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Using an Amylase Pretreatment of Pig Manure to Enhance Biogas Production

Jianchang Li, Kewei Sun, Juan He, Qiuling Chen

National Engineering Center of Solid Waste Resources Recovery Kunming University of Science and Technology Kunming 650093, PR. China
AbstractAn experimental study was conducted that promoted the efficiency of anaerobic digestion by hydrolyzing pig manure in advance with amylase before loading it into the digester. A modified Gompertz equation was applied to determinate the kinetic parameters of biogas production. The results showed that the removal percentage of TS and VS, the biogas production potentials and rates have been improved through an amylase pre-treatment. Especially through amylase and -amylase pretreatment together, the removal percentage of TS and VS, as compared with control group, was increased by10.84% and 11.11% respectively, biogas production potential increased by 13.10%, and specific biogas production rate increased by 9.30%. Experimental results also indicate that the digestive process goes through four stages: lag stage, peak stage, sub-peak stage, and fade stage. Different stages which occurred in this experiment might reflect the characteristics of sequential biodegradation of different organic substrates from easy and simple to difficult and complex. Key words: Biogas production; Anaerobic digestion; Hydrolyses pretreatment; Amylase pretreatment

Jianchang Li
Provincial Key Laboratory of Rural Energy Engineering Yunnan Normal University Kunming 650092, PR. China Some researchers believe that hydrolysis is the rate-limiting step in the process of anaerobic digestion of organic wastes[5-7]. Several methods have been used to increase biogas yield, such as accelerating the hydrolysis of organic matter by increasing temperature[8] or putting biogas bio-additive into the digester[9]. Others researchers reported the methanogenic step is a ratelimiting step. As a result, a lot of good quality inoculums are put into biogas plants in the initial start-up phase, which also is a way to cure disorder in biogas digesters[9]. Obviously, although the opinions are different, all these attempts contribute to increasing biogas yield and /or rate. Hydrolase as a biocatalyst has been widely used in predegradation of fats, protein, starch, cellulose and semicellulose. However, using a hydrolase pre-treatment for anaerobic fermentation is novel and rarely reported. Goel investigated the enzymatic behavior of activated sludge under anaerobic and aerobic conditions. The specific enzyme activities of -glucosidase and protease did not change significantly during the anaerobic and the aerobic phases of sequencing batch reactor (SBR)[10]. Guellil tested the enzymatic activity of exopolymeric substances (EPSs) extracted from activated sludges for their ability to hydrolyse the organic colloidal fraction of wastewater[4]. Ubukata investigated the mechanisms of hydrolysis and the absorption of starch by activated sludge, hydrolysis of starch to maltose and maltotriose and not to glucose, appears to be the ratedetermining step during the removal of starch[11]. Zhang has researched the relationship between hydrolase and biogas yield in the process of anaerobic fermentation using pig manure as feedstock. The results showed that enzyme activity is related to biogas yield, and adding hydrolase can enhance the biogas yield[12]. Therefore, based on the concept of hydrolysis rate-limiting steps, this investigation will try to develop an approach to hydrolase pre-treatment in digestion, and focu on amylase pretreatment of pig manure, a main feedstock used in China, to enhance biogas yield and minimize fermentative time. II. MATERIAL AND METHODS



Anaerobic digestion is a biochemical process, in which a variety of microorganisms work together to break down complex organic wastes resulting in biogas production. The process of decomposing organic matter by microorganisms is a consecutive process and is divided into three steps, as described by Lawrence and McCarty in 1967[1-3]: hydrolysis, acetogenesis and hydrogen formation, and methane formation, which involves hydrolytic and fermentative bacteria, hydrogenproducing and acetate-forming bacteria, and methaneproducing bacteria. The first step is the breakdown of particulate matter to soluble organic constituents that can be processed through the bacterial cell wall. This step is carried out by a variety of hydrolytic and fermentative bacteria through the release of extra-cellular enzymes that reside in close proximity to the bacteria[4]. The soluble organic materials that are produced through hydrolysis consist of sugars, fatty acids, and amino acids. In the second step, those soluble constituents are further converted to carbon dioxide, hydrogen and a variety of short chains of organic acids by hydrogen and acid forming bacteria. In a final step, one group of methane-producing bacteria reduce the hydrogen to produce ammonia, hydrogen sulfide, and methane; another group convert acetic acid to methane gas.

A. Reactor A batch anaerobic reactor, as shown in Fig.1, is made up of three parts: a temperature controller, a water bath made of glass and a set of digestive units that include one 500 mL flask as a fermentative bottle, another 500 mL flask as a gas measuring

Supported by the National Engineering Center of Solid Waste Resources Recovery in Kunming University of Science and Technology, China Corresponding author: Kewei Sun, E-mail:

978-1-4244-6255-1/11/$26.00 2011 IEEE

liquid displacement aspirator bottle and a 1,000 mL measuring cylinder to determine the gas volume by water displacement.

TABLE I. Procedures

EXPERIMENTAL PROCEDURES CG 90 230 45 90 100 450 EG1 90 230 0.5 45 90 100 450 EG2 90 230 45 0.25 90 100 450 EG3 90 230 0.5 45 0.25 90 100 450

Figure 1. Schematic representation of an anaerobic reactor: 1. temperature controller; 2. temperature probe; 3. heater; 4. fermentative flask; 5. water bath; 6. biogas container; 7. 3-way stopcock; 8. measuring cylinder

Pig manure/g Water/mL -Amylases/g Heating at 80C/min -Amylases/g Heating at 60C/min Inoculums/g Total mixture weight /g

B. Feedstock Fresh pig manure, taken from a rural household barn in Kunming City and stored in a low temperature refrigerator (21C) was used as feedstock. Its total solid (TS) was 28.45%, its volatile solid (VS) was 22.31% and its pH value was 6.80. C. Innoculums Inoculums were taken from a biogas digester in the biomass energy research group of the Provincial Key Laboratory of Rural Energy Engineering, Yunnan Normal University. When pig manure digestion was completed, without bigas production, the anaerobic activated sludge deposited in the bottom of digester was used as inoculums. It had a TS of 13.4%, a VS of 9.86% and a pH value of 7.19. D. Amylase -Amylase (EC, alternative names: 1,4--D-glucan glucanohydrolase) is produced by Beijing Aoboxing Biotech Company Ltd. It has an optimum pH value of 6.0, a temperature range of 60-70C might even be used at 90C, and its enzyme activity is 4,000 unit/g. -Amylase(EC, alternative names: 1,4--D-glucan glucohydrolase) is produced by Zhangjiagang Jinyuan Biochemical Co., Ltd. It has an optimum pH value range of 4.0-4.5, a suitable temperature of 60C, and its enzyme activity is 400,000 unit/g. In this experiment enzymatic dose, reaction time and temperature were referred to within the parameters studied by Hao and Gu[13,14]. E. Experimental procedure 1) Experimental groups To investigate the effect of amylase pretreatment on the pig manure anaerobic digestion four groups were formed: one control group (CG) and three experimental groups (EG) 1, 2 and 3. With the exception of adding amylase, all groups were subjected to the same experimental conditions. The pretreatment procedure is shown in table 1. Pig manure in CG was directly used as substrate without amylase pretreatment, whereas, pig manure in EG was pre-treated in advance by amylase, and then used as substrate. Pig manure in EG1 was hydrolyzed by -amylase, in EG2 by -amylase and in EG3 by -amylase and -amylase together.

2) Preparation of feedstock Feedstock was prepared at a TS concentration of about 8%: 90.0 g of fresh pig manure with a TS concentration of 28.45% was weighed in an electric tray balance, was put into a beaker with a volume of 500 mL, and then 230 mL tap water was added. The concentration of TS was about 8%. 3) Amylase pre-treatment All beakers filled with feedstock were placed into a water bath at a temperature of 80C, starting a magnetic stirrer to stir feedstock. In the first step 0.5 g -amylases were added to beakers labeled EG2, and EG4, and kept the temperature for 45 minutes. Then temperature was lowered to 60C; 0.25 g amylase was added to beakers labeled EG3 and EG 4, and temperature kept stable for 90 minutes. 4) Anaerobic digestion starting-up Firstly the treated feedstock was transferred into digestive flasks, 100 g inoculums were added, some water was supplied until total weight of digestive mixture was up to 450g. Secondy digestive flask, gas-container and measuring cylinder were connected together with plastic gas pipes and 3-way stopcocks. Finally digestive flasks were placed into a water bath. 5) Fermentative temperature Fermentative process was carried out at a mesophilic temperature of 35C, controlled by a temperature controller. F. Analytical methods 1) TS and VS TS and VS (based on wet) of fresh pig manures, inoculums and digested residues were measured by the gravimetric techniques, referred to General Analytical Methods of Biogas Digestion (Biogas Institute of Chengdu, 1984).. 2) pH value The measure of pH value was made by an Orion pH-meter. 3) Biogas yield and rate Biogas yield was recorded through water displacement, and biogas rate was calculated in term of the biogas yield per day. 4) Methane content Methane content was analyzed by a gas chromatograph (HP4890D) equipped with a thermal conductivity detector (TCD) and two 1.5 m 2 mm stainless-steel columns in a series packed with 6FT Porapack 80/100 mesh in the first and 10FT molecular sieve 13X 45/100 mesh in the second. Injector, column and detector temperatures were kept at 150C, 80C and 250C, respectively. Argon was used as the carrier gas. The injection volume of the gas sample was 0.5 mL at a time.



A. Removal percentage of TS and VS TS and VS of pig manures and inoculums were directly obtained by test, but TS and VS of digestive mixture (feedstock and inoculums) were calculated according to the quantities of fresh pig manure and inoculums and the total weight of digestive mixture. The calculative formula is (1) and (2).
TS= W1 TS1+W2 TS2 100% W0

analyze the value of TS, VS and pH, and then they were compared with their previous values. The results are shown in the Table 2. In this table, TS and VS represented the removal percentage of TS and VS, respectively, while TS and VS referred as to the EG variation of TS and VS compared with CG. The calculation formulas as follows (3), (4), (5) and (6). The minus value of TS and VS indicated the decreasing value of them after digestion, while the minus value of TS and VS presented the decreasing percentage of TS and VS. But when discussed, it was explained using words for convenience.
TS = TSafter TSbefore 100 TSbefore

(1) (2)

W1 VS1+W2 VS2 VS= 100% W0

(3) (4) (5) (6)

Where W0 is total weight of mixture material (450 g); W1 is weight of fresh pig manure (90 g); W2 is weight of inoculums (100 g); TS1 is total solid of fresh pig manure (28.45%); VS1 is volatile solid of feedstock (22.31%); TS2 is total solid of inoculums (13.40%); VS2 is volatile solid of inoculums (9.86%). Once the digestive process ended and no more biogas was produced, digested samples were immediately taken out to

VS =

VSafter VSbefore 100 VSbefore

TS =

TS of CG TS of CG 100 TS of CG VS of EG VS of CG 100 VS of CG

VS =


Before fermentation 8.67 8.67 8.67 8.67 6.65 6.65 6.65 6.65 6.92 6.90 6.91 6.93




Variable VS -3.60 -3.95 -3.93 -4.00




4.15 3.72 3.80 3.66

3.05 2.7 2.72 2.65

7.15 7.20 7.18 7.17

-4.52 -4.95 -4.87 -5.01

-52.13 -57.09 -56.17 -57.78

0.00 9.51 7.74 10.84

-54.14 -59.40 -59.10 -60.15

0.00 9.72 9.17 11.11

0.23 0.3 0.27 0.24

Table 2 showed the removal percentages of TS and VS from the pig manures. The values of the removal percentages of TS and VS of pig manures in CG were 52.13% and 54.14% respectively, while the values in EG1 with -amylase pretreatment were 57.09% and 59.40%, increased by 9.51% and 9.72% respectively. If pig manure was hydrolyzed by amylase in advance in EG2, its removal percentage of TS and VS were 56.17% and 59.10%, increased by 7.74% and 9.17% respectively. If pig manure was pretreated by -amylase and amylase together in EG3, the removal percentage of TS and VS were 57.78% and 60.15%, increased by 10.84% and 11.11%. The results indicated that higher removal percentage of TS and VS can be obtained using amylase pretreatment in all experimental groups, EG1 prior to EG2, and inferior to EG3. The reason for these results is relative to the properties of amylases hydrolysis starch. There is an amount of starch in pig manure. Each starch molecule is a long chain of up to a thousand glucose molecules bonded either in a straight line or branching. The Alpha enzyme attacks the branch points and reduces the long molecule chain into individual segments, while the gamma enzyme attacks the ends of each branch and cuts off individual glucose molecules. When pig manure is hydrolyzed by amylases as mentioned above, short-chain starch molecule segments and/or individual glucose was obtained in feedstock pre-treated, which makes the hydrolytic step of anaerobic digestion easy to conduct greatly eliminating the

hydrolytic limitation factors. As a result, fermentative time was minimized and the removal percentage of TS and VS promoted. B. Daily biogas production rate Fig.2 clearly indicates that the biogas production rate in the process divided into four stages: lag stage (0-2 day), peak stage (3-8 day), sub-peak stage (12-19 day), and fade stage (after 20 day). During the lag stage, also called the start-up stage, the biogas production rate was lower because there was so much oxygen trapped in digestive flasks at the beginning of digestion that limited anaerobe growth and biogas production. Anaerobic digestion needs a strict anaerobic condition for anaerobes, however at the beginning of digestion there is not a strict anaerobic condition but a facultative situation. The top layer of fermentative mixture was aerobic, while the middle is anaerobic, and bottom layer is strict anaerobic. So it is very clear that biogas microorganisms in such situations neither grow fast nor produce much more biogas. With oxygen consumed by the facultative anaerobe, the fermentative condition gradually changed from aerobic to facultative and then to a strict anaerobic condition, which means the rapid growth of anaerobic microorganisms and high

production of biogas will begin, and the fermentative process will rapidly turn into next stage. During the peak stage, due to the rapid growth of anaerobic microorganisms and the high concentration of substrates, a very high biogas production rate has been reached. The maximum biogas production rate reached in all groups was from 3 to 5 days. CG was 1320 mL/d on the 4th day, EG1 1500 mL/d on the same day, EG2 1480 mL/d on the 5th day, and EG3 1580 mL/d on the 4th day. Obviously, biogas production rate of EG3 ranked the first, EG1 second, EG2 third, and CG fourth. The reason for this result seems to be relative to the concentration of easily degradable materials. Because through the amylase pretreatment there is much more sugar or glucose in the EGs the microorganisms can easily use the glucose. After peak gas production, the biogas production rate quickly decreased and sub-peak stage occurred. This stage lasted 12 to 19 days. During this sub-peak stage, the submaximum biogas production rate was 640 mL/d for CG on the 14th day, 650 mL/d for EG 1 on the 13th and 14th day, 680 mL/d for EG 2 on the 14th day, and 650 mL/d for EG 3 on the 13th day. Those values have a little difference. At this time microorganisms are almost depleted of easily degradable materials, and have to choose the difficult degradable materials such as cellulose or semi-cellulose. This results in a relatively lower biogas production rate. With the gradual depletion of all kinds of substrates by the end of sub-peak stage, the process went into a fade stage and from the 20th day ceased production of biogas.

R ie H (t ) = H exp exp ( t ) + 1 H


The relation between Gompertz equation and Modified Gompertz equation is: H(t) = y(x); H=a; R=ca/e; = (b-1)/c. H(t) is accumulative biogas production yield (mL) at time t; H is maximum accumulative biogas production yield (mL); R is maximum biogas production rate (mL/d); is lag-phase time (d); and e is exp(1), i.e. 2.71828.
S = 381 .220 616 46 r = 0. 996 2032 2
0 00 14

cummlu Yield (ml)

000 12 00 100 00 80 00 60 0 400 0 200

a=11512.56 b=1.279 c =0.205

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34


time (d)

S = 381.220616 46 r = 0. 996 20322

00 140

cummlu Yield (ml)

00 120 00 100 0 800 0 600 0 400 0 200 0

a=13056.41 b=1.182 c =0.186

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34


time (d)

S = 392.636168 37 r = 0. 995 95022

00 140

cummlu Yield (ml)

00 120 00 100 0 800 0 600 0 400 0 200 0

a=12949.94 b=1.185 c =0.180

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34


time (d)

S = 405.628096 91 r = 0. 995 64145

00 140

cummlu Yield (ml)

00 120 00 100 0 800 0 600 0 400 0 200 0

Figure 2. Biogas production per day as a function of time

a=13013.84 b=1.172 c =0.195

C. Biogas production potential and specific biogas production rate The accumulative biogas production curve as shown in Fig.3a-d was obtained through using CurveExpert software to analyze experimental data; then the modified Gompertz equation was applied to determine the kinetic parameters of biogas production[15,16]: maximum accumulative biogas production yield, maximum biogas production rate, and lagphase time. Gompertz equation:















time (d)

Figure 3. Accumulative biogas production based on modified Gompertz equation: 4a, CG; 4b, EG1; 4c, CG2; 4d, CG3

y( x) = a exp[ exp(b cx)]

Modified Gompertz equation:


The estimated kinetic parameters based on the modified Gompertz equation are listed in Table 3. Biogas production was well correlated to the modified Gompertz equation (r>0.99). The maximum accumulative biogas production yield (H) for CG, EG1, EG2, and EG3 were 11512, 13037, 12950, and 13014 mL respectively; maximum biogas production rate estimated (R) for CG was 866 mL/d, EG1 892 mL/d, EG2 859 mL/d and EG3 935 mL/d; the estimated lag times () were 1.36, 0.98, 1.03, and 0.88 days respectively in CG, EG1, EG2, and EG3. Biogas production potentials and specific biogas production rates were calculated from H and R divided by the VS weight of substrates respectively. The biogas production

potentials of EG1, EG2, and EG3 were 649, 645 and 648 mL/gVS respectively, compared with CG (573 mL/d), which has been increased by 13.26%, 12.57% and 13.10%. The specific biogas production rates of EG1, EG2, and EG3 were
TABLE III. Groups CG EG 1 EG 2 EG 3 H(mL) 11512 13037 12950 13014

44, 43 and 47 mL/gVSd respectively, compared with CG (43 mL/gVSd), which has been increased by 2.3%, 0%, 9.30%. Obviously accumulative biogas productions in all EGs were higher than that in CG.

ESTIMATED PARAMETERS OF GOMPERTZ EQUATION FOR BIOGAS PRODUCTION (d) 1.36 0.98 1.03 0.88 Biogas production potential
(mL/gVS) increment

R (mL/d) 866 892 859 935

Specific biogas production rate

(mL/gVSd) increment

r 0.9963 0.9962 0.9960 0.9956

573 649 645 648

0 13.26% 12.57% 13.10%

43 44 43 47

0 2.30% 0 9.30%



Compared with CG, the removal percentage of TS and VS, biogas production potentials and rates in all EGs have been significantly improved through amylase pre-treatment. Especially through -amylase and -amylase pretreatment together, the removal percentage of TS and VS, as compared with control group, was increased by10.84% and 11.11% respectively, biogas production potential increased by 13.10%, and specific biogas production rate increased by 9.30%. Experimental results also indicated that the digestive process goes through four stages: lag stage, peak stage, subpeak stage, and fade stage. The different stages that occurred in this experiment may reflect the characteristics of sequential degradation of different organic substrates from easy and simple to difficult and complex. Hydrolase pre-treatment applied to anaerobic digestion contributes to promoting the degradability of feedstock and increasing the yield of biogas. However, it is difficult for this way to be used in practical or commercial operation, due to the added cost when hydrolase was directly used for raw material pretreatment. A solution might be to use a cheap solid-state fermentative mixture or bio-additive contained the components of hydrolase, instead of pure hydrolase. ACKNOWLEDGMENT This experimental investigation was funded by the National Engineering Center of Solid Waste Resources Recovery, Kunming University of Science and Technology, PR. China. We would like to Mr.Emilio Campana (Switzerland), civil dipl. engineer ETH/SIA for sincere language corrections. REFERENCES
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