The objective of this study was to evaluate the performance of anaerobic digestion (AD) within the intermediate zone, specifically at 45°C. Single-stage batch anaerobic digestion system was developed in the lab and performance was monitored for more than 2 years. The AD system was able to achieve high biogas production with about 62% - 67% methane content. The digester exhibited high acetate accumulation, but sufficient buffering capacity was observed as the pH, alkalinity and volatile fatty acids-to-alkalinity ratio were within recommended values. The system achieved 36.5% reduction of total solids (TS) and 47.8% reduction of volatile solids (VS), which exceeded the required VS destruction efficiency for Class A biosolids. The pathogen counts were less than 1000 MPN/g total solids in the effluent, which also satisfied Class A biosolids requirements. The accumulation of acetate was presumably due to the high temperature which contributed to high hydrolysis rate. Consequently, it produced large amount of toxic salts that combined with the acetate, making them not readily available to be consumed by methanogens. The slower degradation of acetate was observed by the kinetic parameters. Accumulation of acetate contributed to 52% to 71% reduction in acetate degradation process, but was not completely inhibitory. The methanogens existing in the system were mostly thermo-tolerant acetate-utilizing methanogens, and specifically from Methanosarcinaceae species.
Anaerobic digestion has been the most widely used method for the degradation and stabilization of domestic and industrial wastes for many years. This process has received increased attention in the recent years due to global energy crisis, as anaerobic digestion has great potential for producing energy-rich biogas and nutrient-rich biosolids. Strict environmental regulations for pollutant minimization also demonstrated the need of anaerobic digestion for safe and effective method of organic waste disposal.
Anaerobic digestion (AD) is a biological process that converts complex substrates into biogas and digestate by microbial action in the absence of oxygen through four main steps, namely hydrolysis, acidogenesis, acetogenesis and methanogenesis [
Although a large number of researchers have addressed the performance of mesophilic and thermophilic anae- robic digestion, very few studies have investigated the possibility of operating anaerobic digestion in the intermediate zone between mesophilic and thermophilic range. This is based on the assumption that limited microbial activity occurs within the intermediate zone of 40˚ - 50˚C due to the fact that neither mesophilic nor thermophilic microorganisms would flourish in the intermediate zone, as the microorganisms are trying to cope with the changing environment, hence causing the digestion to slow down [
According to U.S. EPA, Class A biosolids can only be produced through treatment processes involving high temperatures such as thermophilic anaerobic digestion [
Two single-stage batch anaerobic digestion systems were constructed and operated in parallel, where the second system served as a duplicate. The digester was made of high density polyethylene (HDPE) 25 L brewery tanks of Hobby Beverage Equipment Company (Temecula, California). The temperature of the digester was maintained at 45˚C with a heating system controlled by a thermostat connected to a temperature sensor inserted into the digester. The digester was covered with aluminum foil and temperature adjustable heating tape was placed on top of the foil to ensure even heat distribution to the digester and to provide protection from the heating tape so that physical failure of the polyethylene would not occur. Gas mixing was applied by circulating the headspace gas to the bottom of the digester by using a peristaltic pump (Cole-Parmer, Vernon Hills, IL). The digester was equipped with a gas collection flask and Wet Tip gas meter (Nashville, TN) to measure volume of gas production.
The anaerobic digester system was initiated by inoculating with seed sludge from well operating mesophilic anaerobic digester in Alexandria Wastewater Treatment Plant, AlexRenew (Alexandria, VA). This was done to benefit from the seed sludge that usually contains abundant amounts of microorganisms such as methane formers and acid forming bacteria. After approximately 3 days of seed feeding, the digester system was fed with raw municipal sludge obtained from Blue Plains Advanced Wastewater Treatment Plant (Washington, DC). The raw sludge was the effluent after the thickening process. During this initial phase, no effluents were taken out as the microorganisms need to have ample time to establish themselves within the new environment. After approximately two weeks, duration that was considered sufficient for microorganisms’ adaptation, the feeding and effluent withdrawing schedules were started. The digester was fed once a day and an equal amount of digested sludge or effluent was withdrawn directly before feeding.
The operational volume of the anaerobic digester was 15 L. The total solids (TS) content of the feed sludge (influent) was 6.5% with an average organic loading rate (OLR) of 5.22 kgVS/m3・d. The feed sludge was acclimated to the desired temperature of 45˚C by incubating for approximately 2 h before feeding. The solid retention time (SRT) of 10 d was selected for the digester, which is regarded as optimum time for the methanogens to complete the digestion process. Because the digester was kept completely mixed throughout the study, and
feeding and wasting were done in equal amounts, solids retention time (SRT) of the reactor was equal to hydraulic retention time (HRT).
Inorganic nutrients in the form of chloride salts were added into the anaerobic digester systems, as an initiative to increase the rate of anaerobic metabolism in term of methane gas production and low VFA accumulation [
The operating parameters of the feed and digested sludge were analyzed regularly for more than 2 years of operations. The pH was measured daily, while the alkalinity, total solids (TS), volatile solids (VS), soluble COD (sCOD), total COD (tCOD), total ammonia nitrogen (TAN) and volatile fatty acids (VFA) were analyzed weekly. Alkalinity, TS, VS, COD and TAN tests were conducted in accordance to Standard Methods 2320 B, 2540 B, 2540 E, 5220 D and 4500-NH3 B & C, respectively [
The effluent from the digester was collected and used as a sample for batch kinetic tests. The effluent was first diluted to eliminate the possible inhibition effects of existing soluble compounds such as ammonia and acetate. Subsequently, the samples were mixed and placed in 500 mL glass bottles. The temperature was controlled and the headspace was flushed with nitrogen gas to create anaerobic condition within the vessel. The substrate (acetic acid) was added in a diluted form of synthetic acetic acid 2.0 N (Fisher Chemical, Pittsburgh, PA) to the samples. The tests were conducted using 400 mL of digester effluent, mixed at least once a day using a magnetic stirrer and incubated at 45˚C. The concentrations of the remaining acetate were observed for every 30 min to 1 h intervals and the tests were conducted until all acetate has been harvested or when there is no further destruction of the remaining acetate.
During the kinetic tests, the concentrations of remaining unionized acetate, S at different sampling time, t was collected. Unionized acetate progress curve was plotted and the model was developed to fit these curves. The kinetic coefficients were determined using a Monod-based model [
The Monod model is shown in Equation (1), and the model proposed by Ref. [
Inorganic nutrients | Concentration (g/L) |
---|---|
FeCl2・4H2O | 35.6 |
ZnCl2 | 2.08 |
NiCl2・6H2O | 4.05 |
CoCl2・6H2O | 4.04 |
MnCl2・4H2O | 3.61 |
where µ, µmax and S are specific growth rate (t−1), maximum specific growth rate (t−1) and concentration of growth-limiting substrate (mg/L), respectively. Ks is half saturation constant, substrate concentration at one-half the maximum growth rate. In the model, the best estimates of the kinetic coefficients and constant can be determined by minimizing differences between model predictions to observed experimental values of sampling time t using Equation (2).
where Y is the yield coefficient (mg/mg), and rmax is the maximum bacterial growth rate (mg/L・d). The model predictions of t were calculated by using the known value of Y together with initial estimates of the parameters that are sought (i.e. Ks, rmax and S0). In this model, S0 was also used as a fitting parameter because it was not known with greater certainty than the other data points, thus, it would not be appropriate to force the best-fit curve through the measured value of S0 [
The inhibition kinetic coefficient, KI and dimensionless inhibition concentration, IHAc, were determined using a Michaelis-Menten model [
where v is the rate of substrate utilization (substrate/unit volume・time), Vm is the maximum specific rate (t−1) and Km is the substrate utilization constant or saturation constant (mg/L). Detail parameter estimation process using the extended version of the model, depending on the type of inhibition, are available in [
The performance of the anaerobic digestion (AD) at 45˚C was assessed based on methane gas production, methane yield, biogas composition, ammonia content, VFA content, buffering capacity, TS and VS reductions, soluble and total COD reductions as well as pathogen destruction. Daily methane production of the anaerobic digester is illustrated in
that as the days progressed, the microbes had established, flourished and expanded their colonies, hence, contributed to the gradual increase of methane production.
The specific methane yield for the AD system was 0.56 ± 0.04 m3・CH4/kgVS removed, which is within the lower end of the recommended range of 0.60 to 1.60 m3・CH4/kgVS removed [
Since inhibition of the anaerobic digestion system is primarily indicated by the levels of total ammonia (TAN) and free ammonia generated within the system, therefore both were measured in the study. The average TAN and free ammonia concentrations were found to be 1735 mg/L and 83 mg/L, respectively. The TAN concentrations were slightly higher than the lower limit of the inhibitory region of 1500 and 3000 mg/L [
Buffering capacity is also a crucial indicator of the well-being of anaerobic digester. The average pH and alkalinity of the digester were 7.41 and 10,082 mg/L, respectively. pH value was within high end of the operable pH range value of 6.6 - 7.6 [
Additionally, other than ammonia and salts, the increase in alkalinity was also attributed to its high
concentration of VFA observed in 45˚C systems. As VFA concentrations began to increase, they are neutralized by the bicarbonate alkalinity, thus, forming volatile acid alkalinity [
With regard to the ability of the digester for reducing TS and VS content, the system was able to achieve 36.5% reduction of TS and 47.8% reduction of VS, which exceed the required average VS destruction efficiency of 45% for Class A biosolids criterion for vector attraction reduction [
The system was able to achieve significantly high reduction of soluble COD (sCOD), total COD (tCOD) and pathogen from the feed, as presented in
Anaerobic digester at 45˚C produced significantly high acetate accumulation, as shown in
The population was most probably a thermo-tolerant acetate-utilizing mesophilic methanogens, a mesophile that can survive at high temperature but whose optimum growth rate occurs at mesophilic temperatures [
Inorganic nutrients | Feed (mg/L) | Digester effluent (mg/L) | % Reduction |
---|---|---|---|
Soluble COD | 11057 ± 1578 | 7012 ± 639 | 37.41 |
Total COD | 74250 ± 6183 | 46232 ± 3380 | 38.25 |
Pathogen count | 7.16 × 106 | 2.52 × 102 | >99.99 |
their methanogenic activity. At 45˚C AD system, the population of true acetate-utilizing mesophilic methanogens or thermo-tolerant acetate-utilizing mesophilic methanogens were gradually diminishing, due to the high ammonia content and high temperature, hence, leaving more acetate unutilized.
As for the presence of propionate, though was commonly believed to also inhibit methanogenesis, the concentrations were well below the tolerable range of 800 mg/L to 3000 mg/L [
A series of kinetic tests were performed in order to investigate the accumulation and inhibitory effect of acetate despite the abundant generation of methane in 45˚C anaerobic digestion system. The comparisons were made between systems containing different sources of acetate, one was found naturally in the system, while the other one, was added in a form of acetic acid to the system. In this study, the former was termed as background acetate (B/g HAc) or salt-originated-acetate and the later was termed as added acetate (HAc) or acetic acid-origi- nated-acetate. Four sets of experiments were conducted varying the concentration of background acetate and added acetate, and corresponding kinetic parameters are summarized in
In anaerobic digestion process, hydrolysis is always rate limiting [
So (mg/L) | rmax (mg/L/day) | Ks (mg/L as HAc) | IHAc | |
---|---|---|---|---|
Run 1 | ||||
B/g HAc + HAc = 500 + 0 mg/L | 0.41 | 0.04 | 101 | 0.48 |
B/g HAc + HAc = 500 + 1000 mg/L | 1.31 | 0.14 | 237 | 1.36 |
Run 2 | ||||
B/g HAc + HAc = 1000 + 0 mg/L | 0.87 | 0.03 | 60 | 0.33 |
B/g HAc + HAc = 1000 + 1000 mg/L | 1.62 | 0.13 | 279 | 1.26 |
Run 3 | ||||
B/g HAc + HAc = 1500 + 0 mg/L | 1.12 | 0.03 | 60 | 0.29 |
B/g HAc + HAc = 1500 + 1000 mg/L | 2.37 | 0.10 | 257 | 0.98 |
Run 4 | ||||
B/g HAc + HAc = 1000 + 0 mg/L | 0.87 | 0.03 | 61 | 0.33 |
B/g HAc + HAc = 0 + 1000 mg/L | 0.90 | 0.10 | 191 | 1.00 |
salts [
Another observation from
which contributed to 52% to 71% reduction in acetate degradation process, could not be considered as completely inhibitory as there was still utilization of acetate at a lower rate.
Methanogen Illumina Assay of the sludge from the 45˚C anaerobic digester verified the presence of the presumptive methanogens. Methanosarcinaceae, a methanogen from mesophilic group was the dominant methanogen found in the system. Comparing the Ks value of the kinetic experiment (Run 4: B/g HAc + HAc = 0 + 1000 mg/L) with Ks values of two dominant mesophilic methanogens, Methanosarcinaceae with Ks of approximately 200 mg/L as HAc and Methanosaetaceae with Ks of approximately 30 mg/L as HAc [
Anaerobic digesters operated at 45˚C for more than 2 years demonstrated high biogas production with methane content of 62% - 67%. The system was able to achieve 47.8% reduction of volatile solids and pathogen count of less than 1000 MPN/g total solids in the digester effluent, satisfying U.S. EPA Class A biosolids requirements. Although the digester showed high acetate accumulation, it was not detrimental as evidenced by adequate buffering capacity and abundant production of methane. The accumulation of acetate produced large amount of toxic salts that combined with the acetate, making them not readily available to be consumed by methanogens, and resulted in 52% to 71% reduction in the acetate degradation process. The methanogens existing in the system were mostly thermo-tolerant acetate-utilizing methanogens with 82.47% Methanosarcinaceae species.
This study was supported by a grant from the United States Department of State under the Pakistan-US Science and Technology Cooperation Program. The authors gratefully acknowledge the assistance of the staff of Blue Plains Advanced Wastewater Treatment Plant in Washington, DC, for providing the feed sludge for the digesters.
Nuruol SyuhadaaMohd,TaqsimHusnain,BaoqiangLi,BaoqiangLi,ArifurRahman,RumanaRiffat, (2015) Investigation of the Performance and Kinetics of Anaerobic Digestion at 45°C. Journal of Water Resource and Protection,07,1099-1100. doi: 10.4236/jwarp.2015.714090