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In the year 2011, the quantity of solid waste in Bangkok (Thailand) was ∼9237 tons per day, an amount equivalent to 21% of the total solid waste generated across the country (43,779 tons per day according to the Pollution Control Department, 2012). The development of biogas production systems from market waste has been growing, especially in Bangkok, where more than 150 vegetable and fruit markets operate today . Most of these markets produce primarily fresh organic waste, for example, rotten fruit and vegetables, scraps, and food leftovers. The disposal of this high-moisture-content putrescible waste creates different environmental problems at the existing treatment sites, such as offensive odors, global warming gas emissions, and leachate disposal. These problems could be tackled by promoting market waste separation at the source for biogas production, an action already included in the plan launched during the Conference of Renewable Energy World-Asia, 2012. This plan primarily aimed to increase the alternative energy participation from 7 to 20% of the total energy demand by 2021.
Anaerobic digestion (AD) is a technology that is useful to treat a variety of biodegradable materials. This process can produce significant amounts of energy and results in environmental benefits by reducing the emission of greenhouse gases, such as carbon dioxide (CO2) and methane (CH4), into the atmosphere. AD processing involves four major phases: hydrolysis, acidogenesis, acetogenesis, and methanogenesis . Each step is performed by a uniquely functioning group of microorganisms: hydrolytic, acetogenic, and methanogenic microorganisms . The growth requirements, growth kinetics, and levels of sensitivity of environmental conditions differ among these microorganisms . The balance between these groups of microorganisms must be maintained during the entire AD process to avoid system failure .
The characteristic features of input material are an important factor that affects the balance of microorganisms and their performances in an anaerobic digester. The digestion of substrate-like market waste can be difficult because this waste contains cellulose and hemicellulose fibers as well as an easily degradable fraction, such as carbohydrates [6, 7]. However, high concentrations of these compounds can affect the digester function. Mata-Alvarez et al.  stated that carbohydrate-rich waste, such as fruit and vegetable waste, causes rapid acid formation during the initial stage of AD, which decreases the pH value in the digester. If this change remains uncorrected, the continued accumulation of acids will inhibit the methanogenic activity, which causes the digester to “sour” and operate inefficiently [9-13].
Volatile fatty acids (VFA) in the form of undissociated (nonionized) acid is toxic to microorganisms due to its ability to penetrate the cell membrane . The undissociated acid (UA) molecules pass through the permeable membrane to affect the intracellular pH of the bacterial cell, which limits microbial growth and activity . In previous studies, van den Heuvel et al.  and Maddox et al.  determined the critical inhibitory concentrations of undissociated butyric acid (50 mM) that affect ATP formation, which affects bacterial growth. Infantes et al.  studied the effect of undissociated acetic on cell growth and substrate consumption. They mentioned that the growth of biomass was inhibited when the UA concentrations ranged from 50 to 70 mM. UA exerts a toxic effect on acetoclastic methanogens 0.26 and 1 mM [19, 20], while Yamaguchi et al.  reported 8.87 mM as the concentration of UA that is toxic to thermophilic methylotrophic methanogens.
Current literatures on VFA inhibition have highlighted the fact that the change in the acid molecular form between dissociated and undissociated is a function of the pH [18, 22, 23]. Thus, this article studies the effects of pH adjustment on the AD of market waste in a single-phase reactor, focusing on the hydrolysis-acidogenesis stage, where UA are produced. The impact of these acids was analyzed over the methane concentration in the outlet gas. To this end, three experimental batch trials were conducted at the laboratory scale using waste with a dry matter content ranging from 8 to 11%. Each experiment was carried out at a different pH regime, the first one without pH control, the second one inducing neutral pH values, and the third one implementing stepwise pH.
MATERIALS AND METHODS
Feedstock and Seed Sludge Preparation
The fresh market waste used in this study was collected from the Tungkru market in Bangkok, Thailand. The compositional variability quartering technique of the market waste was analyzed several times using manual sorting on weekdays and weekend days. Feedstock market waste, which contained vegetable and fruit residues at a ratio of 80:20 (wet weight basis), consisted of the following components: 30% cabbage, 20% Chinese kale and Chinese white cabbage, 30% other vegetables, and 20% seasonal fruit. This composition represented the actual composition of the largest fruit and vegetable market, Seimummuang, in Thailand . A mixture of fruit and vegetable waste was shredded to particle size smaller than 5 cm and homogenized after first removing any nonbiodegradable matter, such as plastic, cans, and inert materials. The anaerobic sludge was taken from the Upflow Anaerobic Sludge Blanket reactors of a pig farm wastewater treatment plant in Ratchaburi, Thailand. The concentrations of total solid (TS) and total volatile solid (VS) in the seed sludge were 34.1 and 22.9 g/L, respectively, with a pH of 7.0.
Experimental Design and Operation
Three sets of experiments, M1 with a neutral pH, M2 with a stepwise pH adjustment and MC without pH adjustment as the control reactor, were conducted at room temperature for 119 days. The batch experiment was performed to investigate the effect of different pH control regimes for neutral pH control and compared with the performance of the stepwise pH reactor, where the system pH was stepwise increased from pH 4.5 to 5.5, 6.0, and 6.5. The changes in the pH during the tests were maintained at the intended level via the manual addition of solid sodium bicarbonate (NaHCO3) as follows: 31 days (days 10–41) for M1 and 29 days (days 1–29) for M2; both reactors were then operated 78 and 90 more days, respectively, without adding any external alkalinity. Shredded market waste and seed sludge at 70:30, v/v, were added to 5-l (3.8-l working volume) closed acrylic pipes in the studied reactor (Figure 1). A 5-cm thick gravel layer covered with nylon screens was placed at the bottom of the reactor to filter the leachate. An anaerobic environment was created by flushing the headspace with nitrogen gas for 5 min prior to sealing. Mixing in the buffered experimental reactors (except for MC) was performed via leachate recirculation from the bottom through the upper part of the reactor in the upward direction once a day using a peristaltic pump (Masterflex model 7520-47) at a flow rate of 12 L/d. To extract the organic content generated from the market waste feedstock, leachate collection, and recirculation was initiated the day after the start-up. Approximately only 10 mL of leachate was sampled for the daily analyses to prevent disturbing the naturally leaching leachate volume in the digester. Liquid samples were taken to analyze the pH, total volatile fatty acid (TVFA), soluble and total chemical oxygen demand (SCOD and TCOD), and individual volatile acids concentration on alternate days at the start of the experiment to once every several days using the method described in section “Analysis.”
The TS, VS, SCOD, and TCOD contents were analyzed according to standard methods . The total VFA content and alkalinity were measured using a titration technique that accounts for ∼70–80% of the total VFA concentration . The pH was measured with a Hach sension 378 pH meter. The individual VFAs were analyzed using gas chromatography (Agilent 6890) equipped with a flame ionization detector and a capillary column type DB-FFAP (30 m × 0.25 mm, 0.25 µm). The flame ionizing detector temperature was 300°C, and the oven temperature was programed from 100 to 200°C at 25°C/min. Helium at 2 mL/min was used as the carrier gas. Liquid samples were first centrifuged at 10,000 revolutions per minute (rpm) for 10 min and then acidified to pH < 2.0 using 0.02 N oxalic acid to ensure that the VFAs were in their acidic forms. The supernatants of the acidified samples were filtered using a 0.45-µm membrane filter and analyzed for their C2-C5 VFA content using a standard curve obtained by injecting a standard mixture of five VFAs of known concentrations. The UA content was determined based on the following equation: [HA] = CT [H+]/KA + [H+], where [HA] is the UA concentration (mg/L), CT is the total acid concentration (mg/L), [H+] is the hydrogen ion concentration (mole/L), and KA is the ionization constant of each acid (acetic acid 1.75 × 10−5, propionic acid 1.40 × 10−5, butyric acid 1.48 × 10−5, and valeric acid 1.60 × 10−5). The daily gas production was measured using a water substitution gas collector connected to a counter (Omron H7EC, Thailand). Biogas samples were withdrawn from the reactor headspace using a 1-mL gas tight syringe (VICI, Precision Sampling) and subsequently injected into the GC. The gas composition (CO2 and CH4) was analyzed using a gas chromatograph (Shimadzu 14B) equipped with a thermal conductivity detector and Porapak Q column (2 m × 3 mm, 80/100 mesh). The injector, column, and detector were operated at 100, 70, and 120°C, respectively. Helium was used as the carrier gas at a flow rate of 30 mL/min. The chemical properties of the market waste feedstock in terms of the total lipids were determined using the Soxhlet method. The protein content was determined based on the total nitrogen content (Micro Kjeldahl method) with a conversion factor of 6.25 . The carbohydrate content was analyzed using the phenol-sulfate method.
RESULTS AND DISCUSSION
Fresh market waste contains high amounts of organic matter, which indicates a high VS content, specifically 88 ± 1% (Table 1) on a dry weight basis (data taken from an average of three samples). The moisture content of market waste is high (90 ± 1%), which facilitates AD, bacterial movement, mass transport, and nutrient assimilation by microorganisms. The total carbon content approached 50%. The C/N ratio was approximately 100:4.4–6.3, which is similar to the result of 100:5.4 obtained by Tubtong (2010). This value was in the optimum C/N ratio for the bioconversion of vegetable biomass to methane, which was 100–128:4 . A basic analysis showed that carbohydrates (17.4–32.4%) and proteins (13.9–19.7%) were the main components, while lipids (2.8–3.9%) were a minor component of the market waste.
Table 1. Characteristics of vegetable and fruit market waste mixtures.
Regulated buffer supplementation to maintain a neutral-pH improved the production of SCOD and TVFA (Figure 2). After introducing buffer starting on day 11, a remarkably high SCOD and VFA production was observed. The SCOD concentration substantially increased from 27.6 to 46.7 g/L. The yield was found to be 0.63 g SCOD/g VS. Similar to the treatment of municipal solid waste reported by Bhattacharyya et al. , ligno-cellulosic material with various alkaline components enhanced the hydrolysis. These investigators found that the addition of strong bases, such as NaOH or Ca(OH)2, can increase the solubilization of organic matter via depolymerization reactions of the lignin-cellulose complex. The TVFA in M1 reached a peak value of 42.7 g HAc/L by day 77 with a generation rate of 0.93 g HAc/L d, which accounted for a yield of 0.62 g HAc/g VS. The addition of high amounts of alkali to maintain a neutral pH can sustain the steady production of high levels of TVFA, which indicates that acidogenesis can be extended.
The yields of VFA and SCOD at the neutral pH condition were 63 and 31% higher than the uncontrolled pH condition, respectively, which suggested that neutral pH control strongly benefited acidification compared with solubilization. The TVFA concentration increased 5.5-fold on day 77, while the TVFA increased only 2.7-fold over the initial condition by day 119 when the pH was not controlled. Compared with the control reactor, the production of VFA was higher in the reactor that was supplied with buffer. In addition, neutral pH benefits the solubilization of SCOD concentration, resulting in a 2-fold increase over the control. The VFA generation rate for the uncontrolled pH reactor was limited to 0.63 g HAc/L d during the first 2 weeks of operation, while the neutral pH reactor could accumulate higher amounts of TVFA. This finding indicates that the addition of buffer can remedy the slowdown in the acid production caused by the depletion of easily digestible substrates and the hydrolysis of cellulose and hemicelluloses .
Single-stage anaerobic digesters that treated market waste without pH control clearly failed. An acid stress phenomenon, indicated by a low pH and VFA accumulation at the beginning of the batch, restricted the acid production, and subsequently affected methanogenesis (see section Undissociated VFA and Methane Production). Therefore, the neutral pH condition can help to overcome the solubilization and acidification limits during start up when processing rapidly biodegradable market waste.
Acid Production with Stepwise pH Control
Stepwise pH control also improved acid production (Figure 2c). However, the acid yield was not comparable to that of the neutral pH control regime. The TVFA concentration increased stepwise during the 29 days of alkali addition (0.3 g NaHCO3/g VSadded or ∼10 times lesser reactor M1); specifically, the TVFA concentration was 8.5, 17.5, 20.6, and 22.4 g HAc/L at pH 4.5, 5.5, 6.0, and 6.5, respectively. The maximum SCOD concentration of 32.5 g/L was recorded on day 16 and remained nearly constant thereafter. The TVFA concentration reached a peak of 22.4 g HAc/L by day 29 (end of buffer addition) and stabilized at 20 ± 1.6 g HAc/L by day 61, when methanogenesis began. The VFA production rate was maximized during the first 13 days of batch runs, after which the VFA content in the stepwise pH control reactor increased by 35.4%, whereas it was only 17.6% in the uncontrolled pH reactor for the next 21 days (days 14–29).
Figure 3 shows the phase transfer performances of the controlled and uncontrolled pH regimes. The TVFA/SCOD represents the amount of solubilized matter that converts to VFA and expresses the degree of success of the acidification . The TVFA/SCOD under neutral pH control approached 0.9 within 31 days. This finding indicates that monometric product did not accumulate or that monomers rapidly ferment when the pH is controlled at a neutral value . A delay in the pH adjustment in a stepwise manner retarded the COD conversion to VFA, reaching a value of 0.87 on day 61. Uncontrolled pH also produced a high TVFA/SCOD ratio, but this regime required long periods of time to reach a TVFA/SCOD value of 0.8 on day 96. The results showed that the degree of acidification, expressed as TVFA/SCOD, was similar for all reactors, but the operation times varied. The slow acidogenesis of the stepwise pH control provided the slow growing methanogens sufficient time to grow and stabilize, which therefore led to the onset of methane production in the single-phase digester.
Controlling the pH can overcome the cessation of acid production that occurs in the uncontrolled pH reactor. Adjusting the pH to neutral values prevented the pH from dropping too low during the startup and led to further VFA production. Both the pH-control digesters (M1 and M2) proved beneficial for acid production, as demonstrated by prolonged acidogenesis and a promotion of phase transfer. However, the difference in the pH control regimes resulted in different acid production profiles. Neutral pH control causes a substantial increase in the VFA production, while the stepwise pH control led to a sustained level of TVFA without blocking methanogenesis (see section “The role of stepwise pH control on the alleviation of acid stress”).
Undissociated VFA and Methane Production
The UA concentration was far away from the threshold limit of 30 mg/L for methanogenesis when the pH was not controlled [20, 33]. The high UA concentrations of 3748–11,968 mg/L were the result of a low pH that shifted the VFA toward its undissociated form (Figure 4). The average methane content was ∼4.5%. The results indicated that methanogenesis was inhibited in the control reactor, which contained high amounts of UA.
Usually, changing the VFA form (dissociated or undissociated) is a function of the pH. As shown in Figure 4, both M1 and M2 demonstrated that the UA content could be controlled. The UA content decreased from 5306 to 81 mg/L after 29 days of stepwise pH control (Table 2) and declined thereafter without further pH adjustment to less than 10 mg/L (data not shown). A methane content of 60% was reached because acid stress from UA that occurs at low pH can be inhibited. The amount of UA for the initiation of methanogenesis in this study was lower than the amounts found in the literature.
Table 2. Variations in the UA from a stepwise increase in the pH, from pH 4.5 to 6.5.
Before buffer addition
After buffer addition
Conversely, although the UA profile of neutral pH control followed the same trend as the stepwise pH control, methanogenesis did not occur. During the early stage, the concentration of UA decreased drastically from 1119 to 89 mg/L and then stabilized, ranging from 9 to 50 mg/L until the end of the run. Although the undissociated VFA concentrations agreed well with the threshold value for methanogenesis, a poor methane content was found (9.1%, max.). Grady et al.  also observed a slight acid inhibition in an anaerobic digester under neutral pH because the UA content is a function of both the total VFA and the pH. This result contradicted the stepwise pH control results observed in this study. This finding implies that different pH control regimes can lead to different methanogenic performances. The lack of methanogenesis at a neutral pH is linked to the UA content as well as other parameters. In this study, we found that a high TVFA concentration occurred at a high TVFA/SCOD ratio, which might be responsible for the lack of methanogenesis.
Relationship Between TVFA/UA and Methane Content
As shown in Figure 4, the UA content can be reduced by adjusting the pH to values close to neutral. Lower UA contents result in a larger difference in the VFA and proportion of UAs. Figure 5 shows the relationships between the methane content and the proportion of TVFA/UA. The methane content and the TVFA/UA proportion strongly correlated for both neutral and stepwise pH control (r = 0.86 and r = 0.94, respectively; P < 0.05).
The TVFA/UA proportion was limited to ∼2000 for neutral pH control (Figure 5a), as a high TVFA level greater than 33 g HAc/L was found (see Figure 6) and resulted in methanogenesis failure, whereas the proportion of TVFA/UA in the stepwise pH control regime gradually increased from 10 to over 10,000. This finding could be attributed to two possible reasons, a shift toward the dissociated form by increasing the pH and decreasing the VFA and the undissociated VFAs after the onset of methanogenesis. In general, because a minimum methane concentration of 60% was required to initiate methanogenesis, the proportion of TVFA/UA was ∼2000 (Figure 5b) and can be considered to be the threshold limit of TVFA/UA for methanogenesis; however, this threshold proportion must be considered along with the TVFA concentration. During methanogenesis, the reduction in the TVFA concentration that was attributed to a progressively increasing proportion of TVFA/UA exceeded 10000. The methane content was poor at the same proportion of TVFA/UA (∼2000) under neutral pH control when the TVFA concentration was high.
The Role of Stepwise pH Control on the Alleviation of Acid Stress
The control reactors with both neutral pH and stepwise increasing pH resulted in a decrease in the undissociated VFA concentration. A neutral pH control led to a relatively high concentration of TVFA, which forced a limit on the TVFA/UA proportion of not greater than 2000. The failure encountered with the neutral pH control could be linked to the disruption of the high accumulated TVFA level. As shown in Figure 6, neutral pH control (M1) led to the high level of TVFA and prolonged acidogenesis, whereas increasing the pH in a stepwise manner (M2) played an important role in maintaining the TVFA level at a safe range for methane formation.
Fruit and vegetable waste could easily result in acidification when the buffering capacity available in the AD system is insufficient. Therefore, the reactor requires externally buffering to enhance the safety of the process, which could be effectively controlled using well-known process parameters, such as the VFA:Alkalinity and FOS/TAC ratios (the ratio between volatile organic acids content and total inorganic carbon content). However, excess VFA production due to alkali addition is a drawback rather than an advantage for a typical rapid biodegradability substrate because it creates an unfavorable environment for methane production.
In fact, an increase in the TVFA and UA concentrations characterized the state of AD stress as well as an acid stress-related parameter in the biogas plant, such as the FOS/TAC value. Our findings suggested that a well-adjusted proportion of TVFA/UA could avoid acid stress in a single-phase digester caused by rapid acidification. To control the rate of VFA release, the proportion of TVFA/UA can be used to estimate the allowable TVFA level for biogas production at which the aforementioned parameters cannot be defined. The use of the TVFA/UA ratio for a single stage reactor at neutralizing conditions was found to be beneficial, which made this ratio the process parameter choice for biogas production when treating highly biodegradable organic wastes. For ratios <2000, methanogenesis was inhibited by VFA accumulation.
In this study, methanogenesis was achieved by maintaining the TVFA level within a defined limit of ∼20 g HAc/L and decreasing the UA content until the TVFA/UA ratio reached 2000. A remarkable biogas production of 60% CH4 was noticed on day 61, when the generation rate was ∼0.36 L/d. The biogas production after 108 days of operation was 23.2 L, 65% of which was methane with a yield of 0.2 L CH4/g VS. Therefore, stepwise pH control was successfully employed to produce biogas from a single-phase system.
The deceleration in acid production can be avoided by maintaining a neutral pH value during the initiation of digesters that process rapidly biodegradable market waste. This process maintains the UA level below the inhibition level for methanogenesis. However, the excess VFA formation during the process can lead to an unfavorable environment for methane production. Stepwise pH control can help prolong the acid accumulation and stabilize the TVFA concentration while minimizing the UA content at an appropriate level. Therefore, stepwise pH control alleviated acid stress problems and induces methanogenesis in a single-stage system. We found that the TVFA concentration and TVFA/UA proportion should be maintained at ∼20 g HAc/L and 2000, respectively, to initiate methane production in both processes that treat rapidly biodegradable material at neutral pH conditions.
The authors gratefully acknowledge to The Joint Graduate School of Energy and Environment of King Mongkut's University of Technology Thonburi and the Center for Energy Technology and Environment, Ministry of Education Thailand, for providing financial assistance. The authors wish to thank Dr. Pawinee Chaiprasert and Dr. Saroch Boonyakitsombut for their valuable suggestions and help with this research.