correlated well with the qualitative enzyme activity determined using API-ZYM kit . The possible reason could be that the enzymes are washed out and high rate of dilutions with the buffer additions in L/S ratio of 1:1. However, higher organic leaching rate in LBR-C and LBR-D might be due the enhanced contact between substrate and cell-free extracellular enzymes achieved through continuous leachate recirculation practices. Protease activity was entirely different from the other two enzymes that showed peak values on day 5 of LBRs operations. Zhang et al. (2007b) also observed maximum protease activity (cell-free) on day 6 and the activities decreased slightly on day 8. The protease activities on day 16 ranged between 0.007 and 0.02 IU/mL in all the LBRs and the maximum value was observed in LBR-D. Parawira et al. (2005) reported 0.01 IU/mL of protease as the maximum value during hydrolysis/acidogenesis of solid potato waste in LBRs. Further, they reported that
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the rise and fall in the enzyme activities may be due to the microbial enrichment, the production/stability of enzymes, availability of substrates, pH and temperature. It is possible that different groups of microorganisms might contribute to the organic waste solubilization at different times in LBRs, and certain enzymes might be stable or active at different times. By comparing the tVFA or COD in the leachate and the enzyme activities, LBR-C showed higher leaching capacity but a lower enzymatic activity than LBR-B or LBR-D. As mentioned earlier, increasing leachate recirculation rate might provide a higher opportunity for hydrolytic microorganisms (and enzymes) to get in contact with the solid surfaces leading to a concomitant increase in biofilm associated enzyme activities along with the cell-free extracellular enzyme activities in LBR-C.
3.3
Microbial community analysis in LBRs Microbial community distributions under different leachate recirculation practices are
depicted in Fig. 5 and listed in Table 3. Neighbor joining method was used and phylogenetic tree was constructed to reveal the relationship between the identified species with their closest similarities (Fig. 5b). The most abundant bands, HR-6 and HR-7, were closely matching with Lactobacillus helveticus and L. panis, respectively. In general, the Lactobacillus sp., dominated under hyperthermophilic conditions in anaerobic reactor fed with kitchen waste (Lee et al., 2008). Other species like L. pontis (HR-1) and Weissella kimchii (HR-3) were also detected in LBR samples at different time intervals. Lü et al. (2009) reported that lactic acid bacteria dominated under acidic pH (5.0) conditions that correlated well with the present study. Lactobacillus pontis and an uncultured β-proteobacteriaum were abundant only at later stages of LBR-C operation. The LBR-C showed maximum organic leaching and the pH was near neutral
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(6.54) at the end of 16th day. Therefore, the near neutral pH might have supported the growth of these two species in LBR-C. Weissella kimchii (HR-3) and an uncultured bacterium (HR-4 and HR-5) were the most commonly found members in all the four LBRs during 1st day and disappeared on 5th day, except in LBR-B. A link between increase in pH and reappearance of the uncultured bacterium on day 9 in LBR-B was observed. Both pH and biochemical components (mainly VFAs) significantly influenced the microbial community distributions, but the effect of pH was more powerful (Lu et al., 2009). The uncultured bacterium HR-5 appeared on day 5 and intensively abundant on day 9 (pH 5.04) in LBR-A, but completely absent in LBR-C and LBR-D. Similarly, L. panis (HR-7) was abundant in LBR-A, LBR-C and LBR-D during day 5 and day 9 and disappeared at later sampling periods. But in LBR-B, the L. panis found to exist until day 16. In LBR-C and LBR-D, γ- proteobacteriaum (HR-9) appeared only on day 16 while not abundant in LBR-A and LBR-B. Abundance of these two different species in LBRs mainly associated with the differential leachate recirculation practices. The reduction of hydraulic retention time (HRT) imposes selective pressure on slow-growing microorganisms, which may result in microbial community shifts both qualitative and quantitatively (Shin et al., 2010). Bifidobacterium sp. (HR-8) and γ- proteobacteriaum (HR-9) were also most commonly abundant in all the LBRs. The proteobacters were abundant in leachate fraction, but not in solid surface or biofilms (Cirne et al., 2007). Uncultured compost bacterium (HR-10) and uncultured archaeon (HR-12) were only observed during the later stages of LBRs operations. Especially on day 9, HR-12 was more abundant in LBR-A and LBR-C with L/S ratio of 1:1. It implies that operation of LBRs with high L/S ratio support the growth of archaea at early stage. Wang et al. (2010) observed that the microbial community distribution in leachate and solid substrates
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(attached biofilms) were distinct. Therefore, the attached biomasses on the solid substrates should also playing a major role in organic leaching from FW in LBRs that was not monitored in this present study. But the community distribution in solid residue and leachate samples might be distinct and requires further investigation. Overall, the maximum number of phylotypes was higher in LBR-A and LBR-B, in which intermittent recirculation was practiced; whereas, LBR-C and LBR-D showed very few phylotypes and only those abundant species played a major role in overall FW degradation process. Furthermore, it is apparent that the microbial community was much more sensitive to environmental changes (especially pH) and affects the distribution of enzymes and metabolites in system was evident.
4.
Conclusion
Results suggested that continuous leachate recirculation practices with 1:1 L/S ratio (i.e., in LBR-C) could be potentially used to enrich the fermentative bacterium in LBRs that leads to higher organic leaching from FW and subsequently can support more CH4 production. The key species are Lactobacillus helveticus, Bifidobacterium thermophilum and Gamma proteobacterium that contributed higher enzyme activities such as α-mannosidase, α-fucosidase, lipase, β-galactosidase and protease resulting in enhanced organic leaching. Enriching these dominant microbes provides a key approach to enhance the hydrolytic-acidogenic performance during food waste digestion.
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Lai, T.E., Nopharatana, A., Pullammanappallil, P.C., Clarke, W.P., 2001. Cellulolytic activity in leachate during leach-bed anaerobic digestion of municipal solid waste. Bioresour. Technol. 80(3), 205-210.
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Lee, M., Hidaka, T., Tsuno, H., 2008. Effect of temperature on performance and microbial diversity in hyperthermophilic digester system fed with kitchen garbage. Bioresour. Technol. 99(15), 6852-6860.
18.
Lü, F., He, P.J., Hao, L.P., Shao, L.M., 2008. Impact of recycled effluent on the hydrolysis during anaerobic digestion of vegetable and flower waste. Water Sci. Technol. 58(8), 1637-1643.
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Lü, F., Shao, L.M., Bru, V., Godon, J.J., He, P.J., 2009. Synergetic effect of pH and biochemical components on bacterial diversity during mesophilic anaerobic fermentation of biomass-origin waste. J. Appl. Microbiol. 106(2), 580-591.
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Parawira, W., Murto, M., Read, J.S., Mattiasson, B., 2005. Profile of hydrolases and biogas production during two-stage mesophilic anaerobic digestion of solid potato waste. Process Biochem. 40(9), 2945-2952.
21.
Romano, R.T., Shang, R., Teter, S., McGarvey, J.A., 2009. The effect of enzyme addition on anaerobic digestion of Jose Tall wheat grass. Bioresour. Technol. 100(20), 4564-4571.
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Selvam, A., Xu, S.Y., Gu, X.Y., Wong, J.W.C., 2010. Food waste decomposition in leachbed reactor: Role of neutralizing solutions on the leachate quality. Bioresour. Technol. 101(6), 1707-1714.
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Shin, S.G., Han, G., Lim, J., Lee, C., Hwang, S., 2010. A comprehensive microbial insight into two-stage anaerobic digestion of food waste recycling wastewater. Water Res. 44(17), 4838-4849.
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Stabnikova, O., Ang, S.S., Liu, X,Y., Ivanov, V., Tay, J.H., Wang, J.Y., 2005. The use of hybrid anaerobic solid-liquid (HASL) system for the treatment of lipid-containing food waste. J. chem. Technol. Biotechnol. 80, 455-461.
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Stabnikova, O., Liu, X.Y., Wang, J.Y., 2008. Anaerobic digestion of food waste in a hybrid anaerobic solid-liquid system with leachate recirculation in an acidogenic reactor. Biochem. Eng. J. 41(2), 198-201.
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Tiquia, S.M., 2002. Evolution of extracellular enzyme activities during manure composting. J. Appl. Microbiol. 92(4), 764-775.
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Wang, H., Vuorela, M., Keranen, A.L., Lehtinen, T.M., Lensu, A., Lehtomäki, A., Rintala, J., 2010. Development of microbial populations in the anaerobic hydrolysis of grass silage for methane production. FEMS Microbiol. Ecol. 72(3), 496-506.
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Wang, J.Y., Zhang, H., Stabnikova, O., Ang, S.S., Tay, J.H., 2005. A hybrid anaerobic solid-liquid system for food waste digestion. Water Sci. Technol. 52(1-2), 223-228.
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Xu, S.Y., Lam, H.P., Karthikeyan, O.P., Wong, J.W.C., 2011. Optimization of food waste hydrolysis in leach bed coupled with methanogenic reactor: Effect of pH and bulking agent. Bioresour. Technol. 102(4), 3702-3708.
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Xu, S.Y., Selvam, A., Wong, J.W.C., 2012. Effect of inoculums to substrate ratio on the hydrolysis and acidification of food waste in leach bed reactor. Bioresour. Technol. 126, 425-430.
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Xu, S.Y, Selvam, A., Wong J.W.C., 2014. Optimization of micro-aeration intensity in acidogenic reactor of a two-phase anaerobic digester treating food waste. Waste Manage. 34, 363-369.
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Yu, Y., Lee, C., Kim, J., Hwang, S., 2005. Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction. Biotechnol. Bioeng., 89, 670-679.
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Zhang, B., He, P.J., Lü, F., Shao, L.M., Wang, P., 2007b. Extracellular enzyme activities during regulated hydrolysis of high-solid organic wastes. Water Res. 41(19), 4468-4478.
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Zhang, R., El-Mashad, H., Hartman, K., Wang, F., Liu, G., Choate, C., Gamble, P., 2007a. Characterization of food waste as feedstock for anaerobic digestion. Bioresour. Technol. 98, 929-935.
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Zhu, M., Lü, F., Hao, L.P., He, P.J., Shao, L.M., 2009. Regulating the hydrolysis of organic wastes by micro-aeration and effluent recirculation. Waste Manage. 29(7), 20422050.
21
Table captions Table 1. Comparison of various leachate recirculation practices employed in previous studies using leach bed reactor Table 2. Performance of LBRs under various leachate recirculation conditions Table 3. Phylogenetic affiliation of the 16S rDNA sequences from DGGE bands
Figure captions Fig. 1. Operational sequence of LBRs. (a) Intermittent leachate recirculation practice with 1:1 and 0.5:1 liquid to solid ratio; (b) continuous leachate recirculation practice with the 1:1 and 0.5:1 liquid to solid ratio. Fig. 2. Changes in (a) pH, (b) volume, (c) COD, (d) TKN, (e) NH4+-N of leachate from LBRs at different time intervals. Fig. 3. Qualitative enzyme distribution under different leachate recirculation regimes and liquid to solid ratios in LBRs. Fig. 4. Qualitative enzyme distribution under different leachate recirculation regimes and liquid to solid ratios in LBRs, (a) alpha-amylase, (b) beta-galactosidase and (c) protease. Fig. 5. Microbial community distribution pattern in LBRs under various leachate recirculation practices, (a) DGGE pattern; and (b) phylogenetic tree constructed using neighbor joining method.
22
Table 1 Comparison of various leachate recirculation practices employed in previous studies using leach bed reactor Reactors (capacity)
Substrate
Operational conditions
Leachate recirculation sequences
Monitoring period and operating temperature
Organic removal (%)
Reference
OF-MSW
Batch mode (SEBAC systems) Batch mode (BIOCELL system) Batch and Semicontinuous modes (HASL system) Batch mode (HASL system)
Interexchange of leachates between the two LBRs (once in a day) UASB (2nd stage) effluent recirculated into the LBRs (1st stage) UASB (2nd stage) effluent recirculated into the LBRs (1st stage)
55 days @ 38oC
NA
Lai et al. (2001)
6 days @ 38oC
70.3-72.5
Han and Shin et al. (2004)
10 and 253 days (for batch) & 36 and 286 days (for semi-continuous) @ ambient temperature
77-78
Wang et al. (2005)
Recirculation of leachate mix (UASB + LBR) in LBR (1st stage) Recirculation of leachate mix (UASB + LBR) in LBR (1st stage)
14 days @ ambient temperature
NA
Stabnikova et al. (2005)
10 days @ ambient temperature
62-63
Lü et al. (2008)
Pre-aeration; methanogenic leachate flooding and recirculation Leachate recirculation from LBR (1st stage) with 50 % replacement of buffer solution
12 days @ 55-60 oC
41-42
Charles et al. (2009)
16 days @ 35oC
69.4 (maximum value)
Present study
1st stage
2nd stage
LBR (200 L) LBR (3.9 L)
LBR (200 L) UASB
Food waste
LBR (5.4 L and 80 L)
UASB
Food waste
LBR (5.4 L)
UASB
Food waste
LBR (1.4 L)
UASB
Vegetable and Fruit waste
Batch mode (Two-phase system)
LBR (7 L)
-
OF-MSW
LBR (6.4 L)
UASB
Food waste
Batch mode (DiCOM system) Batch mode (HASL system)
23
Table 2. Performance of LBRs under various leachate recirculation conditions Intermittent leachate recirculation
Continuous leachate recirculation
LBR-A
LBR-B
LBR-C
LBR-D
Liquid solid ratio (L/S)
1:1
0.5:1
1:1
0.5:1
Leachate volume (L)
8.34
4.20
8.50
4.07
Particulars
Leachate parameters COD (g/L)
12.54–36.79 (avg. 19.76)
11.41–58.80 (avg. 38.28)
5.41–61.32 (avg. 30.97)
19.77–96.29 (avg. 49.98)
CODcumulative (g/kg VS)
163.78
159.57
240.16
184.81
tVFA (g/L)
1.77–14.55 (avg. 10.02)
4.82–26.02 (avg. 13.73)
5.41–28.4 (avg. 15.84)
9.28–28.29 (avg. 21.03)
tVFAcumulative (g/kg VS)
51.01
34.13
82.08
49.58
67.8
45.4
69.4
54.0
79.3
44.2
79.7
63.8
36.2
17.9
40.9
31.2
0.50
0.43
0.65
0.44
0.13
0.09
0.22
0.14
Solid waste parameters VS removal (%) TOC removal (%) TKN removal (%)
Overall performance Specific COD yield (g/gVSrem) Specific VFAs yield (g/gVSrem)
24
Table 3. Phylogenetic affiliation of the 16S rDNA sequences from DGGE bands Band no.
Class matching with cultured clone
HR1
Lactobacillus pontis
100
Fermentation system
HR2
Uncultured beta proteobacterium
99
River sediments
HR3
Weissella kimchi
99
Vegetable food waste
HR4
Uncultured bacterium
99
Lake sediments
HR5
Uncultured bacterium
99
Two-phase AD system
HR6
Lactobacillus helveticus
100
Fermentation system
HR7
Lactobacillus panis
99
Fermentation system
HR8
Bifidobacterium thermophilum
100
Two-phase AD system
HR9
Gamma proteobacterium
99
Soil system
HR10
Uncultured compost bacterium
100
Composting system
HR11
Beta proteobacterium
100
Composting system
HR12
Uncultured archaeon
99
Sea sediments
Similarity (%)
Isolated sources
25
LBR-A
LBR-B
1kg SFW
1kg SFW
+
+
1L Water
0.5L Water
(a) Intermittent leachate recirculation LBR-C
LBR-D
1kg SFW
1kg SFW
+
+
1L Water
0.5L Water
(b) Continuous leachate recirculation Figure 1.
26
LBR-A LBR-C
7
LBR-B LBR-D
(a)
pH
6 5 4 3
Volume (mL)
(b) 1200 800 400 0
COD (g/L)
120
(c)
90 60 30 0
(d)
TKN (mg/L)
2400 1600 800 0
(e)
300 200
+
NH4 -N (mg/L)
400
100 0 0
2
4
6
8
10
12
14
16
Time (d)
Figure 2.
27
N Al ap ka th lin ol -A e S- A Ph C BI cid os on -p P p tro ho ho ha l sp sp tas ho ha e hy ta d s Es E rola e te st se ra er Le se as uc Li e in pa Va e a s L ry ip e l i n C e la a ys a m se tin ry id e l am as ar i e yla da Al m se ph id Al aa ph ch T se a- ym ryp Be ga o si ta la tri n Be -ga cto psi ta la sid n - c N a -a Al gul tos se ve ph uc id ty r a l-b B a-g on se et et luc ida a a- - o se g g si Al luc ul co das ph os s e a- am ida m Al a inid se ph nn a a- os se f u id c o as si e da se
Relative activity unit Relative activity unit
Relative activity unit
Relative activity unit
Day 1 Day 9 Day 16
5
5
4
5
4
(a) LBR- A
4
3
2
1
0
(b) LBR- B
3
2
1
0
(c) LBR- C
4
3
2
1
0 5
(d) LBR- D
3
2
1
0
Figure 3.
28
Amylase (units/mL)
25
LBR-A LBR-B LBR-C LBR-D
(a)
20 15 10 5
Beta-glactodisase (units/mL)
0 1.0
(b)
0.8 0.6 0.4 0.2 0.0
Protease (units/mL)
0.05
(c)
0.04 0.03 0.02 0.01 0.00 0
2
4
6
8
10
12
14
16
Time (d) Figure 4.
29
(a)
30
(b) HR7 AJ422032 Lactobacillus pontis 61 FJ749718 Lactobacillus pontis
HM218420 Lactobacillus pontis HR1
97
AF371487 Uncultured bacterium FN667444 Uncultured compost FN667300 Uncultured compost ba
50
GQ505035 Uncultured bacterium
90 32
NR 026310 Lactobacillus panis
95
AY763430 Lactobacillus acidoph GQ505062 Uncultured bacterium HR6 HM218419 Lactobacillus helveti
100
94
AY675251 Lactobacillus suntory GU138588 Lactobacillus helveti 72 HR3
AY281294 Weissella kimchii 60
GQ479964 Uncultured bacterium 98
GQ480139 Uncultured bacterium AB593356 Weissella cibaria
96
HR5 EU828368 Uncultured bacterium
60
M59113 CLORR16SAB Clostridium
99
GQ477905 Uncultured bacterium 99
GU227148 Clostridium tyrobutyr AM902695 Bifidobacterium sp PF AB540215 Bifidobacterium sp JC HR8 97
GU361834 Bifidobacterium therm GQ272611 Bifidobacterium therm EU828410 Uncultured bacterium
HR9
44
AY922098 Uncultured gamma prot
94
EF634040 Azotobacter chroococc
69
EF634039 Gamma proteobacterium 65
EF100152 Azotobacter beijerinc HM152743 Uncultured Pseudomona
94
99
FJ546375 Uncultured bacterium HR10 FN667521 Uncultured compost ba
44
EF621530 Uncultured gamma prot 97
EF174257 Uncultured bacterium 14
HQ132426 Uncultured beta prote HR2 FJ202963 Uncultured bacterium 85 95
EU376166 Uncultured bacterium DQ395661 Uncultured organism c GQ115961 Uncultured bacterium EF562102 Uncultured Comamonada
58
GQ916170 Uncultured bacterium AJ617882 Uncultured bacterium HR11 HM480181 Uncultured beta prote
94
AF368755 Pseudomonas saccharop GU323672 Uncultured bacterium AB441675 Beta proteobacterium HR4 EF488764 Raoultella terrigena 72 Y17658 Klebsiella terrigena
GU562475 Uncultured bacterium FM242723 1 Uncultured bacteriu 100 72
HR12 FM242736 1 Uncultured archaeon
0.2
Figure. 5.
31
Highlights 1. Continuous leachate recirculation is ideal for food waste hydrolysis in LBRs 2. L/S ratio of 1:1 and buffer addition selectively enriches hydrolysing bacteria 3. Key enzymes in LBRs are α-mannosidase, α–fucosidase, lipase and β- galactosidase 4. Lactobacillus sp., found to be predominant in food waste treating LBRs.
32
S0960-8524(14)00668-3 http://dx.doi.org/10.1016/j.biortech.2014.05.009 BITE 13417
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
21 January 2014 30 April 2014 3 May 2014
Please cite this article as: Xu, S.Y., Karthikeyan, Obuli.P., Selvam, A., Wong, J.W.C., Microbial community distribution and extracellular enzyme activities in leach bed reactor treating food waste: effect of different leachate recirculation practices, Bioresource Technology (2014), doi: http://dx.doi.org/10.1016/j.biortech.2014.05.009
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Microbial community distribution and extracellular enzyme activities in leach bed reactor treating food waste: effect of different leachate recirculation practices Su Yun Xu1,2,4, Obuli. P. Karthikeyan,2,3,4, Ammaiyappan Selvam2 and Jonathan W.C. Wong2,*
1
Department of Environmental & Low-Carbon Science, School of Environment and Architecture,
University of Shanghai for Science and Technology, Shanghai, China 2
Sino-Forest Applied Research Centre for Pearl River Delta Environment and Department of Biology,
Hong Kong Baptist University, Hong Kong SAR 3
School of Marine and Tropical Biology, Faculty of Engineering, James Cook University, Townsville,
Queensland, Australia 4
Contributed equally
*Corresponding author.
Prof. Jonathan W.C. Wong Ph.D., MH Sino-Forest Applied Research Centre for Pearl River Delta Environment and Department of Biology Hong Kong Baptist University Hong Kong SAR Tel.: +852 3411 7056 Fax: +852 3411 2355 E-mail: [email protected]
1
Abstract
This study aimed at understanding the relationship between microbial community and extracellular enzyme activities of leach bed reactor (LBR) treating food waste under different leachate recirculation practices (once per day and continuous) and liquid to solids (L/S) ratios (1:1 and 0.5:1). Microbial community analysis using PCR-DGGE revealed that Lactobacillus sp., Bifidobacter sp., and Proteobacteria were the most abundant species. Number of phylotypes were higher in LBRs with intermittent recirculation; whereas, a lower number of phylotypes dominated by the key players of degradation was observed with continuous recirculation. The L/S ratio of 1:1 significantly enhanced the volatile solids removal compared with 0.5:1; however, this effect was insignificant under once a day leachate recirculation. Continuous leachate recirculation with 1:1 L/S ratio significantly improved the organic leaching (240 g COD/kg volatile solid) and showed distinct extracellular enzyme activities suitable for food waste acidogenesis.
Key words: Food waste; leach bed reactor; hydrolysis; extracellular enzymes; microbial community; DGGE
1. Introduction In Hong Kong, food waste (FW) represented about 36% of the municipal solid waste stream disposed in landfills during 2012 (HKEPD, 2014). The FW are generally characterized with high moisture (> 70%), volatile solids (85-92% of total solids) contents and carbon to nitrogen (C/N) ratio of 14.5-20.0% (Browne et al., 2013; Zhang et al., 2007a). Therefore, anaerobic digestion of
2
FW is considered a more feasible option due to its energy recovery benefits and associated greenhouse gas mitigation. Due to technical simplicity and high organic loading/conversion rates, dry anaerobic digestion technology (> 20% of total solids) is considered to be more advantageous over the wet technology (<10% of total solids) to treat the highly biodegradable waste components like FW (Karthikeyan and Visvanathan, 2013). The leach led reactor (LBR) is conceptualized by Ghosh (1981) for treating high-solid organic substrate and more commonly used for FW treatment under single or two-phase configurations in recent studies (Browne et al., 2013; Selvam et al., 2010; Stabnikova et al., 2008; Wang et al., 2005; Xu et al., 2012; Zhu et al., 2009). Advantages of two-phase anaerobic digestion of FW using LBR and UASB (up-flow anaerobic sludge blanket) reactors were well documented in earlier publications (Browne et al., 2013; Lü et al 2008; Wang et al., 2005; Xu et al., 2011). However, hydrolysis of FW is a critically rate limiting step depending on the solids retention time (SRT) in LBRs and subsequent biomethanation of organics in UASB, which is essential to be well understood to improve the process rate. Leachate recirculation is the most commonly used approach (as detailed in Table 1) to improve the rate of hydrolysis/acidogenesis in LBRs that redistributes the available nutrient contents and buffer the system, leading to a more effective microbial activity. In many cases, combination of leachate recirculation with other process controls namely, particle size reduction (Kim et al., 2008), pH adjustment (Selvam et al., 2010, Xu et al., 2011), micro-aeration (Xu et al., 2014; Zhu et al., 2009), enzyme addition (Romano et al., 2009), inoculum addition (Charles et al., 2009) and temperature control (Lee et al., 2008), were also considered but most of these studies did not have a clear understanding of metabolic complexity during hydrolysis/acidogenesis of FW in LBRs.
3
Each of the above mentioned process variables are expected to positively influence the distribution of microbial communities and associated extracellular enzyme activities within the LBRs under various stages of operation/waste stabilization (Cirne et al., 2007; Dearman et al., 2006; Lü et al., 2009; Wang et al., 2010). Therefore, a clear understanding is thus required for further control of the reactors positively. Lü et al. (2009) found divergence in microbial community and metabolites in anaerobic batch reactors due to the effect of pH. Dearman et al. (2006) found that the methane production rate is significantly correlated with the bacterial community distribution structure within the old and new LBRs operated in a sequential mode. The development and distribution of microorganisms in LBRs treating grass silage with continuous leachate recirculation revealed that bacteria belonging to Bacteroidetes, Betaproteobacteria, Alphaproteobacteria, and Gammaproteobacteria were the dominant ones (Wang et al., 2010). In addition, they found Archaea (hydrogenotrophic genus Methanobacterium) in the 10th and 17th day of leachate samples. Members of the phylum Firmicutes, Actinobacteria, Chloroflexi and Flavobacterium were reported from 1st stage LBRs while treating energy crops (Cirne et al., 2007). Thus, it is very clear that the favorable physical and chemical conditions namely pH, buffering capacity and metabolite re-distribution through leachate recirculation, and feedstock characteristics were probably of equal importance for microbial distribution and effective enzyme production in LBRs. However, the available literature on the distribution of microbial diversity and enzyme activities associated with solid organic substrates under various liquid recirculation regimes are inadequate. Thus, the main challenge for maturation of two phase technology for FW treatment is the inadequate information on the microbial dynamics under optimized acidogenic LBR.
4
Particularly, the microbial metabolic complexity i.e., microbial composition, enzymes and metabolites distribution pattern in LBRs, as this governs the degradation and gas production rates, needs to be investigated. Therefore, this study aimed to investigate the metabolic complexity of hydrolytic/acidogenic LBRs treating FW under various leachate recirculation conditions (buffering) mainly to establish the correlation between microbial community and enzyme distribution patterns to address the existing knowledge gaps.
2.
Methods
2.1
Food waste and inoculum FW was prepared using bread, boiled rice, cabbage and cooked meat at 35, 25, 25 and 15 %
(on wet weight basis), respectively. Particle size of the FW was reduced to less than 10 mm before feeding into the reactor. The total solids (TS) and volatile solids (VS) contents of the FW were 39.5±1.3% and 97.1±0.8% of TS, respectively; and total organic carbon and total nitrogen contents were 56.4% and 4.5%, respectively. The active inoculum used as the seed in LBRs was collected from the anaerobic sludge digester at Shek Wu Hui wastewater treatment plant, Hong Kong and stored at 4oC before use.
2.2
Reactor design, loading and operational sequences Four identical LBRs, as reported previously (Xu et al., 2011), were used in this study. Each
reactor was initially loaded with 1 kg of FW and 0.2 kg of anaerobic sludge as inoculum. About 75 g of wood chips, as a bulking agent, was mixed with the FW in all the LBRs to avoid the substrate compaction and channeling of leachate. Bulk density of the substrate mixture was 0.65
5
kg/L. Organic acids were found to be effectively leached from the FW with wood chips in the LBRs (Demirer and Chen, 2008, Xu et al., 2011). The LBRs were loaded with food waste at two different liquid to solid (L/S) ratios of 1:1 (LBR-A and LBR-C) and 0.5:1 (LBR-B and LBR-D) and received intermittent (LBR-A and LBR-B) and continuous (LBR-C and LBR-D) leachate recirculation (Fig. 1). Initially, LBR-A and LBR-C were added 1.0 L tap water, while LBR-B and LBR-D were added 0.5 L to achieve the determined L/S ratio. In LBR-A and LBR-B, the percolated leachate was collected at the bottom and manually recycled back into the LBRs once a day. Whereas, leachate recirculation was practiced continuously at a flow rate of 5 mL/min using a peristaltic pump in LBR-C and LBR-D. Exactly 50% of the total leachate volume was replaced with a buffer solution (0.5 N NaHCO3) to adjust the pH to 6.0 (once in a day) in all the LBRs. The LBRs were continuously operated and monitored for 16 days at 35oC. Leachate samples were collected at regular intervals and characterized for pH, COD, total Kjeldahl nitrogen (TKN) and ammonium nitrogen following Standard methods (APHA, 2005). The residual solid materials after digestion were analyzed at the end of the operation to determine VS reduction.
2.3
Enzyme activities Qualitative and quantitative enzyme assays were performed to understand the
hydrolysis/acidogenesis of FW in the LBRs operated under various conditions and also to interpret with the microbial community distributions. Three replicate samples for each treatment were collected during 1st, 9th and 16th day of LBRs operation, and API ZYMTM strips (BioMerieux, Marcy l' Etoile, France) were used for qualitative enzyme assay. Each API ZYMTM strip had 20 micro-well containing dehydrated chromogenic substrates of phosphatases (3),
6
esterases (3), aminopeptidases (3), proteases (2), glycosyl-hydrolases (8) and 1 well without any substrates for control measurement. About 65 µL of leachate samples were pipetted out into each micro-well and strips were incubated at 37 oC for 4 h. After that, one drop each of ZYM A and ZYM B reagents was added into well and illuminated under light for 30-60 sec for color development. A numerical value of 1-5 (from low, 5 nmol, to high, >20, nmol) was assigned to each micro-well according to the color chart provided by the manufacturer. This kit has been successfully used for monitoring extracellular enzyme activities in anaerobic digestion study (Charles et al., 2009). Three major enzymes, namely α-amylase, β-galactosidase and protease were also assayed quantitatively. Samples collected on day 1, 2, 3, 5, 9, 13 and 16 of LBRs operations were assayed for these enzyme activities. All the chemicals and reagents used for enzyme assays were obtained from Sigma (St. Louis, USA). Amylase activity was assayed by determining the amount of reducing sugar released from the starch substrate according to the method of Bernfeld (1955). The β-galactosidase activity in leachate samples were determined by reducing lactose into glucose (Bergmeyer and Bernt, 1974) while protease was quantified from L-tyrosine release from casein during peptide bond cleavage (Folin and Ciocalteu, 1927). Enzyme unit (IU/mL) in each case was defined as the amount of enzyme, which releases 1 µmol of end products under the assay conditions per minute.
2.4
Microbial community distribution analysis Leachate samples collected on day 1, 5, 9 and 16 of LBR operations were used for
microbial community analysis. Leachate samples, 2 mL, were centrifuged at 10,000 g for 3 minutes and the biomass pellet was used for the extraction of DNA.
7
2.4.1 DNA extraction and PCR amplification Genomic DNA was extracted from the pellet samples using the Soil MasterTM DNA extraction kit (Epicenter® Biotech., WI, USA) as per the manufacturer’s instructions. The quality of the DNA was checked with 0.8% (w/v) agarose gel electrophoresis and further quantified using Nanodrop® ND-1000 spectrophotometer. Besides ratio of 260/280 and 260/230 wavelengths were used to assess the contamination of protein and humic substances, respectively. RedsafeTM nucleic acid staining solution (iNtRON biotechnology Inc., Korea) was used for staining the agarose gel. The extracted DNA was used as a template for polymerase chain reaction (PCR) amplifications. Each PCR reaction mixture contained 25 µL of 2× PCR master mix (Promega, USA), 2 µL each of forward (BAC-338F: 5’- GC clampACTCCTACGGGAGGCAG-3’) and reverse (BAC-805R: 5’GACTACCAGGGTATCTAATCC-3’) primers for bacteria (Yu et al., 2005), 2 µL of genomic DNA and made up to 50 µl with nuclease free water. The 5’ end of the forward primer was capped with 40 bp GC-clamp to stabilize the melting behavior of the DNA. The PCR reaction mixtures were amplified using DNA Engine Gradient thermal cycler (Bio-Rad, USA) under the programmed conditions of: initial denaturation at 94oC for 3 min; followed by 35 cycles of denaturation at 94 oC for 30 s, annealing at 55 oC for 20 s and extension at 72 oC for 45 s and a final extension at 72 oC for 7 min.
2.4.2 Denaturing gradient gel electrophoresis (DGGE), Sequencing and BLAST analysis For DGGE analysis, the PCR products were separated using DCodeTM Universal Mutation Detection System (Bio-Rad Laboratories, Inc., California, USA) as follows: 50 µL of the PCR
8
product, mixed 6× loading dye (Promega, USA), was loaded onto 6.5% polyacrylamide gel (acrylamide-bisacrylamide, 37.5:1) containing a linear gradient between 40% and 60%, where 100% denaturants is a mixture of 7 M urea and 40% (v/v) formamide. Electrophoresis was run at a constant voltage of 150 for 7 h in 1× TBE buffer (at 60 oC) and the gel was stained with RedsafeTM Nucleic Acid staining solution for 60 min. Gel image was captured using a Bio-Rad gel documentation system (ChemiDoc XRS, USA) and analyzed using the Quantity One 4.5.0 program (Bio-Rad image) for qualitative analysis. Bands that were present in relatively high abundance in different samples, appeared in most of the samples or only appeared in distinct samples were selected for excision and cloning. Selected DGGE bands were excised from the gel and the DNA were eluted using 200 µL DI water under overnight incubation at 4 oC. Eluted DNA samples were further PCR-amplified using corresponding forward and reverse primer without GC-clamp. The PCR-products were cloned in Escherichia coli (JM109) using pGEM-T Easy Vector system (Promega, WI, USA) according to the manufacturer’s instructions. Positive clones were isolated randomly from the LB agar plate (with 100 µg/mL of Ampicilin, 100 mM IPTG, 50 mg/mL of X-gal) and cultured in LB broth with ampicillin. Plasmid DNA were extracted from the clones using Wizard® plus mini preparation kit (DNA purification systems, Promega) and used as templates for sequencing (Tech Dragon Ltd., Hong Kong). The BLASTn tool was then used to search for closely related sequences in the GenBank at the National Center for Biotechnology Information (NCBI) Database (http://www.ncbi.nlm.nih.gov/BLAST).
3.
Results and Discussion
3.1
Hydrolysis/acidogenesis of FW under various recirculation regimes in the LBRs
9
Hydrolysis/acidogenesis of FW in the LBRs under different recirculation regimes were monitored over 16 days and the changes in the leachate characteristics are depicted in Fig. 2, while the data are summarized in Table 2. The cumulative leachate productions were 8.3 and 8.5 L from LBR-A and LBR-C, respectively; whereas, LBR-B and LBR-D produced 4.2 and 4.1 L of leachate, respectively. Further, the leachate production was almost equal to that of buffer (pH 6.0) replacement in all the four reactors, indicating that the LBRs were performed efficiently without any clogging problems. Even though the acidic leachate from LBRs were replaced (50% of leachate volume) with pH 6.0 buffer solution from day 1, the average pH values were 5.04±0.42, 4.71±1.62, 5.14±1.41 and 4.71±0.58 in LBR-A, LBR-B, LBR-C and LBR-D, respectively. Leachate pH was too acidic (3.94 to 4.17) during initial periods and increased slightly during the later stages in all the LBRs that could be linked to the rapid acidification rate of FW. After 16 days, the leachate pH values were between 5.20 and 5.40, except LBR-C, in which a near neutral pH of 6.54 was observed (Fig. 2). The possible reason could be that the continuous leachate recirculation with the L/S ratio of 1:1 in LBR-C may have continuously flushed out the organic acids (from hydrolysis/acidogenesis of FW) and buffered the system efficiently than the intermittent recirculation practices and lower L/S ratio (0.5:1) practiced in other LBRs. The total VFA production was significantly high in LBR-C, followed by LBR-A and LBR-D with almost similar quantities, and LBR-B showed the lowest leaching quantities (Table 2). It is also evident that pH is one of the major factors governing the product spectrum, especially VFA (data not shown), of hydrolysis/acidogenesis in LBRs along with the leachate recirculation regimes. Kim et al. (2008) reported that the major end product of acidogenesis of FW was acetate at the pH 4.5-5.5 and lactate at the pH 3.3-3.4. Similar results were observed in the present study (data not
10
shown), in which lactate dominated during the initial periods and acetate dominated during the later stage of LBRs operations with respect to pH changes. Furthermore, continuous leachate recirculation (in LBR C and D) was superior than daily leachate recirculation practices (in LBRA and LBR-B) in accelerating the hydrolysis/acidogenesis through; (i) maintaining FW completely wet in LBRs, (ii) uniform buffering of the system, (iii) improving mass transportation, and (iv) minimizing nutrient deficiency for microbial activity as reported earlier (Browne et al., 2013; Kusch et al., 2012; Lü et al. 2008). Similar to total VFA production, cumulative COD production was maximum (240 g/kg FW) in LBR-C and minimum in LBR-B (159 g/kg FW). The peak COD was observed during 2nd, 12th and 13th day in all the four LBRs and initial peak (2nd day) may be due to simple washout of organics (Fig. 2). Wang et al. (2005) observed maximum COD production on 8th day of pilotscale LBR operation with FW as a substrate. Lai et al. (2001) reported that the initial high COD values in LBRs were mainly contributed by the readily available organic particulates and partially decomposed organics from MSW. Further, they stated that the generation of COD from approximately day 10 onwards can be taken as a true indicator for the particulate solubilization in LBRs due to enzymatic activities. Overall rates of hydrolysis/acidogenesis in LBRs under different leachate recirculation regimes were calculated and compared in Table 2. The initial FW and residual digested solids from LBRs were also analyzed for VS, TOC and TKN contents and differences were accounted as removal. The results showed that the continuous leachate recirculation in LBR-C and LBR- D showed slightly higher VS, TOC and TKN removal than LBR-A and LBR-B. Lü et al. (2008) observed that the partial recirculation of hydrolytic/acidogenic leachate was favorable for the hydrolysis of recalcitrant biomass and achieved maximum TS (60.7 %), VS (62.9 %) and C (58.4
11
%) removal efficiencies with 1:3 of hydrolytic: methanogenic leachate mix. While, the present study showed better hydrolysis/acidogenesis in LBR-C (69.4% VS reduction) with continuous leachate recirculation with 50% replacement of pH 6.0 buffer solution. The specific yields of COD and VFA were the highest and significantly higher than other treatments. Therefore, understanding the metabolic network, i.e., enzyme activity and microbial community distribution, of LBR-C in comparison with the other LBRs appears important for further process improvements.
3.2
Enzyme activities in LBRs Enzyme profiles at different time intervals of the LBR operations were determined
qualitatively using API ZYMTM strips and the results are shown in Fig. 3. Activities of aminopeptidases (leucine, valine and cystein arylamidases), esterases (lipase, esterase-lipase and esterase) and proteases (trypsin and chymotrypsin) were in low levels throughout the study in all the LBRs, while, the phosphatases (acid and alkaline phosphatases) and glycosyl-hydrolases (βgalactosidase, α-glucosidase and glucosaminidase) activities were higher. Especially, LBR-B and LBR-D with the lower L/S ratio of 0.5 showed higher activities than those with an L/S ratio of 1.0 (LBR-A and LBR-C). The maximum enzyme activities were recorded on day 9 and 16 in all LBRs, except LBR-D (Fig. 3), in which the enzyme activity on day 1 was higher for phosphatases and glycosyl-hydrolases. It was observed that the distribution of iso-valeric and iso-butyric acids in LBR-D was 3-4 times higher than the other LBRs on day 1, while caproic acid was comparatively higher in LBR-B on days 9 and 16 than the other treatments (data was not shown). Enzyme activities mainly depends on the substrate availability, in the other words few of the above mentioned enzyme activities are substrate-inducible (Tiquia, 2002). The
12
enzyme profile of LBR-C showed higher activities of α-mannosidase, α–fucosidase, lipase and βgalactosidase. Therefore, it is very clear from the results that the enzyme activities are depend mainly on individual substrate distribution and LBR operational sequences. Further, the quantitative analysis of major hydrolytic enzymes, namely α-amylase, βgalactosidase and protease at different time intervals of the LBR operations are presented in Fig. 4. During hydrolysis, complex insoluble organic materials are hydrolyzed by extracellular enzymes secreted by hydrolytic microorganisms. In general, these cell-free extracellular enzyme activities were found to be relatively higher than the cell-bound extracellular enzyme activities in LBRs (Parawira et al., 2005; Zhang et al., 2007b). Therefore, in the present study only cell-free enzyme activities were considered. Initial enzyme activities in the LBRs were higher and then decreased to lower levels with respect to time, except for protease, which showed the peak value on day 5. The α-amylase, β-galactosidase and protease activities for the L/S of 1.0 was comparatively lower than the L/S of 0.5 and correlated well with the qualitative enzyme activity determined using API-ZYM kit . The possible reason could be that the enzymes are washed out and high rate of dilutions with the buffer additions in L/S ratio of 1:1. However, higher organic leaching rate in LBR-C and LBR-D might be due the enhanced contact between substrate and cell-free extracellular enzymes achieved through continuous leachate recirculation practices. Protease activity was entirely different from the other two enzymes that showed peak values on day 5 of LBRs operations. Zhang et al. (2007b) also observed maximum protease activity (cell-free) on day 6 and the activities decreased slightly on day 8. The protease activities on day 16 ranged between 0.007 and 0.02 IU/mL in all the LBRs and the maximum value was observed in LBR-D. Parawira et al. (2005) reported 0.01 IU/mL of protease as the maximum value during hydrolysis/acidogenesis of solid potato waste in LBRs. Further, they reported that
13
the rise and fall in the enzyme activities may be due to the microbial enrichment, the production/stability of enzymes, availability of substrates, pH and temperature. It is possible that different groups of microorganisms might contribute to the organic waste solubilization at different times in LBRs, and certain enzymes might be stable or active at different times. By comparing the tVFA or COD in the leachate and the enzyme activities, LBR-C showed higher leaching capacity but a lower enzymatic activity than LBR-B or LBR-D. As mentioned earlier, increasing leachate recirculation rate might provide a higher opportunity for hydrolytic microorganisms (and enzymes) to get in contact with the solid surfaces leading to a concomitant increase in biofilm associated enzyme activities along with the cell-free extracellular enzyme activities in LBR-C.
3.3
Microbial community analysis in LBRs Microbial community distributions under different leachate recirculation practices are
depicted in Fig. 5 and listed in Table 3. Neighbor joining method was used and phylogenetic tree was constructed to reveal the relationship between the identified species with their closest similarities (Fig. 5b). The most abundant bands, HR-6 and HR-7, were closely matching with Lactobacillus helveticus and L. panis, respectively. In general, the Lactobacillus sp., dominated under hyperthermophilic conditions in anaerobic reactor fed with kitchen waste (Lee et al., 2008). Other species like L. pontis (HR-1) and Weissella kimchii (HR-3) were also detected in LBR samples at different time intervals. Lü et al. (2009) reported that lactic acid bacteria dominated under acidic pH (5.0) conditions that correlated well with the present study. Lactobacillus pontis and an uncultured β-proteobacteriaum were abundant only at later stages of LBR-C operation. The LBR-C showed maximum organic leaching and the pH was near neutral
14
(6.54) at the end of 16th day. Therefore, the near neutral pH might have supported the growth of these two species in LBR-C. Weissella kimchii (HR-3) and an uncultured bacterium (HR-4 and HR-5) were the most commonly found members in all the four LBRs during 1st day and disappeared on 5th day, except in LBR-B. A link between increase in pH and reappearance of the uncultured bacterium on day 9 in LBR-B was observed. Both pH and biochemical components (mainly VFAs) significantly influenced the microbial community distributions, but the effect of pH was more powerful (Lu et al., 2009). The uncultured bacterium HR-5 appeared on day 5 and intensively abundant on day 9 (pH 5.04) in LBR-A, but completely absent in LBR-C and LBR-D. Similarly, L. panis (HR-7) was abundant in LBR-A, LBR-C and LBR-D during day 5 and day 9 and disappeared at later sampling periods. But in LBR-B, the L. panis found to exist until day 16. In LBR-C and LBR-D, γ- proteobacteriaum (HR-9) appeared only on day 16 while not abundant in LBR-A and LBR-B. Abundance of these two different species in LBRs mainly associated with the differential leachate recirculation practices. The reduction of hydraulic retention time (HRT) imposes selective pressure on slow-growing microorganisms, which may result in microbial community shifts both qualitative and quantitatively (Shin et al., 2010). Bifidobacterium sp. (HR-8) and γ- proteobacteriaum (HR-9) were also most commonly abundant in all the LBRs. The proteobacters were abundant in leachate fraction, but not in solid surface or biofilms (Cirne et al., 2007). Uncultured compost bacterium (HR-10) and uncultured archaeon (HR-12) were only observed during the later stages of LBRs operations. Especially on day 9, HR-12 was more abundant in LBR-A and LBR-C with L/S ratio of 1:1. It implies that operation of LBRs with high L/S ratio support the growth of archaea at early stage. Wang et al. (2010) observed that the microbial community distribution in leachate and solid substrates
15
(attached biofilms) were distinct. Therefore, the attached biomasses on the solid substrates should also playing a major role in organic leaching from FW in LBRs that was not monitored in this present study. But the community distribution in solid residue and leachate samples might be distinct and requires further investigation. Overall, the maximum number of phylotypes was higher in LBR-A and LBR-B, in which intermittent recirculation was practiced; whereas, LBR-C and LBR-D showed very few phylotypes and only those abundant species played a major role in overall FW degradation process. Furthermore, it is apparent that the microbial community was much more sensitive to environmental changes (especially pH) and affects the distribution of enzymes and metabolites in system was evident.
4.
Conclusion
Results suggested that continuous leachate recirculation practices with 1:1 L/S ratio (i.e., in LBR-C) could be potentially used to enrich the fermentative bacterium in LBRs that leads to higher organic leaching from FW and subsequently can support more CH4 production. The key species are Lactobacillus helveticus, Bifidobacterium thermophilum and Gamma proteobacterium that contributed higher enzyme activities such as α-mannosidase, α-fucosidase, lipase, β-galactosidase and protease resulting in enhanced organic leaching. Enriching these dominant microbes provides a key approach to enhance the hydrolytic-acidogenic performance during food waste digestion.
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21
Table captions Table 1. Comparison of various leachate recirculation practices employed in previous studies using leach bed reactor Table 2. Performance of LBRs under various leachate recirculation conditions Table 3. Phylogenetic affiliation of the 16S rDNA sequences from DGGE bands
Figure captions Fig. 1. Operational sequence of LBRs. (a) Intermittent leachate recirculation practice with 1:1 and 0.5:1 liquid to solid ratio; (b) continuous leachate recirculation practice with the 1:1 and 0.5:1 liquid to solid ratio. Fig. 2. Changes in (a) pH, (b) volume, (c) COD, (d) TKN, (e) NH4+-N of leachate from LBRs at different time intervals. Fig. 3. Qualitative enzyme distribution under different leachate recirculation regimes and liquid to solid ratios in LBRs. Fig. 4. Qualitative enzyme distribution under different leachate recirculation regimes and liquid to solid ratios in LBRs, (a) alpha-amylase, (b) beta-galactosidase and (c) protease. Fig. 5. Microbial community distribution pattern in LBRs under various leachate recirculation practices, (a) DGGE pattern; and (b) phylogenetic tree constructed using neighbor joining method.
22
Table 1 Comparison of various leachate recirculation practices employed in previous studies using leach bed reactor Reactors (capacity)
Substrate
Operational conditions
Leachate recirculation sequences
Monitoring period and operating temperature
Organic removal (%)
Reference
OF-MSW
Batch mode (SEBAC systems) Batch mode (BIOCELL system) Batch and Semicontinuous modes (HASL system) Batch mode (HASL system)
Interexchange of leachates between the two LBRs (once in a day) UASB (2nd stage) effluent recirculated into the LBRs (1st stage) UASB (2nd stage) effluent recirculated into the LBRs (1st stage)
55 days @ 38oC
NA
Lai et al. (2001)
6 days @ 38oC
70.3-72.5
Han and Shin et al. (2004)
10 and 253 days (for batch) & 36 and 286 days (for semi-continuous) @ ambient temperature
77-78
Wang et al. (2005)
Recirculation of leachate mix (UASB + LBR) in LBR (1st stage) Recirculation of leachate mix (UASB + LBR) in LBR (1st stage)
14 days @ ambient temperature
NA
Stabnikova et al. (2005)
10 days @ ambient temperature
62-63
Lü et al. (2008)
Pre-aeration; methanogenic leachate flooding and recirculation Leachate recirculation from LBR (1st stage) with 50 % replacement of buffer solution
12 days @ 55-60 oC
41-42
Charles et al. (2009)
16 days @ 35oC
69.4 (maximum value)
Present study
1st stage
2nd stage
LBR (200 L) LBR (3.9 L)
LBR (200 L) UASB
Food waste
LBR (5.4 L and 80 L)
UASB
Food waste
LBR (5.4 L)
UASB
Food waste
LBR (1.4 L)
UASB
Vegetable and Fruit waste
Batch mode (Two-phase system)
LBR (7 L)
-
OF-MSW
LBR (6.4 L)
UASB
Food waste
Batch mode (DiCOM system) Batch mode (HASL system)
23
Table 2. Performance of LBRs under various leachate recirculation conditions Intermittent leachate recirculation
Continuous leachate recirculation
LBR-A
LBR-B
LBR-C
LBR-D
Liquid solid ratio (L/S)
1:1
0.5:1
1:1
0.5:1
Leachate volume (L)
8.34
4.20
8.50
4.07
Particulars
Leachate parameters COD (g/L)
12.54–36.79 (avg. 19.76)
11.41–58.80 (avg. 38.28)
5.41–61.32 (avg. 30.97)
19.77–96.29 (avg. 49.98)
CODcumulative (g/kg VS)
163.78
159.57
240.16
184.81
tVFA (g/L)
1.77–14.55 (avg. 10.02)
4.82–26.02 (avg. 13.73)
5.41–28.4 (avg. 15.84)
9.28–28.29 (avg. 21.03)
tVFAcumulative (g/kg VS)
51.01
34.13
82.08
49.58
67.8
45.4
69.4
54.0
79.3
44.2
79.7
63.8
36.2
17.9
40.9
31.2
0.50
0.43
0.65
0.44
0.13
0.09
0.22
0.14
Solid waste parameters VS removal (%) TOC removal (%) TKN removal (%)
Overall performance Specific COD yield (g/gVSrem) Specific VFAs yield (g/gVSrem)
24
Table 3. Phylogenetic affiliation of the 16S rDNA sequences from DGGE bands Band no.
Class matching with cultured clone
HR1
Lactobacillus pontis
100
Fermentation system
HR2
Uncultured beta proteobacterium
99
River sediments
HR3
Weissella kimchi
99
Vegetable food waste
HR4
Uncultured bacterium
99
Lake sediments
HR5
Uncultured bacterium
99
Two-phase AD system
HR6
Lactobacillus helveticus
100
Fermentation system
HR7
Lactobacillus panis
99
Fermentation system
HR8
Bifidobacterium thermophilum
100
Two-phase AD system
HR9
Gamma proteobacterium
99
Soil system
HR10
Uncultured compost bacterium
100
Composting system
HR11
Beta proteobacterium
100
Composting system
HR12
Uncultured archaeon
99
Sea sediments
Similarity (%)
Isolated sources
25
LBR-A
LBR-B
1kg SFW
1kg SFW
+
+
1L Water
0.5L Water
(a) Intermittent leachate recirculation LBR-C
LBR-D
1kg SFW
1kg SFW
+
+
1L Water
0.5L Water
(b) Continuous leachate recirculation Figure 1.
26
LBR-A LBR-C
7
LBR-B LBR-D
(a)
pH
6 5 4 3
Volume (mL)
(b) 1200 800 400 0
COD (g/L)
120
(c)
90 60 30 0
(d)
TKN (mg/L)
2400 1600 800 0
(e)
300 200
+
NH4 -N (mg/L)
400
100 0 0
2
4
6
8
10
12
14
16
Time (d)
Figure 2.
27
N Al ap ka th lin ol -A e S- A Ph C BI cid os on -p P p tro ho ho ha l sp sp tas ho ha e hy ta d s Es E rola e te st se ra er Le se as uc Li e in pa Va e a s L ry ip e l i n C e la a ys a m se tin ry id e l am as ar i e yla da Al m se ph id Al aa ph ch T se a- ym ryp Be ga o si ta la tri n Be -ga cto psi ta la sid n - c N a -a Al gul tos se ve ph uc id ty r a l-b B a-g on se et et luc ida a a- - o se g g si Al luc ul co das ph os s e a- am ida m Al a inid se ph nn a a- os se f u id c o as si e da se
Relative activity unit Relative activity unit
Relative activity unit
Relative activity unit
Day 1 Day 9 Day 16
5
5
4
5
4
(a) LBR- A
4
3
2
1
0
(b) LBR- B
3
2
1
0
(c) LBR- C
4
3
2
1
0 5
(d) LBR- D
3
2
1
0
Figure 3.
28
Amylase (units/mL)
25
LBR-A LBR-B LBR-C LBR-D
(a)
20 15 10 5
Beta-glactodisase (units/mL)
0 1.0
(b)
0.8 0.6 0.4 0.2 0.0
Protease (units/mL)
0.05
(c)
0.04 0.03 0.02 0.01 0.00 0
2
4
6
8
10
12
14
16
Time (d) Figure 4.
29
(a)
30
(b) HR7 AJ422032 Lactobacillus pontis 61 FJ749718 Lactobacillus pontis
HM218420 Lactobacillus pontis HR1
97
AF371487 Uncultured bacterium FN667444 Uncultured compost FN667300 Uncultured compost ba
50
GQ505035 Uncultured bacterium
90 32
NR 026310 Lactobacillus panis
95
AY763430 Lactobacillus acidoph GQ505062 Uncultured bacterium HR6 HM218419 Lactobacillus helveti
100
94
AY675251 Lactobacillus suntory GU138588 Lactobacillus helveti 72 HR3
AY281294 Weissella kimchii 60
GQ479964 Uncultured bacterium 98
GQ480139 Uncultured bacterium AB593356 Weissella cibaria
96
HR5 EU828368 Uncultured bacterium
60
M59113 CLORR16SAB Clostridium
99
GQ477905 Uncultured bacterium 99
GU227148 Clostridium tyrobutyr AM902695 Bifidobacterium sp PF AB540215 Bifidobacterium sp JC HR8 97
GU361834 Bifidobacterium therm GQ272611 Bifidobacterium therm EU828410 Uncultured bacterium
HR9
44
AY922098 Uncultured gamma prot
94
EF634040 Azotobacter chroococc
69
EF634039 Gamma proteobacterium 65
EF100152 Azotobacter beijerinc HM152743 Uncultured Pseudomona
94
99
FJ546375 Uncultured bacterium HR10 FN667521 Uncultured compost ba
44
EF621530 Uncultured gamma prot 97
EF174257 Uncultured bacterium 14
HQ132426 Uncultured beta prote HR2 FJ202963 Uncultured bacterium 85 95
EU376166 Uncultured bacterium DQ395661 Uncultured organism c GQ115961 Uncultured bacterium EF562102 Uncultured Comamonada
58
GQ916170 Uncultured bacterium AJ617882 Uncultured bacterium HR11 HM480181 Uncultured beta prote
94
AF368755 Pseudomonas saccharop GU323672 Uncultured bacterium AB441675 Beta proteobacterium HR4 EF488764 Raoultella terrigena 72 Y17658 Klebsiella terrigena
GU562475 Uncultured bacterium FM242723 1 Uncultured bacteriu 100 72
HR12 FM242736 1 Uncultured archaeon
0.2
Figure. 5.
31
Highlights 1. Continuous leachate recirculation is ideal for food waste hydrolysis in LBRs 2. L/S ratio of 1:1 and buffer addition selectively enriches hydrolysing bacteria 3. Key enzymes in LBRs are α-mannosidase, α–fucosidase, lipase and β- galactosidase 4. Lactobacillus sp., found to be predominant in food waste treating LBRs.
32