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substrate ratio on methane yield and production rate from straw
Biological Wastes 28 (1989) 247-255
Effect of Inoculum[Substrate Ratio on Methane Yield and Production Rate from Straw A n d r e w G. H a s h i m o t o Agricultural EngineeringDepartment, Oregon State University,
Corvallis, Oregon 97331-3906, USA (Received 8 August 1988; revised version received 10 October 1988; accepted 13 October 1988)
ABSTRACT This study evaluated the effect of inoculum/substrate ratio on methane yield and production rate in small-batch fermentors. Four trials were conducted with substrate concentrations of 10, 20, 30 or 40 g ball-milled straw diluted with tap water to a total weight of 1 kg. Inoculum concentrations were: 100 (control), 90, 70, 50, 30 and 20 or 10% by volume. Results showed that the ultimate methane yield was drastically lower at inoculum/substrate ratios (on a volatile solids basis) below 0.25. Methane production rate increased at a decreasing rate up to an inoculum/substrate ratio o f two, after which it remained relatively constant.
INTRODUCTION The bioconversion of complex organic substrates such as biomass into methane requires that sufficient inoculum be present to complete the process. Since methanogenesis requires at least three distinct groups of bacteria, it is apparent that care must be taken to assure that the needed bacteria are present in sufficient numbers to degrade the primary and intermediate products of fermentation. Grady (1985), in discussing the microbial basis and methods of measuring biodegradation, emphasized the importance of having sufficient concentrations of necessary microorganisms, enzymes and growth factors to degrade complex compounds. A widely used method to assess the potential methane yield from biomass is to ferment the material under batch conditions with an appropriate 247 Biological Wastes 0269-7483/89/$03-50 O 1989 Elsevier Science Publishers Ltd, England. Printed in Great Britain
248
Andrew G. Hashimoto
i n o c u l u m until n o m o r e m e t h a n e is p r o d u c e d . I n o c u l u m size h a s r a n g e d f r o m 2 % o f the f e r m e n t o r w o r k i n g v o l u m e ( O w e n et aL, 1979) to 6 7 % o f the w o r k i n g v o l u m e ( H a s h i m o t o et al., 1981). T h e p u r p o s e o f this s t u d y w a s to d e t e r m i n e the effect o f i n o c u l u m / s u b s t r a t e r a t i o o n the u l t i m a t e m e t h a n e yield a n d the m e t h a n e p r o d u c t i o n rate.
METHODS
Experiment design F o u r trials were c o n d u c t e d w i t h s u b s t r a t e c o n c e n t r a t i o n s o f 10, 20, 30 o r 40 g ball-milled s t r a w (BMS) diluted with t a p - w a t e r to a t o t a l weight o f 1 kg. W i t h i n e a c h trial, the v o l u m e o f s u b s t r a t e s u s p e n s i o n used r a n g e d f r o m 0 to
TABLE 1 Protocol for Experiment to Determine Effect of Inoculum/Substrate Ratio on Bo and K a Trial
Substra~ concn (g B M S kg- 1)
1
10 10 10 10 10 10 20 20 20 20 20 20 30 30 30 30 30 30 40 40 40 40 40 40
2
3
4
Inoculum Substra~ (%) (ml) 100 90 70 50 30 10 100 90 70 50 30 10 100 90 70 50 30 20 100 90 70 50 30 20
0 5 15 25 35 45 0 5 15 25 35 45 0 5 15 25 35 40 0 5 15 25 35 40
lnoculum (ml)
Duration (days)
No. of gas rneasuremen~
50 (control) 45 35 25 15 5 50 (control) 45 35 25 15 5 50 Control) 45 35 25 15 10 50 (control) 45 35 25 15 10
150
12
150
13
152
13
148
16
a Each treatment was replicated with three batch fermentors.
Effect o f inoculum/substrate ratio on straw
249
45 ml. The inoculum constituted the remainder of the 50-ml working volume of the fermentor. Table 1 shows the protocol used in the experiment. Each treatment was replicated (n = 3). Substrate and inoculum The substrate used in this experiment was wheat straw (Triticum aestivum, Bennett variety) grown in Clay County, Nebraska. The straw was passed through a hammer mill equipped with a 1-cm-opening mesh screen and ballmilled for 24 h in a 5-liter porcelain jar charged with 2-cm-diameter by 2-cmlong porcelain cylinders and rotated at 52 rpm. The BMS was previously analyzed to contain 35% cellulose, 26.6% hemicellulose and 8% lignin on a dry matter basis. At the start of each trial, appropriate amounts (10, 20, 30 or 40 g) of BMS were placed in a beaker and tap-water was added to make a total suspension weight of 1 kg. The BMS suspension was mixed for 1 h, using a magnetic stirrer, before being pipetted to the fermentors. The inoculum was obtained from a 3-liter working volume, mesophilic (35°C) fermentor fed beef-cattle manure. The fermentor was not fed for 3 weeks before the start of each trial so that most of the substrate in the fermentor would be utilized. At the start of each trial, 1 liter of fermentor contents was removed, passed through a no. 10 sieve (2-mm-square openings), and collected in a flask. The contents of the flask were continuously purged with O2-free nitrogen passed through copper filings heated to 300°C. Table 2 shows the concentrations of the inoculum and BMS used in each trial. Procedures Serum bottles served as fermentors. Each fermentor had an effective volume of 119.1 _ 0 . 6 m l when a 1-cm-thick black butyl rubber stopper (Bellco Glass, Vineland, New Jersey) was sealed in place. At the start of each trial, TABLE 2 Inoculum and BMS Constituents at Start of Batch Fermentations
B M S concn (g kg - x)
10 20 30 40
pH
COD (g litre- l)
TS (g litre- x)
VS (g litre 1)
Inoculum
BMS
lnoculum
BMS
Inoculum
BMS
Inoculum
BMS
7"97 7'84 7'53 7'84
7-98 7-86 7-65 7-65
14"9 15-7 15"3 15'1
11"6 22"3 35-6 47-4
15-9 15-6 16-6 16-1
9"4 19-2 28-4 37-9
10"3 10"2 9"3 9"3
8"5 17"1 25-7 33"9
250
Andrew G. Hashimoto
appropriate volumes of inoculum were pipetted using a 25-ml, wide-mouth serological pipette (Bellco Glass, Vineland, New Jersey). During the pipetting operation, the serum bottle was purged with O2-free nitrogen. The bottles were stoppered, sealed with a 20-mm tear-off aluminum seal (Wheaton Scientific, Millville, New Jersey) and then incubated at 35 + I°C in a Fischer Econotemp incubator (Model 55D). Gas production and constituent analyses were performed throughout the fermentations. At the end of the trials, the Constituents from the three replicates were combined and analyzed for total solids (TS), volatile solids (VS), chemical oxygen demand (COD) and pH.
Analytical methods The TS, VS and pH were determined using the standard methods for wastewater analyses (American Public Health Association, 1980), and COD was determined according to the ampule method (OI Corporation, Texas). Gas volume was measured by inserting the needle of a distilled waterlubricated glass syringe directly into the serum bottles. Displacement of the syringe plunger, which was positioned horizontally, was used as a measure of the gas production. Gas production was corrected to standard temperature (0°C), pressure (1 atm), and zero water-vapor pressure. Methane and carbon dioxide concentrations were determined with a Packard 428 gas chromatograph (GC) equipped with a thermal conductivity detector (TCD). A stainless steel column (1.83 m by 6.4 mm) packed with 60/80 Chromosorb 102 (Supelco) was used. Helium was used as the carrier gas at a flow rate of 60 ml min- 1. The temperatures of the injector, column and detector were 150, 70 and 150°C, respectively. Certified gas standard gases were used for calibration of methane and carbon dioxide. Methane yield (B, ml CH4g-1 VS) was calculated by subtracting a proportional amount of the methane produced by the controls (average production from three controls) from the methane production of each treatment fermentor, then dividing the difference by the amount of VS in the BMS. For example, when 25 ml of inoculum and 25 ml of BMS were used, only 50% of the control's methane production was subtracted from the total treatment methane production since the control contained 50ml of inoculum. Ultimate methane yield (B o, ml CH4g-1 VS) and methane production rate (K, per day) were calculated using least-squares fit of the data (PRIMOS version of SAS release 5-03, 1985) to the following equation: B = Bo(l -- exp (-- Kt)) where t is the time in days.
(1)
Effect of inoculum/substrate ratio on straw
251
300 A "~
03
--~> 200
~Z E
TNOC • I0
%
I00
O~
• 30 • 70
50
6'0
9'0
I20
150
Time (doys)
Fig. l.
Cumulative methane yield from ball-milled straw concentration of 10 g kg-a and various inoculum concentrations.
RESULTS A N D DISCUSSION Figure 1 is a representative graph showing the changes in B with fermentation time at various inoculum concentrations and a BMS concentration of 10 g kg- 1. Figure 1 shows the mean data points, and curves described by eqn (1) using mean B 0 and K values for each treatment from Table 1. Table 3 shows the fermentor constituents at the end of the batch fermentations along with the mean B o values for each treatment. The results show that, for each BMS concentration, B o increased as the inoculum/ substrate ratio increased. It is apparent that the fermentations were stressed at 10% inoculum for the 10 and 20 g BMS kg-x treatments, and at 20 and 30% inoculum for the 30 and 40 g BMS kg- 1 treatments. This is indicated by the lower Bo and pH, and higher COD and VS concentrations relative to the other fermentations at the same BMS concentrations. Figure 2 shows a dramatic increase in B o as the inoculum/substrate ratio increases to 0.25, then a more gradual increase as the ratio increases above 0-25. This gradual increase in B o is statistically significant, as indicated in Table 3. The mean Bo from the treatments that produced the most methane (Table 3: 10g BMS kg- 1 and 90% inoculum, 10 g BMS kg- 1 and 70% inoculum, and 20 g BMS k g - 1 and 90% inoculum) was 326 + 4 ml CH4 g - 1 VS (243 + 6 ml CH4 g - x COD). This value from the 50-ml working-volume serum bottles is similar to the B o for the same straw fermented in 3-1itre working-volume
Andrew G. Hashimoto
252
TABLE 3 Fermentor Constituents at Termination and Methane Yields and Production Rates
BMS concn lnoculum I/S ratio pH COD VS Bo K (gkg -1) (% v/v) (VSbasis) (glitre -1) (glitre -~) (mlCH4g -1 VS)(day -~) I0 10 10 10 10 10
100 90 70 50 30 10
NA 10.91 2-83 1.21 0.52 0-19
7"73 7.61 7.41 7.23 7-01 4.95
15.9 16-0 13.1 10.3 8.3 11.9
9-8 9-0 7.7 6-2 4.9 7.1
NA 331 a 323 a 313 b 313 b 18e
NA 0-17 b,c 0.18 b,c 0-13c 0-08 d 0.16 b
20 20 20 20 20 20
100 90 70 50 30 10
NA 5.34 1-38 0.59 0.25 0.07
7.75 7-56 7.32 7.17 6.90 4.77
14.7 14.8 12.9 12-6 10-5 21.3
9.1 9.2 8.3 7.4 6-6 12.5
NA 324 a 312 b 309 ¢ 299 c 7y
NA 0.24 a 0-18b 0.09 ¢ 0.04 d 0-10c
30 30 30 30 30 30
100 90 70 50 30 20
NA 3.27 0.85 0.36 0-16 0-09
7.84 7-61 7.42 7.27 5.09 4.77
14.4 13.1 13.4 13.4 26-8 28.3
8:6 8.5 8-2 8.1 13.6 15-5
NA 313 b 304 b,c 305 b,c 33d 22 e
NA 0.24 0.13 c 0-07d 0.244 0.16 b,c
40 40 40 40 40 40
100 90 70 50 30 20
NA 2-48 0.64 0.28 0.12 0.07
7.81 7.52 7.37 7.19 4-93 4.73
12.1 12-1 12.1 13-2 30.8 34.5
8-5 8.8 8.9 9.3 18.4 22.3
NA 313 b 311 b 307 b'c 23 ~ 13e.y
NA 0.2M 0.10c 0-04d 0.24" 0.14 b'c
a I Means in columns with different superscripts differ (P < 0"01). NA, Not applicable. fermentors (304_10ml CH4g -1 VS or 242_+16ml CH4g -1 COD; H a s h i m o t o , 1986). T h i s d e m o n s t r a t e s t h a t t h e s e r u m - b o t t l e t e c h n i q u e d o e s produce results similar to the larger-scale batch fermentations. T a b l e 3 a n d F i g . 3 s h o w t h e effect o f t h e I / S r a t i o o n K. D i s r e g a r d i n g t h e d a t a f o r t h e s t r e s s e d f e r m e n t a t i o n (as i n d i c a t e d a b o v e ) , K t e n d e d t o i n c r e a s e as the I/S ratio increased. The data for the stressed fermentations should be disregarded because these fermentations approached s a t u r a t i o n (i.e. methane producton stopped) much sooner than the uninhibited fermenta t i o n s a n d , t h u s , g a v e a n u n r e p r e s e n t a t i v e h i g h K. T h i s is i l l u s t r a t e d b y t h e 1 0 % i n o c u l u m c u r v e i n F i g . 1. The results from this study clearly show that the I/S ratio and fermentation time are important factors when estimating the B 0 of biomass.
253
Effect o f inoculum/substrate ratio on straw I000
..................................
I00 (•
>-.
o
=_..~ /
BMS (g/I)
I0
I0
• • • •
I .01
0.1
I
20 50 40
I00
IO
[nocuium/Substrate Ratio (gVS I/g VS BMS)
Fig. 2.
Relationship between ultimate methane yield and inoculum/substrate ratio.
For example, the methane yield after 30 and 150 days of fermentation would be 69.9 and 99.8% of the B o, respectively, at an I/S ratio of 0"3 (K = 0"04 day- 1, Fig. 3). However, at an I/S ratio of 2.0 (K = 0.2 day- 1, Fig. 3), 30 and 150 days of fermentation would produce 99.8 and 100-0% of the Bo, respectively. I.O0
A 0 "t3
~, 0.I0 el.
0010. I
.S ........
i
........
BMS (g/I) • • • •
I'(3
I0 2O 50 40
.......
I00
Inoculum/Subslrote Ratio (gVS I / g VS BMS)
Fig. 3. Relationship between methane production rate constant and inoculum/substrate ratio.
254
Andrew G. Hashimoto
These results may explain some of the conflicting data reported in the literature relative to the effectiveness of various pretreatments in increasing the biodegradability of biomass. To illustrate this point, data from two studies are compared to show how differences in I/S ratios may have caused the large difference in pretreatment effectiveness reported. In the first study (Study 1), Pavlostathis & Gossett (1985) reported that Wiley-milled wheat straw that was pretreated with 536g N a O H k g - 1 VS for 24h at room temperature produced about two times more methane than the untreated Wiley-milled straw (263 ml CH 4 g - 1 COD for the treated straw, and 120 ml CH 4g-1 COD for the untreated straw). In the second study (Study 2), Hashimoto (1986) reported only a 28% increase in B o when ball-milled wheat straw was pretreated with 80g N a O H k g - ~ VS for 1 h at 90°C compared with untreated ball-milled straw (322 ml CH4 g-1 COD for the treated straw, and 239 ml CH 4 g - ~ COD for the untreated straw). Although the effectiveness of the pretreatment appeared to be much less in Study 2, both the untreated and treated Bo values were significantly higher than those from the respective treatments of Study 1. One possible explanation for the much higher B o values in Study 2 may be the different type and amount of inoculum and fermentation times used in the two studies. Study 1 used inoculum acclimated to primary sewage sludge at a level of ,-~2% of the working volume of the fermentor, and used fermentation times of ,~ 30 days. Study 2 used inoculum acclimated to beef cattle manure at levels ranging from 33 to 67% of the fermentor working volume, and used fermentation times in excess of 100 days. In conclusion, the present study showed that inoculum size and fermentation time can significantly affect the methane yield and the value of Bo. It is important that the effects ofinoculum size and fermentation time are addressed whenever studies are undertaken to determine the methane yield from complex organic substrates such as biomass, to ensure that accurate results are obtained. ACKNOWLEDGMENTS This study was conducted at the R o m a n L. Hruska US Meat Animal Research Center, Clay Center, Nebraska. Technical support of Steve Robinson and Dave Sypherd is greatly appreciated. REFERENCES American Public Health Association (1980). Standard Methods for the Examination of Water and Wastewater, 15th Edn. American Public Health Association, New York.
Effect of inoculum/substrate ratio on straw
255
Grady, C. P. L., Jr (1985). Biodegradation: its measurement and microbiological basis. Biotechnol. Bioengng, 27, 660-74. Hashimoto, A. G. (1986). Pretreatment of wheat straw for fermentation to methane. Biotechnol. Bioengng, 28, 1857-66. Hashimoto, A. G., Varel, V. H. & Chen, Y. R. (1981). Ultimate methane yield from beef cattle manure: effect of temperature, ration constituents, antibiotics and manure age. Agric. Wastes, 3, 241-56. Owen, W. F., Stuckey, D. C., Healy, J. B., Young, L. Y. & McCarty, P. L. (1979). Bioassay for monitoring biochemical methane potential and anaerobic toxicity. Water Res., 13, 485-92. Pavlostathis, S. G. & Gossett, J. M. (1985). Alkaline treatment of wheat straw for increasing anaerobic biodegradability. BiotechnoL Bioengng, 27, 334-44.
Effect of Inoculum[Substrate Ratio on Methane Yield and Production Rate from Straw A n d r e w G. H a s h i m o t o Agricultural EngineeringDepartment, Oregon State University,
Corvallis, Oregon 97331-3906, USA (Received 8 August 1988; revised version received 10 October 1988; accepted 13 October 1988)
ABSTRACT This study evaluated the effect of inoculum/substrate ratio on methane yield and production rate in small-batch fermentors. Four trials were conducted with substrate concentrations of 10, 20, 30 or 40 g ball-milled straw diluted with tap water to a total weight of 1 kg. Inoculum concentrations were: 100 (control), 90, 70, 50, 30 and 20 or 10% by volume. Results showed that the ultimate methane yield was drastically lower at inoculum/substrate ratios (on a volatile solids basis) below 0.25. Methane production rate increased at a decreasing rate up to an inoculum/substrate ratio o f two, after which it remained relatively constant.
INTRODUCTION The bioconversion of complex organic substrates such as biomass into methane requires that sufficient inoculum be present to complete the process. Since methanogenesis requires at least three distinct groups of bacteria, it is apparent that care must be taken to assure that the needed bacteria are present in sufficient numbers to degrade the primary and intermediate products of fermentation. Grady (1985), in discussing the microbial basis and methods of measuring biodegradation, emphasized the importance of having sufficient concentrations of necessary microorganisms, enzymes and growth factors to degrade complex compounds. A widely used method to assess the potential methane yield from biomass is to ferment the material under batch conditions with an appropriate 247 Biological Wastes 0269-7483/89/$03-50 O 1989 Elsevier Science Publishers Ltd, England. Printed in Great Britain
248
Andrew G. Hashimoto
i n o c u l u m until n o m o r e m e t h a n e is p r o d u c e d . I n o c u l u m size h a s r a n g e d f r o m 2 % o f the f e r m e n t o r w o r k i n g v o l u m e ( O w e n et aL, 1979) to 6 7 % o f the w o r k i n g v o l u m e ( H a s h i m o t o et al., 1981). T h e p u r p o s e o f this s t u d y w a s to d e t e r m i n e the effect o f i n o c u l u m / s u b s t r a t e r a t i o o n the u l t i m a t e m e t h a n e yield a n d the m e t h a n e p r o d u c t i o n rate.
METHODS
Experiment design F o u r trials were c o n d u c t e d w i t h s u b s t r a t e c o n c e n t r a t i o n s o f 10, 20, 30 o r 40 g ball-milled s t r a w (BMS) diluted with t a p - w a t e r to a t o t a l weight o f 1 kg. W i t h i n e a c h trial, the v o l u m e o f s u b s t r a t e s u s p e n s i o n used r a n g e d f r o m 0 to
TABLE 1 Protocol for Experiment to Determine Effect of Inoculum/Substrate Ratio on Bo and K a Trial
Substra~ concn (g B M S kg- 1)
1
10 10 10 10 10 10 20 20 20 20 20 20 30 30 30 30 30 30 40 40 40 40 40 40
2
3
4
Inoculum Substra~ (%) (ml) 100 90 70 50 30 10 100 90 70 50 30 10 100 90 70 50 30 20 100 90 70 50 30 20
0 5 15 25 35 45 0 5 15 25 35 45 0 5 15 25 35 40 0 5 15 25 35 40
lnoculum (ml)
Duration (days)
No. of gas rneasuremen~
50 (control) 45 35 25 15 5 50 (control) 45 35 25 15 5 50 Control) 45 35 25 15 10 50 (control) 45 35 25 15 10
150
12
150
13
152
13
148
16
a Each treatment was replicated with three batch fermentors.
Effect o f inoculum/substrate ratio on straw
249
45 ml. The inoculum constituted the remainder of the 50-ml working volume of the fermentor. Table 1 shows the protocol used in the experiment. Each treatment was replicated (n = 3). Substrate and inoculum The substrate used in this experiment was wheat straw (Triticum aestivum, Bennett variety) grown in Clay County, Nebraska. The straw was passed through a hammer mill equipped with a 1-cm-opening mesh screen and ballmilled for 24 h in a 5-liter porcelain jar charged with 2-cm-diameter by 2-cmlong porcelain cylinders and rotated at 52 rpm. The BMS was previously analyzed to contain 35% cellulose, 26.6% hemicellulose and 8% lignin on a dry matter basis. At the start of each trial, appropriate amounts (10, 20, 30 or 40 g) of BMS were placed in a beaker and tap-water was added to make a total suspension weight of 1 kg. The BMS suspension was mixed for 1 h, using a magnetic stirrer, before being pipetted to the fermentors. The inoculum was obtained from a 3-liter working volume, mesophilic (35°C) fermentor fed beef-cattle manure. The fermentor was not fed for 3 weeks before the start of each trial so that most of the substrate in the fermentor would be utilized. At the start of each trial, 1 liter of fermentor contents was removed, passed through a no. 10 sieve (2-mm-square openings), and collected in a flask. The contents of the flask were continuously purged with O2-free nitrogen passed through copper filings heated to 300°C. Table 2 shows the concentrations of the inoculum and BMS used in each trial. Procedures Serum bottles served as fermentors. Each fermentor had an effective volume of 119.1 _ 0 . 6 m l when a 1-cm-thick black butyl rubber stopper (Bellco Glass, Vineland, New Jersey) was sealed in place. At the start of each trial, TABLE 2 Inoculum and BMS Constituents at Start of Batch Fermentations
B M S concn (g kg - x)
10 20 30 40
pH
COD (g litre- l)
TS (g litre- x)
VS (g litre 1)
Inoculum
BMS
lnoculum
BMS
Inoculum
BMS
Inoculum
BMS
7"97 7'84 7'53 7'84
7-98 7-86 7-65 7-65
14"9 15-7 15"3 15'1
11"6 22"3 35-6 47-4
15-9 15-6 16-6 16-1
9"4 19-2 28-4 37-9
10"3 10"2 9"3 9"3
8"5 17"1 25-7 33"9
250
Andrew G. Hashimoto
appropriate volumes of inoculum were pipetted using a 25-ml, wide-mouth serological pipette (Bellco Glass, Vineland, New Jersey). During the pipetting operation, the serum bottle was purged with O2-free nitrogen. The bottles were stoppered, sealed with a 20-mm tear-off aluminum seal (Wheaton Scientific, Millville, New Jersey) and then incubated at 35 + I°C in a Fischer Econotemp incubator (Model 55D). Gas production and constituent analyses were performed throughout the fermentations. At the end of the trials, the Constituents from the three replicates were combined and analyzed for total solids (TS), volatile solids (VS), chemical oxygen demand (COD) and pH.
Analytical methods The TS, VS and pH were determined using the standard methods for wastewater analyses (American Public Health Association, 1980), and COD was determined according to the ampule method (OI Corporation, Texas). Gas volume was measured by inserting the needle of a distilled waterlubricated glass syringe directly into the serum bottles. Displacement of the syringe plunger, which was positioned horizontally, was used as a measure of the gas production. Gas production was corrected to standard temperature (0°C), pressure (1 atm), and zero water-vapor pressure. Methane and carbon dioxide concentrations were determined with a Packard 428 gas chromatograph (GC) equipped with a thermal conductivity detector (TCD). A stainless steel column (1.83 m by 6.4 mm) packed with 60/80 Chromosorb 102 (Supelco) was used. Helium was used as the carrier gas at a flow rate of 60 ml min- 1. The temperatures of the injector, column and detector were 150, 70 and 150°C, respectively. Certified gas standard gases were used for calibration of methane and carbon dioxide. Methane yield (B, ml CH4g-1 VS) was calculated by subtracting a proportional amount of the methane produced by the controls (average production from three controls) from the methane production of each treatment fermentor, then dividing the difference by the amount of VS in the BMS. For example, when 25 ml of inoculum and 25 ml of BMS were used, only 50% of the control's methane production was subtracted from the total treatment methane production since the control contained 50ml of inoculum. Ultimate methane yield (B o, ml CH4g-1 VS) and methane production rate (K, per day) were calculated using least-squares fit of the data (PRIMOS version of SAS release 5-03, 1985) to the following equation: B = Bo(l -- exp (-- Kt)) where t is the time in days.
(1)
Effect of inoculum/substrate ratio on straw
251
300 A "~
03
--~> 200
~Z E
TNOC • I0
%
I00
O~
• 30 • 70
50
6'0
9'0
I20
150
Time (doys)
Fig. l.
Cumulative methane yield from ball-milled straw concentration of 10 g kg-a and various inoculum concentrations.
RESULTS A N D DISCUSSION Figure 1 is a representative graph showing the changes in B with fermentation time at various inoculum concentrations and a BMS concentration of 10 g kg- 1. Figure 1 shows the mean data points, and curves described by eqn (1) using mean B 0 and K values for each treatment from Table 1. Table 3 shows the fermentor constituents at the end of the batch fermentations along with the mean B o values for each treatment. The results show that, for each BMS concentration, B o increased as the inoculum/ substrate ratio increased. It is apparent that the fermentations were stressed at 10% inoculum for the 10 and 20 g BMS kg-x treatments, and at 20 and 30% inoculum for the 30 and 40 g BMS kg- 1 treatments. This is indicated by the lower Bo and pH, and higher COD and VS concentrations relative to the other fermentations at the same BMS concentrations. Figure 2 shows a dramatic increase in B o as the inoculum/substrate ratio increases to 0.25, then a more gradual increase as the ratio increases above 0-25. This gradual increase in B o is statistically significant, as indicated in Table 3. The mean Bo from the treatments that produced the most methane (Table 3: 10g BMS kg- 1 and 90% inoculum, 10 g BMS kg- 1 and 70% inoculum, and 20 g BMS k g - 1 and 90% inoculum) was 326 + 4 ml CH4 g - 1 VS (243 + 6 ml CH4 g - x COD). This value from the 50-ml working-volume serum bottles is similar to the B o for the same straw fermented in 3-1itre working-volume
Andrew G. Hashimoto
252
TABLE 3 Fermentor Constituents at Termination and Methane Yields and Production Rates
BMS concn lnoculum I/S ratio pH COD VS Bo K (gkg -1) (% v/v) (VSbasis) (glitre -1) (glitre -~) (mlCH4g -1 VS)(day -~) I0 10 10 10 10 10
100 90 70 50 30 10
NA 10.91 2-83 1.21 0.52 0-19
7"73 7.61 7.41 7.23 7-01 4.95
15.9 16-0 13.1 10.3 8.3 11.9
9-8 9-0 7.7 6-2 4.9 7.1
NA 331 a 323 a 313 b 313 b 18e
NA 0-17 b,c 0.18 b,c 0-13c 0-08 d 0.16 b
20 20 20 20 20 20
100 90 70 50 30 10
NA 5.34 1-38 0.59 0.25 0.07
7.75 7-56 7.32 7.17 6.90 4.77
14.7 14.8 12.9 12-6 10-5 21.3
9.1 9.2 8.3 7.4 6-6 12.5
NA 324 a 312 b 309 ¢ 299 c 7y
NA 0.24 a 0-18b 0.09 ¢ 0.04 d 0-10c
30 30 30 30 30 30
100 90 70 50 30 20
NA 3.27 0.85 0.36 0-16 0-09
7.84 7-61 7.42 7.27 5.09 4.77
14.4 13.1 13.4 13.4 26-8 28.3
8:6 8.5 8-2 8.1 13.6 15-5
NA 313 b 304 b,c 305 b,c 33d 22 e
NA 0.24 0.13 c 0-07d 0.244 0.16 b,c
40 40 40 40 40 40
100 90 70 50 30 20
NA 2-48 0.64 0.28 0.12 0.07
7.81 7.52 7.37 7.19 4-93 4.73
12.1 12-1 12.1 13-2 30.8 34.5
8-5 8.8 8.9 9.3 18.4 22.3
NA 313 b 311 b 307 b'c 23 ~ 13e.y
NA 0.2M 0.10c 0-04d 0.24" 0.14 b'c
a I Means in columns with different superscripts differ (P < 0"01). NA, Not applicable. fermentors (304_10ml CH4g -1 VS or 242_+16ml CH4g -1 COD; H a s h i m o t o , 1986). T h i s d e m o n s t r a t e s t h a t t h e s e r u m - b o t t l e t e c h n i q u e d o e s produce results similar to the larger-scale batch fermentations. T a b l e 3 a n d F i g . 3 s h o w t h e effect o f t h e I / S r a t i o o n K. D i s r e g a r d i n g t h e d a t a f o r t h e s t r e s s e d f e r m e n t a t i o n (as i n d i c a t e d a b o v e ) , K t e n d e d t o i n c r e a s e as the I/S ratio increased. The data for the stressed fermentations should be disregarded because these fermentations approached s a t u r a t i o n (i.e. methane producton stopped) much sooner than the uninhibited fermenta t i o n s a n d , t h u s , g a v e a n u n r e p r e s e n t a t i v e h i g h K. T h i s is i l l u s t r a t e d b y t h e 1 0 % i n o c u l u m c u r v e i n F i g . 1. The results from this study clearly show that the I/S ratio and fermentation time are important factors when estimating the B 0 of biomass.
253
Effect o f inoculum/substrate ratio on straw I000
..................................
I00 (•
>-.
o
=_..~ /
BMS (g/I)
I0
I0
• • • •
I .01
0.1
I
20 50 40
I00
IO
[nocuium/Substrate Ratio (gVS I/g VS BMS)
Fig. 2.
Relationship between ultimate methane yield and inoculum/substrate ratio.
For example, the methane yield after 30 and 150 days of fermentation would be 69.9 and 99.8% of the B o, respectively, at an I/S ratio of 0"3 (K = 0"04 day- 1, Fig. 3). However, at an I/S ratio of 2.0 (K = 0.2 day- 1, Fig. 3), 30 and 150 days of fermentation would produce 99.8 and 100-0% of the Bo, respectively. I.O0
A 0 "t3
~, 0.I0 el.
0010. I
.S ........
i
........
BMS (g/I) • • • •
I'(3
I0 2O 50 40
.......
I00
Inoculum/Subslrote Ratio (gVS I / g VS BMS)
Fig. 3. Relationship between methane production rate constant and inoculum/substrate ratio.
254
Andrew G. Hashimoto
These results may explain some of the conflicting data reported in the literature relative to the effectiveness of various pretreatments in increasing the biodegradability of biomass. To illustrate this point, data from two studies are compared to show how differences in I/S ratios may have caused the large difference in pretreatment effectiveness reported. In the first study (Study 1), Pavlostathis & Gossett (1985) reported that Wiley-milled wheat straw that was pretreated with 536g N a O H k g - 1 VS for 24h at room temperature produced about two times more methane than the untreated Wiley-milled straw (263 ml CH 4 g - 1 COD for the treated straw, and 120 ml CH 4g-1 COD for the untreated straw). In the second study (Study 2), Hashimoto (1986) reported only a 28% increase in B o when ball-milled wheat straw was pretreated with 80g N a O H k g - ~ VS for 1 h at 90°C compared with untreated ball-milled straw (322 ml CH4 g-1 COD for the treated straw, and 239 ml CH 4 g - ~ COD for the untreated straw). Although the effectiveness of the pretreatment appeared to be much less in Study 2, both the untreated and treated Bo values were significantly higher than those from the respective treatments of Study 1. One possible explanation for the much higher B o values in Study 2 may be the different type and amount of inoculum and fermentation times used in the two studies. Study 1 used inoculum acclimated to primary sewage sludge at a level of ,-~2% of the working volume of the fermentor, and used fermentation times of ,~ 30 days. Study 2 used inoculum acclimated to beef cattle manure at levels ranging from 33 to 67% of the fermentor working volume, and used fermentation times in excess of 100 days. In conclusion, the present study showed that inoculum size and fermentation time can significantly affect the methane yield and the value of Bo. It is important that the effects ofinoculum size and fermentation time are addressed whenever studies are undertaken to determine the methane yield from complex organic substrates such as biomass, to ensure that accurate results are obtained. ACKNOWLEDGMENTS This study was conducted at the R o m a n L. Hruska US Meat Animal Research Center, Clay Center, Nebraska. Technical support of Steve Robinson and Dave Sypherd is greatly appreciated. REFERENCES American Public Health Association (1980). Standard Methods for the Examination of Water and Wastewater, 15th Edn. American Public Health Association, New York.
Effect of inoculum/substrate ratio on straw
255
Grady, C. P. L., Jr (1985). Biodegradation: its measurement and microbiological basis. Biotechnol. Bioengng, 27, 660-74. Hashimoto, A. G. (1986). Pretreatment of wheat straw for fermentation to methane. Biotechnol. Bioengng, 28, 1857-66. Hashimoto, A. G., Varel, V. H. & Chen, Y. R. (1981). Ultimate methane yield from beef cattle manure: effect of temperature, ration constituents, antibiotics and manure age. Agric. Wastes, 3, 241-56. Owen, W. F., Stuckey, D. C., Healy, J. B., Young, L. Y. & McCarty, P. L. (1979). Bioassay for monitoring biochemical methane potential and anaerobic toxicity. Water Res., 13, 485-92. Pavlostathis, S. G. & Gossett, J. M. (1985). Alkaline treatment of wheat straw for increasing anaerobic biodegradability. BiotechnoL Bioengng, 27, 334-44.