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Techset Composition Ltd, Salisbury

Environmental Technology, 2013 http://dx.doi.org/10.1080/09593330.2013.810294

TENT810294

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Biological pretreatment of non-occulated sludge augments the biogas production in the anaerobic digestion of the pretreated waste activated sludge
J. Merrylina , S. Adish Kumarb , S. Kaliappanb , Ick-Tae Yeomc and J. Rajesh Banua
a Department

of Civil Engineering, Regional Centre of Anna University, Tirunelveli, India; b Department of Civil Engineering, Ponjesley College of Engineering, Nagercoil, India; c Department of Civil and Environmental Engineering, Sungkyunkwan University, Seoul, South Korea (Received 26 February 2013; nal version received 23 May 2013 ) High-eciency resource recovery from municipal solid waste (MSW) has been a focus of attention. The objective of this research is to develop a bio-pretreatment process for application prior to the anaerobic digestion of MSW to improve methane productivity. Bacillus licheniformis was used for pretreating MSW (non-occulated with 0.07% citric acid), followed by anaerobic digestion. Laboratory-scale experiments were carried out in semi-continuous bioreactors, with a total volume of 5 L and working volume of 3 L. Among the nine organic loading rates (OLRs) investigated, the OLR of 0.84 kg SS m3 reactor day1 was found to be the most appropriate for economic operation of the reactor. Pretreatment of MSW prior to anaerobic digestion led to 55% and 64% increase of suspended solids (SS) and volatile solids reduction, respectively, with an improvement of 57% in biogas production. The results indicate that the pretreatment of non-occulated sludge with Bacillus licheniformis which consumes less energy compared to other pretreatment techniques could be a cost-eective and environmentally sound method for producing methane from MSW. Keywords: anaerobic digestion; methane production; municipal solid waste; biochemical methane potential; bacterial pretreatment

Q1

1. Introduction Anaerobic digestion is one of the more commonly used stabilization processes in sludge management, and provides eective pathogen destruction, reduction of volatile solids (VS) and odour potential, and an energy source in the form of biogas.[1] Much progress has been made in the fundamental understanding and control of the anaerobic digestion process, and the design and application of equipment. This process is one of the dominant processes for stabilizing sludge because of the energy conservation and recovery. Anaerobic digestion of municipal wastewater sludge can in many cases produce sucient digester gas to meet most of the energy needs for plant operation.[2] Microorganisms and microbialenvironment conditions are crucial in this process, as in other biological treatment processes. During the anaerobic digestion of biosolids, the rst stage in the degradation of particulate organic matter is the solubilization and enhanced hydrolysis of complex polymeric organic carbon structures. The slow degradation rate of sludge in an anaerobic digester is due to the rate limiting step of sludge hydrolysis. This is caused by the low biodegradability of the cell walls and extracellular biopolymers in sludge. It is important to reduce the amount of sludge produced and to reduce its residual organic content. The methods of

improvement of biodegradability of a particular substrate are mainly based on better accessibility of the substrate for microbes. Pretreatment can improve the subsequent anaerobic digestion. One advantage of sludge solubilization prior to anaerobic treatment is that increasing the amount of released soluble substrate signicantly enhances volatile fatty acids (VFAs) generation for improved gas production during anaerobic digestion. Second, pretreatment decreases the viscosity of the sludge, permitting a greater solids concentration to be fed to an anaerobic digester. A higher solids concentration in the feed either enables increased digestion times in an existing digester, or allows for a small digester volume.[3] Various sludge pretreatment methods have achieved signicant results in the lysis or disintegration of municipal sludge, and have thereby enhanced biogas production. Several methods have been studied to treat municipal sludge, including thermal,[4] chemical,[5] ultrasonic [6] and mechanical methods.[7] In addition, biological-microbial enzymes or bacterial methods,[8,9] and the combination of such processes, have also been used.[10] Bacteria are usually contained within a occulated matrix of exopolymeric substances.[11,12] Extracellular polymeric substances (EPS) as a network detains the extracellular hydrolytic enzymes.[13] Researchers have

Corresponding

author. Email: rajeshces@gmail.com

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J. Merrylin et al. 2.3. Bacterial pretreatment Bacillus licheniformis, a thermophilic protease producing bacterium, was used to pretreat the deocculated sludge. This bacteria was mass cultivated in a nutrient broth at a temperature of 55 C in a 5 L fermenter, and the cells were harvested from the pure cell culture at the early stationary phase (20 h). These cells served as the inoculum. These cells were innoculated in a concentration of 77 106 bacterial cells mg1 of SS. The inoculated sludge was pretreated for 24 h and this pretreated sludge was used for further studies. 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224

Q2

also suggested that a higher concentration of EPS protein could bring more negatively charged amino groups,[14,15] and could thus strengthen the electrostatic interactions with cations. So the extraction of EPS from sludge could lead to a reduction in sludge volume and mass.[16] When enzymes are added to sludge, they are often adsorbed on to the sludge matrix, which can lead to inactivation. Treating the sludge with a cation binding agent prior to enzymatic pretreatment improves the organic matter in the sludge as a substrate for the enzymes through making components previously protected by the EPS structure more available to be degraded.[17] The use of microbial enzymes for the enhancement of degradation of WAS is the basis for another process called the enzymatic hydrolysis process.[18,19] The primary benet is the killing of pathogens, but a further benet is the enhancement of biogas production in anaerobic digestion. A 10% improvement in biogas production was found in laboratory trials.[19] When a laboratoryscale mesophilic anaerobic reactor received this pretreated municipal WAS, a 1.5-fold increase in biogas production compared with a system receiving unconditioned sludge was found. In this study, a mesophilic anaerobic reactor is considered, which is fed with pretreated municipal WAS.

2. Materials and methods 2.1. Sludge sampling Municipal sewage sludge was collected from secondary clarier in a local wastewater treatment plant in Trivandrum, Kerala in a sterilized container. The collected sample was aerated continuously at room temperature to prevent settling of the solids and to prevent constant changes in the biological sludge. The amount of biogas production from the sludge depends on the characteristics of the waste. Sludge characteristics such as protein, carbohydrates, total solids (TS), VS contents, and pH should be investigated before pretreatment. The pH of the raw sludge was 6.5, the CODS was 200 mg L1 , the total COD was 12 g L1 , the TS content was 13 g L1 , the VS content was 8 g L1 and the suspended solids (SS) content was 10 g L1 .

2.2.

Removal of EPS

EPS is removed from sludge before pretreatment using the optimized citric acid concentration of 0.07%, according to the procedure by Merrylin et al.[20] The dosage of citric acid used is indicated as the gram equivalent, i.e 0.07 gm of citric acid is added to 100 mL of sludge. The mixture was kept at 4 C for 3 h with constant agitation to ensure proper mixing.[20] Through EPS removal, the sludge was dispersed and deocculated. This deocculation accelerates the bacterial activity. This deocculated sludge is further subjected to bacterial pretreatment.

2.4. Biochemical methane potential A biochemical methane potential (BMP) test was used to evaluate biogas recovery from sludge after bacterial pretreatment. In this test, biogas production of raw and pretreated sludge samples (both occulated and deocculated) was initially determined by batch tests at 35 C in three reactors. In reactor 1, 50 mL of deocculated sludge (EPS removed and bacterially pretreated) was inoculated with 150 mL of cow rumen and fed into the 300 mL reactor. In reactor 2, 50 mL of occulated sludge (bacterially pretreated) was inoculated with 150 mL of cow rumen and was fed into a 300 mL reactor. Reactor 3 was run as a control in which 50 mL and 150 mL of inoculum (cow rumen) was used to determine endogenous amount of biogas production. Rumen bacteria from the digestive tract of a cow were used as an innoculum. The rumen is an exclusive organ of ruminant animals in which the digestion of cellulose and other polysaccharide molecules occur through the activity of specic microbial populations. The capacity of cellulose digestion that these animals possess is related to the presence of anaerobic microorganisms in its rumen, which decompose up the glucose polymer chains to acetate. Methanogenic microorganisms that convert acetate into methane and carbon dioxide also exist in rumen.[21] The better performance of the inoculated reactors may be related to the potential increase in the number of indigenous anaerobic microorganisms of rumen, which contributed substantially to the degradation of the organic material in the reactor. After adding the substrates and the inoculum, the head space above the liquid in the assay reactors is purged with 30% CO2 and 70% N gas to enhance the anaerobic conditions in the reactor. The gas mixture was introduced into the reactor at the rate of 1 L min1 for 5 min. Then, the reactor was sealed with a rubber septum and aluminium to make it air tight, and then placed on a shaker (150200 rpm). The biogas was measured by inserting a needle into the septum. The gas pressure in the reactor was allowed to displace the syringe plunger, and the displaced volume is recorded. The cumulative gas production was measured using a water displacement method. The reactors were continuously shaken to allow sucient blending. The methane content in the biogas was measured using a gas chromatograph. The modied Gompertz equation was to study the cumulative biogas

Environmental Technology 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280

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Figure 1.

Semi-continuous anaerobic reactor set-up.

generation and the kinetics of biogas production. Bt = B exp exp Rb B exp( t ) + 1 , (1)

where Bt is the cumulative biogas produced (mL) at any time (t ), B is the biogas production potential (mL), Rb is the maximum biogas production rate (mL day1 ), and is the lag phase (days), which is the minimum time taken to produce biogas, or the time taken for bacteria to acclimatize to the environment, in days. The constants B, Rb and were determined using the non-linear regression approach with the aid of Polymath software. In many biological elds, the basic knowledge of phenomena is insucient to build a mechanistic model. In this study, the eect of EPS removal in sludge to produce methane in the anaerobic digester was analysed using a Gompertz model,[22] as shown in Equation (1). This equation was utilized by researchers to study the cumulative methane production in biogas production.[23] 2.5. Anaerobic semi-continuous reactor Two identical semi-continuous reactors (control and experimental) were run. The control reactor (CR) was fed with the raw sludge and the experimental reactor (ER) was fed with pretreated sludge. Their total volume was 5 L, and their working volume was 3 L, the reactor set-up is shown in Figure 1. The reactors were inoculated with the activated slurry collected from a local biogas plant in India. Organic loading rate (OLR) of 0.16, 0.26, 0.34, 0.4, 0.5, 0.58, 0.66, 0.83 and 1 kg SS m3 reactor d1 with the solids retention

times (SRT) ranging from 30 to 10 days were sequentially applied to investigate the performance of the anaerobic digestion of the pretreated sludge. Moreover, in order to preserve the nitrication capability of the activated sludge, high SRT were applied to the biomass in the activated sludge process (10 days). The rst three loadings were carried out with dierent mixed liquor SS (MLSS) of 5000, 7500 and 10,000 at the constant SRT of 30 days. The rest of the loadings were carried out with dierent SRT of 25, 20, 17, 15, 12 and 10 days with a constant MLSS of 10,000. The CR was also run under the same conditions as that of the ER. Feeding and withdrawals were carried out each day using peristaltic pumps in a semi-continuous mode. Biogas production was measured by the liquid displacement method, in which the dierence in the water level in a cylinder linked to the reactors is observed. The displaced liquid is therefore considered to have the same volume as the produced biogas. 2.6. Analytical parameters The SS, VS and alkalinity parameters were measured according to the standard methods [24] to determine the sludge reduction eciency for each reactor. The pH was measured using a digital pH meter. VFAs were analysed by distillation-titration method, and the result was expressed in acetic acid. The extent of MLSS reduction during the experiment was calculated by the equation mentioned below: MLSS reduction (%) = Initial Final 100. Initial (2) Q3

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J. Merrylin et al. biogas production potential of 114 mL at a maximum biogas production rate of 86 mL h1 , with a lag phase of 4 days. In reactor 3, which contained the raw sludge, the biogas production potential was 21 mL at a maximum biogas production rate of 17 mL h1 . The modied Gompertz equation was observed to adequately describe biogas production with R2 values of 0.9917, 0.9909 and 0.9962 for reactors 1, 2 and 3, respectively. 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448

The methane content in the biogas was analysed using a Baroda gas chromatograph equipped with a thermal conductivity detector and porapack Q column with hydrogen as the carrier gas at a ow rate of 40 mL min1 .

3.

Results and discussion

3.1. Bacterial pretreatment Sludge deocculated with 0.07% citric acid was pretreated with a protease secreting bacteria, Bacillus licheniformis.[25] Sludge disintegration in biological systems incorporates the mechanism of cell lysis transforming the cell content into the medium, the breakdown of EPS fractions in the occulated matrix, and the biodegradation of the end products by microbial metabolism.[26] The eciency of pretreatment was measured in terms of COD solubilization and was found to be in the range of 1012% throughout the study (data not shown). The anaerobic degradability of the pretreated sludge was assayed using the BMP test.

3.2. BMP test The biogas production from non-occulated, occulated and raw sludge was examined in three reactors. The estimated kinetic constants using non-linear regression and other characteristics of the reactors 13 are shown in Table 1. A graphical representation of cumulative biogas production is shown in Figure 2. Biogas production is slow at the beginning and at the end of observation; this indicates that the biogas produced under batch condition corresponds to specic growth rate of methanogenic bacteria.[27] To analytically quantify the parameters of the batch growth curve, a modied Gompertz equation was tted to the cumulative biogas production data. Values of parameters obtained are listed in Table 1. The best t to the Gompertz equation is compared with the experimental data in Figure 2. During the rst 5 days, biogas production is low due to the lag phase of microbial growth. After 7 days, the biogas production increased signicantly due to exponential growth of microorganisms. After 20 days, the biogas production stabilized due to the stationary phase of microbial growth. At the end of 21 days, reactor 1 produced the highest cumulative biogas production potential (B) of 195 mL at a maximum biogas production rate (Rb) of 127.37 mL h1 , with a lag phase () of 1.8 days. Reactor 2 had an estimated
Table 1.

3.3. Semi-continuous anaerobic digesters 3.3.1. Start-up of the reactor During the acclimatization phase the biologically pretreated sludge was fed at an OLR of 0.16 kg SS m3 reactor d1 and the set-up was continued until a stable period was achieved. Stable periods were dened by the process showing a fairly constant performance in terms of biogas production, VFA concentration and pH in the reactor, without showing symptoms of any process unbalance or failure (i.e. cease in biogas production, VFA accumulation or pH drop for at least one loading rate). With this assessment after 50 days, the uctuations in these parameters were less than 10% and it was believed that steady state was achieved. After normal operation had been established, fresh solids were added to the digester by mixing them with the digesting sludge, which greatly improves the rate of digestion.

3.3.2. OLR variation The OLR is a measure of the biological conversion capacity of the anaerobic digestion system. Feeding the system above its sustainable OLR, results in low biogas yield due to biomass concentration and the mass transfer rate of substrates to bacteria.[28] In such a case, the feeding rate to the system must be reduced. OLR is a particularly important control parameter in continuous systems. Both the biogas yield and methane production have a close relation with OLR, so special emphasis should placed on reactor performance in steady-state conditions in the OLR that do not result in reactor failure.[29] In this study, during a total period of 300 days, the performance of the semi-continuous anaerobic reactors was evaluated at dierent OLRs. The feeding was initialized with a low OLR of 0.16 kg SS m3 reactor d1 and the corresponding SRT was 30 days. When the OLR was increased to 0.26 kg SS m3 reactor d1 , the SRT was maintained at 30

Kinetic parameters calculated from the theoretical model for various samples. Modied Gompertz parameters (model)

Digester Control (1) Flocculated (2) Non-Flocculated (3)

B, L/(gVS) 0.395313 2.02478 3.571262

Rb, L/(gVS d) 17.5008 106.9286 127.37811

, d 4.216581 3.594685 1.845676

R2 0.996254 0.990903 0.99175

RMSD 0.000132 0.001179 0.001882

Environmental Technology 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504
0.30 Non-flocculated-exp 0.25 Non-flocculated fit Flocculated-exp 0.20 Flocculated-fit 0.15 Control-exp Control-fit 0.10

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Specific gas production (L/gVS)

0.05

0.00 0 5 10 15 20 25

Days

Figure 2.

Modied Gompertz model t to the experimental data.

days but the MLSS in the feed sludge was increased from 5000 to 7500 mg L1 . Similarly for the consecutive OLR (0.34 kg SS m3 reactor d1 ), the MLSS was increased from 7500 to 10,000 mg L1 . The OLR was shifted to the next higher value, once the reduction eciencies of SS and VS were found to be consistent with a particular OLR. An increase in the OLR increases the extent of the reactions, and vice versa. Each time sludge is withdrawn, a fraction of the bacterial population is removed, thus implying that the cell growth must at least compensate for the cell removal to ensure steady-state operation and to avoid process failure.[30] 3.3.3. SS reduction SS reduction is an indication of sludge stability, and it is used for assessing the eectiveness of a process in stabilizing the sludge.[31] It is observed in Figure 3(a) that the overall performance eciency in terms of reduction in the SS increases as the SRT was reduced. SRT has a signicant eect on the digester performance. At individual SRTs, steady-state operations were achieved, and the results mentioned are the average of ve consecutive consistent readings. The SRT is determined by the average time it takes for organic material to be digested completely, as measured by the chemical and biological oxygen demand of the exiting euent. Speeding up the process will make the process more ecient. Microorganisms that consume organic material control the rate of digestion that determines the time for which the substrate must remain in the digestion chamber. Figure 3(a) shows the variation of SS in the digested sludge. There was an SS reduction of about 26% in the ER, and 11% SS reduction in the CR. SS analysis shows that there was an increase of 55% in the amount of SS reduction in the ER

compared to the CR. This indicates that the biological pretreatment with Bacillus licheniformis has a great advantage over the non-pretreated sludge (control). It is also observed in the gure that the maximum SS reduction was achieved when the loading rate was 0.84 kg SS m3 reactor d1 , and the reduction % declined with further increases in OLR. 3.3.4. VS reduction VS reduction was examined as well to evaluate the reactor performance and stability of the digestate. During the digestion process, volatile solids are degraded to a certain extent and converted into biogas. The sludge volume is thereby reduced. The degree of stabilization is often expressed as the per-cent reduction in volatile solids.[2] It was observed from Figure 3(b) that with the increase in OLR, there is an increase in VS reduction. A maximum VS degradation value of 30% was achieved when operating with loading rate of 0.84 kg SS m3 reactor d1 . On the other hand, when the loading rate was increased further, there was a decrease in VS reductions, and only 28.6% was obtained as illustrated in Figure 3(b). Comparably, these VS reduction were lower than the results by Castillo et al. who also reported that VS removal eciency is decreased with decreases in the retention time.[32] 3.3.5. Biogas production Figure 4 shows the total biogas production and methane production at dierent OLRs and SRTs. Initially, the biogas production was 24 mL g1 with addition of VS, Q4 and then it followed a zig-zag path for quite some time during the acclimatization period. During each phase of OLR, the total biogas production in the digester showed

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J. Merrylin et al.

(a)

5 OLR Experimental reactor (ER)-SS % Control reactor (CR)-SS %

30.00 25.00 20.00 15.00 10.00 5.00 0.00 70 105 140 175 Time (days) 210 245 280 SS reduction (%)

4.5

4 3.5 3 2.5 2 1.5 1 0.5 0 35

(b) 5
OLR 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 35 85 135 185 Time (days) 235 285 Control reactor (CR)-VS % Experimental reactor (ER)-VS %

35.00 30.00 25.00 20.00 15.00 10.00 5.00 0.00

Figure 3.

(a) Removal prole of SS of digested sludge at dierent OLRs. (b) Removal prole of VS of digested sludge at dierent OLRs.
200.00 180.00 Daily biogas - ER Daily biogas - CR 500 450 400 350 300 250 200 150 100 50 0 35 60 85 110 135 160 185 Time (days) 210 235 260 285

Daily biogas (mL/g VSS Added)

160.00 140.00 120.00 100.00 80.00 60.00 40.00 20.00 0.00

Methane - ER Methane - CR

Figure 4.

Total biogas and methane production in the dierent reactors.

617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672

OLR (KgSS/m 3 reactor day)

OLR (KgSS/m 3 reactor day)

Methane (mL)

VS reduction (%)

Environmental Technology 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 appreciable increases until a stage when methanogenesis could not work quickly enough to convert acetic acid into methane. The results showed that compared with the control, the methane amount and the biogas production were increased by 57% and 53%, respectively, due to the biological pretreatment. In contrast to the present study, in works with physical and chemical pretreatment, signicantly higher levels of biogas production in the range of 8488% were obtained.[33] However these pretreatment techniques had intensive energy demands and high operation costs.[23] Biogas production was higher with 0.84 kg SS m3 reactor d1 , but with an increase in OLR to 1 kg SS m3 reactor d1 , it was decreased, which may have been due to either a lack of a critical requirement of inoculum to take the load of additional OLR, or that a lag period proportional to the growth inhibitorrich recalcitrant chemical composition was required.[34] The cumulative biogas produced during the entire reactor operation period of the ER was 51 L, and the cumulative biogas produced during the entire reactor operation period of the CR was 22 L. The decreasing methane content can also be attributed to the higher OLR, which favours a higher growth rate of the acid-forming bacteria over the methanogenic bacteria. Thus, the methane conversion process was adversely aected by reducing the methane content, which led to the formation of carbon dioxide at a higher rate.[35] Therefore, the methane yield was reduced at higher OLR due to the lower methane content. Thus, at OLR greater than 0.84 kg SS m3 reactor d1 , the methane production was decreased. The methane gas production rate increased appreciably and ranged from 20 to 412 mL day1 , as the methane content in the biogas was varied from 65% to 70%.[2] The higher gas production in the bacterially pretreated non-occulated sludge evidently indicated that it hydrolysed more organic material solution, which was immediately used by anaerobic bacteria, and eventually facilitated the digestion processes. With a decrease in SRT from 12 to 10 days, there was a decrease in biogas production. A shorter SRT results in decreasing biogas production.[23] Therefore, considering both biogas and methane production, an SRT of 12 days was found to be a suitable retention time for eective sludge degradation. Therefore, the digester eciency (kg of VS fed and its conversion to methane) and the digestion eciency as a function of OLR were optimal at 0.84 kg SS m3 reactor d1 . 3.3.6. pH During the start-up of the digester, the pH was in the range of 8.2, which gradually decreased with continuous feeding. The decrease in pH can be explained by the formation of acidic compounds through the depolymerization of the organic matter by enzymes produced by microorganisms present in mainly the secondary sludge.[36] After the acclimatization period, the pH of the euent from the

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semi-continuous digester remained steady in the range of 7.38.2 but dropped when the OLR increased from 0.84 to 1 kg SS m3 reactor d1 , as shown in Figure 5. It was also observed that during every shift to the next lower SRT, the pH dropped. The pH of a digester may drop to below 6.6 if there is an excessive accumulation of VFAs, due to excessive organic loading or the presence of toxic materials in the digester. Low pH has inhibitory eects on the methanogen.[37] The operational range of pH should be between 6.6 and 7.6, with the optimum range being 7.0 7.2.[38] It is evident from the graph that with an increase in OLR, there is decrease in pH. The results also reveal that there were uctuations, but the pH remained in the methanogenic range, which proves that the digester could maintain its pH to some extent, even though higher organic loading was supplied. 3.3.7. Alkalinity pH cannot be an eective measure of the stability of an anaerobic process when there is a high buering capacity.[39] Under this condition, alkalinity levels reveal a potential anaerobic process performance directly. Calcium, magnesium, and ammonium bicarbonates are examples of buering substances found in a digester. The digestion process produces ammonium bicarbonate from breakdown of protein in the pretreated sludge feed. The concentration of alkalinity in a digester is, to a great extent, proportional to the solids feed concentration. A wellestablished digester has a total alkalinity of 20005000 mg L1 .[2] The variation of alkalinity and pH with digestion time in the present study is displayed in Figure 5. The total alkalinity in this digester was in the range of 30005000 mg L1 . The alkalinity of feed sludge and digested sludge varied signicantly as pretreatments were used. An increase in alkalinity is normally due to the activity of methanogenic bacteria, which can produce alkalinity in the form of carbon dioxide, ammonia, and bicarbonate.[40] Carbon dioxide is produced in the fermentation and methanogenesis phases of the digestion process. Due to the partial pressure of gas in a digester, the carbon dioxide solubilizes and forms carbonic acid. The carbon dioxide concentration in the digester gas is therefore reective of the alkalinity requirements. 3.3.8. Volatile fatty acid VFA is an important intermediate produced during the anaerobic digestion process. VFA reduces the pH of the system, which has an inhibitory eect on the growth of the methanogenic bacteria. VFAs should be less than 250 mg L1 for satisfactory performance of the anaerobic process. In the present study, VFA in the initial stage was above 250 mg L1 , which was in turn neutralized by increases in alkalinity. Higher levels of VFA in the reactor during the rst phase of operation indicate the prevalence of acid fermentation. Furthermore, with the decrease in SRT

8 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840
6,000 5,500 5,000 4,500

J. Merrylin et al.
16 Alkalinity pH 14

12 Alkalinity (mg/L) 4,000 3,500 3,000 8 2,500 2,000 6 1,500 1,000 35 70 105 140 175 Time (days) 210 245 280 pH

10

Figure 5.

Variation of pH and alkalinity in the digested sludge at various OLRs.

400 VFA VFA/Alkalinty 350 0.120 300 0.100 VFA/Alkalinity 0.140 0.160

250

0.080

0.060 200 0.040 150 0.020

100 35 70 105 140 175 Time (days) 210 245 280

0.000

Figure 6.

Variation of VFA and VFA/alkalinity in the digested sludge at various OLRs.

there was decrease in VFA and it was within 250 mg L1 resulting in the better performance of the anaerobic digestion (Figure 6). The ratio of VFA to total alkalinity was in the range of 0.020.07 (Figure 6). The range of 0.020.08

for the ratio of VFAs to total alkalinity ratio is a good working range of an anaerobic process.[41] VFAs are the main precursors of methane formation. Studies have shown that 70% of the methane in an anaerobic system is produced from

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VFA (mg/L)

Environmental Technology 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952

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7.5 7.5 7.5 7.5 7.5 7.5 7.3 7.4 7.3 7.2 7.0 6.9 3650 3600 3500 3350 2980 2970 6.9 8.3 10.4 11.3 11.4 11.7 5.7 7.2 9.2 10.2 10.3 10.6 52 83 106 143 189 185 38 50 60 72 84 83 366 377 383 375 360 350

18 24 0.58

15 24 0.66

VFAs in the form of acetic acid. During digestion, VFAs are formed and consumed in the hydrolysis or acidogenic phases. It is therefore dicult to determine the exact VFA concentration during digestion, since the formation rate is expected to be equal to the further disappearance during methanogenesis.[30] During digestion, organic substances degraded by microorganisms are the only carbon source, and the basic source of easily assimilated organic carbon comprises short chain carboxylic acids. 4. Selection of the signicant OLR and SRT The results obtained after reactor stabilization are reported in Table 2. The optimal loading rates and retention time for methanogenic digestion will depend on the quality of the feedstock and on the desired eciency of the overall process. For the most cost-eective operation, it is important to operate a reactor at the highest loading rate possible. A high organic loading will normally result in excessive VFA production in the digester with a consequent decrease in pH, and will adversely aect the methanogenic bacteria. A low organic loading will not provide a sucient quantity of biogas for other uses, but will make the digester unnecessarily large. An SRT that is too short will not allow sucient time for anaerobic bacteria to metabolize the wastes, especially for methane-forming bacteria. Excessive SRT could result in the excessive accumulation of digested materials in the digester, and the construction of a digester that is too large.[42] An optimum range of SRT for suspended growth digesters falls within the range 1060 days, while attached growth and high-rate digesters can be operated at much shorter SRTs.[43] It was identied from the results that an OLR of 0.84 kg SS m3 reactor d1 operated with 12 days SRT is the most appropriate OLR for ecient and economical operation of the digester. The OLR and retention times reported in various studies on municipal solid waste digestion have ranged from 0.07 to 0.35 lb VS ft3 day and from 10 to 30 days, respectively. Most of these studies have included co-digestion with raw sewage sludge. Uma et al.[23] have reported OLRs of 3.555.9 g VS L1 day with retention times of 20, 15 and 12 days, and it was indicated that good performance of the reactor was achieved with an SRT of 15 days while using dairy sludge. 5. Conclusions The inuence of bacterial pretreatment (after EPS removal) prior to anaerobic digestion was studied. BMP assay results of pretreated sludge conrmed that the reactors fed with bacterially pretreated sludge indicated better performance in terms of organic matter stabilization and gas production compared with the CR. BMP results also indicated that there is more gas production in the reactor with non-occulated pretreated sludge compared with the occulated pretreated sludge. Thus, non-occulated pretreated sludge was chosen for semi-continuous digesters, and by

CR

Digesters performance after stabilization.

Feed pH Outlet pH Alkalinity (mg L1 ) SS removal (%) VS removal (%) Methane production (mL) Total biogas (mL gVS1 added) VFA production (mL day1 )

SRT (days) Experimental run (days) OLR (kg SS m3 reactor d1 )

Parameters

Table 2.

30 121 (a) 0.16 (b) 0.26 (c) 0.33 6.9 7.9 4500 10.1 12.4 67 60 246

6.9 6.9 6.9 6.9 6.9 6.9 7.4 7.8 7.6 7.8 7.3 6.9 4000 4400 4200 4500 3800 3845 13.9 17 21.1 25.7 25.8 24.9 17.2 20.1 24.1 24.8 29.5 28.7 106 161 216 325 412 393 78 96 123 162 184 175 244 247 243 245 240 239

ER

25 24 0.4

20 25 0.5

18 24 0.58

15 24 0.66

12 24 0.84

10 17 1

30 121 (a) 0.16 (b) 0.26 (c) 0.33 7.5 7.4 3700 5.1 3.9 32 29 364

25 24 0.4

20 25 0.5

12 24 0.84

10 17 1

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J. Merrylin et al.
[16] Tian Y. Behavior of bacterial extracellular polymeric substances from activated sludge: a review. Int J Environ Pollut. 2008;32:7889. [17] Wawrzynczyk J, Szewczyk E, Norrlw O, Dey SE. Application of enzymes, sodium tripolyhospate and cation exchange resin for the release of extracellular polymeric substance from sewage sludge characterization of the extracted polysaccharide/glycoconjugates by panel of lection. J Biotechnol. 2007;130:274281. [18] Mayhew M, Le M, Brade C, Harrison D. The united utilities enzymic hydrolysis process validation of phased digestion at full-scale to enhance pathogen removal. In: WEF Proceedings of the Residuals and Biosolids Conference, Baltimore (MD); 2003. [19] Mayhew M, Le M, Ratcli R. A novel approach to pathogen reduction in biosolids: the enzymic hydrolyser. Water Sci Technol. 2002;46:427434. [20] Merrylin J, Adish Kumar S, Kaliappan S, Yeom IT, Rajesh BJ. Eect of extracellular polymeric substances on sludge reduction potential of Bacillus licheniformis. Int J Environ Sci Technol. 2013;10:8592. [21] Brock TD. Biology of microorganisms. 10th ed. NJ: Q5 Prentice-Hall; 2002. p. 1104. [22] Elaiyaraju P, Partha N. Biogas production from co-digestion of orange peel waste and jatropha de-oiled cake in an anaerobic batch reactor. Afr J Biotechnol. 2012;11: 3393345. [23] Uma R, Adish KS, Kaliappan S, Ick TY, Rajesh BJ. Low temperature thermo-chemical pretreatment of dairy waste activated sludge for anaerobic digestion process. Bioresour Technol. 2011;103:415424. [24] APHA. Standard methods for the examination of water and wastewater. 21st ed. Washington (DC): American Public Health Association; 2005. [25] Merrylin J, Adish Kumar S, Kaliappan S, Yeom IT, Rajesh BJ. Eect of EPS removal on the sludge reduction potential of Bacillus licheniformis on its optimized pH conditions. Water Q6 Environ J. 2012. doi/10.1111/wej1204. [26] Ayol A, Filibeli A, Sir D, Kuzyaka E. Aerobic and anaerobic bioprocessing of activated sludge: oc disintegration by enzymes. J Environ Sci Health A Tox Hazard Subst Environ Eng. 2008;43:15281535. [27] Luengo PL, Alvarez JM. Inuence of temperature, buer, composition and straw particle length on the anaerobic digestion of wheat straw-pig manure mixtures. Resour Conserv Recycl. 1988;1:2737. [28] Vandevivere P, De Baere L, Verstraete W. Biomethanization of the organic fraction of municipal solid wastes. J. MataQ7 Alvarez. London: IWA; 2003. pp. 111140. [29] Linke B. Kinetic study of thermophilic anaerobic digestion of solid wastes from potato processing. Biomass Bioenerg. 2006;30:892896. [30] Appels L, Dewil R, Baeyens J. Ultrasonically enhanced anaerobic digestion of municipal sewage. Int J Sust Eng. 2008;1:94104. [31] Gholamreza M, Hassan A, Akram J. Eect of ozonation on reduction of volume and mass of waste activated sludge. J Appl Sci Res. 2008;4:122127. [32] Castillo MEF, Cristancho DE, Arellano VA. Study the operational condition for anaerobic digestion of urban solid waste. Waste Manage. 2006;26:546556. [33] Zhang HJ. Sludge treatment to increase biogas production. Trita-LWR Degree Project 1020; 2010. [34] Kirtane RD, Suryawanshi PC, Patil MR, Chaudhari AB, Kothari RM. Optimization of organic loading rate for different fruit wastes during biomethanization. J Sci Ind Res. 2009;68:252255.

combining pretreatment with anaerobic digestion, it led to 56% and 58% of SS and VS reduction, respectively, with a 57% improvement in biogas production when operated at an ecient OLR of 0.84 kg SS m3 reactor d1 . Acknowledgements
The authors are thankful to the Department of Biotechnology, India, for nancial assistance to this project (BT/PR13124/GBD/ 27/192/2009) under their scheme Rapid Grant for Young Investigator.

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