Potential biodiesel and biogas production from corncob by anaerobic fermentation and black soldier fly

Accepted Manuscript Potential biodiesel and biogas production from corncob by anaerobic fermentation and black soldier fly Wu Li, Longyu Zheng, YuanYuan Wang, Jibin Zhang, Ziniu Yu, Yanlin Zhang, Qing Li PII: DOI: Reference:

S0960-8524(15)00903-7 http://dx.doi.org/10.1016/j.biortech.2015.06.112 BITE 15192

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

23 April 2015 21 June 2015 22 June 2015

Please cite this article as: Li, W., Zheng, L., Wang, Y., Zhang, J., Yu, Z., Zhang, Y., Li, Q., Potential biodiesel and biogas production from corncob by anaerobic fermentation and black soldier fly, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.06.112

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Potential biodiesel and biogas production from corncob by anaerobic fermentation and black soldier fly

Wu Lia, Longyu Zhengb, YuanYuan Wanga, Jibin Zhangb, Ziniu Yub, Yanlin Zhanga, *, Qing Li c,*

a

College of Engineering, Huazhong Agricultural University, 430070 Wuhan, PR China

b

State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, 430070 Wuhan, P. R. China

c

College of Science, Huazhong Agricultural University, Wuhan, 430070 P. R. China

*Corresponding Authors. Tel.: +86 27 87280802; fax: +86 27 87393882(Q. Li), Tel. /fax: +86 27 87283099(Y. Zhang). E-mail addresses: [email protected] (Q. Li), [email protected] (Y. Zhang)

ABSTRACT： ： Bioenergy has become attractive as alternatives of gradually exhausted fossil fuel. Obtaining high grade bioenergy from lignocellulose is attractive that can gradually meet the demand. This study reported biogas and biodiesel were produced from corncob by a two-step bioprocess, biogas was produced from corncob by anaerobic fermentation, then biogas residue was converted by black soldier fly larvae, and then biodiesel was produced from larvae grease. 86.70L biogas was obtained from 400g corncob with the accumulation of biogas yield of 220.71 mL/g VSadded by anaerobic digestion. Besides, 3.17g of biodiesel was produced from grease after inoculating black soldier fly larvae into 400g biogas residue. Meanwhile, the results showed that the addition of black soldier fly larvae could be effective for the degradation of 1

lignocellulose and the accumulation of grease. Keywords: Corncob；Black soldier fly；Anaerobic fermentation；Biogas；Biodiesel

1. Introduction With the development of the world economy and the deepening of industrialization, the demand for energy is growing rapidly. Faced with limited fossil energy, governments began to search for new alternative energy. Compared with traditional fossil fuels, lignocellulose materials, as biomass energy source, are land-based biomass instead of fossil energy deposits, processing and conversion of carbohydrates instead of hydrocarbons. Therefore, lignocellulose materials, as for its abundant, renewable and environmental friendly features, could be a feasible choice for the replacement of fossil fuels [Panwar et al., 2012]. To meet sustainability requirements, biological treatments for organic waste have been found to be a good way to recover the hidden energy. Renewable energy can be successfully released from potential lignocellulose materials such as crop straw with the production of biogas [Zhong et al., 2011, Guo et al., 2011]. As an abundant agricultural by-product of food crops, corncob is considered to be a potential raw material for energy recycling because its hemicellulose content is the highest among all of the agricultural by-products [Yuan et al., 2004]. Presently, amount of corncobs with the total yield of more than 20 million tons annually in China are used for the production of xylitol, furfural, hydrogen, ethanol and other value-added products [Fan et al., 2014;Yang et al., 2010;Cheng et al., 2010;Liu et al., 2010;Zhang et al., 2012], unfortunately, there are still approximately half million tons of corncob residues becoming solid wastes with limited application, causing environmental pollution and renewable resource waste [Gu et al., 2014]. Biogas is produced by anaerobic digestion with anaerobic bacteria or fermentation of biodegradable

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materials such as lignocellulose. Before anaerobic fermentation, pretreatment is very important for the degradation of lignocellulose. Currently, much work for pretreatment and hydrolysis of agricultural biomass resources have been done by biological and chemical ways, such as using acid, base, microbial and ammonia individually and in combinations [Chen et al., 2010;Li et al., 2010;Zheng et al., 2014;Lee et al., 2010], which can destroy the anti-degradation lignocellulose, make it easier for anaerobic bacteria and enzyme adhesion on cellulose and hemicelluloses, conducive to the acidification and hydrolysis, effectively improve the biogas production of subsequent fermentation [Zhao et al., 2010;Teghammar et al., 2012]. Additionally, choosing appropriate inoculation ratio and fermentation temperature to match the properties of raw material are benefit for maintaining stable, efficient operation of the anaerobic fermentation system [Qu et al., 2014]. Like other lignocellulose, corncob could be a source of biogas for energy recycling. Biogas residue is always directly spread in the fields and the excessive effect of fertilizer can easily destroy the farmland. So exploring a new energy recovering method for biogas residue could be the reasonable use of organic wastes. Fortunately, a kind of insect called black soldier fly (Diptera: Stratiomyidae) which has been found with the characteristic of converting organic matter from the wastes such as animal manure or plant material into insect biomass with high levels of protein and grease [Li et al., 2011a], may help convert extra energy from organic residue and obtain biomass accumulation, it can alleviate energy and environment problems. The black soldier fly larvae (BSFL) could be used as a high protein feed while grease extracted from BSFL can be a low cost biodiesel feedstock [Zheng et al., 2012b]. In this study, the black soldier fly is a new attempt for the traditional anaerobic fermentation system for further energy recycling. At present, the methods for biogas production from lignocellulose materials by anaerobic fermentation are common, and the methods for converting organic wastes by BSFL for biodiesel are also reported [Li et al., 2011a; Li et al., 2011b; Zheng et al., 2012a; Zheng et al., 2012b]. But there are no reports 3

about the method for the continuous conversion of biogas residue for biodiesel production. A two-step bioprocess was investigated for recovering biogas and biodiesel from corncob combined anaerobic fermentation and BSFL in this study. Batch experiments on anaerobic fermentation were conducted to evaluate effects of different ratios between corncob and pig manure for biogas generation, then biogas residue was converted by inoculating BSFL for the accumulation of grease. And the grease was extracted for biodiesel. Furthermore, the lignocellulose changes in two-step bioprocess were evaluated. All the methods aimed to evaluate the potential of using low grade raw materials for high grade bioenergy production.

2. Methods 2.1. Raw materials The anaerobic sludge was taken from an anaerobic digester at college of engineering, Huazhong Agricultural University, Wuhan, China. The anaerobic digester is keep working for more than 6 years by adding moderate amount of pig manure every 2 months. Pig manure (PM) was provided by the breeding farm at Huazhong Agricultural University, and corncob was collected after harvest from experimental fields at Huazhong Agricultural University. Corncob was chopped and grinded into small particles less than 5 mm in size, then pretreated by mixing with microbial agents production by Beijing green lily company, add appropriate water, piled in stacks in 35±2℃ for 5 days until white mycelium appeared. The main characteristics of anaerobic sludge, pig manure and corncob were determined before being used for the study and showed in Table 1. BSFL were obtained from the Huazhong Agricultural University colony. This colony was established in 2005 from the eggs of a colony at the Texas Agricultural Experiment Station, Stephenville, TX, GA, USA. BSFL were fed for about 6 days with standard colony diet before being used for this study. 4

2.2. Experimental design Fig.1 describes the production of high grade bioenergy by converting biomass wastes to biogas and biodiesel, the details of the experiments are as follows. The experiments for evaluating the biogas production ratios were performed in 2.5L glass bottles with working volume 2L. Glass bottles were used as anaerobic digestion vessel in this study. The bottles were sealed with rubber stoppers with drilled holes, and a slender glass tube with a silicone tube was inserted to export gas for biogas volume and composition measurement. Biogas production was recorded by quantifying water displacement everyday. Each experiment contained about 30% inoculums. However, ratios between corncob and PM (based on VS) were 5:2, 2:1, 3:2, 1:1 and 2:3, with constant 150g of PM with inoculums, filled water into glass bottles until 2000g. Meanwhile, there was a control with 150g of PM with inoculums in glass bottle. Then put all the experiments in water bath and controlled the temperature at 35±2℃for anaerobic fermentation. The experiments were terminated when gas production was observed less than 500 ml per day. After the anaerobic fermentation of the experiments described above, three of 5L glass bottles with working volume of 4L with the selected biogas production ratio were performed for biogas production and preparing biogas residue for BSFL. The anaerobic fermentation conditions were the same as above experiments. The biogas residues from the bottles were dried at 105℃ until the weight kept the same. Based on preliminary trials of inoculating BSFL, 500 of 6 days old larvae (about 3 mg/larva) were inoculated into mixture of 400 g corncob biogas residue for insect biomass accumulation. The experiments were carried out in a greenhouse at 27℃ with 70% air moisture. Conversion was terminated when prepupae accounted for about half of the larvae. Then the larvae were separated, washed, and inactivated at 110℃ for 10 min and dried at 65℃ until constant weight was gained. The larval grease content was extracted twice for 5

8 h in Soxhlet extractor with 200 mL petroleum ether. Then the crude larval grease was obtained from a rotary evaporator combining the leaching liquor and evaporating petroleum ether, and then calculated by weight [Zheng et al., 2012]. Biodiesel was produced in a 100 mL reactor equipped with a reflux condenser, a thermometer, a mechanical stirrer, and a sampling outlet by the processes of acid-catalyzed esterification of free fatty acids and alkaline-catalyzed transesterification [Li et al., 2011]. All the experiments were conducted in triplicates.

2.3. Analytical methods Total solids (TS) and total volatile solids (VS) were determined by standard techniques [APHA, 1998]. pH was determined with a pHS-3C pH meter made by Shanghai Precision& Scientific Instrument Co., Ltd. Biogas production was measured by water displacement. Methane content in biogas was analyzed by gas chromatograph (GC9790II, ZheJiang, China), fitted with a TCD and 1.5 m stainless steel column, and with 5A molecular sieve packed. Injector, detector and oven were maintained at 55℃, 100℃ and 50℃. Experimental data were analyzed and curves were drawn by the Origin software version 8.0. The modified Gompertz equation was utilized to the observed cumulative CH4 production curves to determine the maximum CH4 production potential (P), CH4 production rate (Rmax), and lag phase (λ) as shown in equation [Gurung et al., 2012]:  ∙

Yt = P ∙ exp −exp 



 −  + 1

Where, Y (t) is the cumulative CH4 production (mL CH4/g VS) at time t; P is the maximum CH4 potential (mL CH4/g VS) at the end of incubation time; t is time (h); Rmax is the CH4 production rate (mL CH4/g VS d); λ is the lag phase (h) and e is exp (1), i.e. 2.71828. Parameters of P, Rmax, and λ were estimated by curve-fitting using Origin software version 8.0. 6

3. Results and discussion 3.1. Effect of different ratios between corncob and PM on biogas production It is known that the concentration of substrates affect the anaerobic fermentation for biogas production. In order to reflect the life value, acidification and methanogenic microbials demand suitable carbon and nitrogen ratio for active metabolism in anaerobic system. As a kind of lignocelluloses, corncob shares the feature of carbon-rich that needs the addition of nitrogen-rich substrates such as animal manure to balance the C/N [Ye et al., 2013]. Then PM was added into the digestion system. Different ratios of corncob and PM led to different carbon to nitrogen (C/N) ratios for anaerobic degradation, and it would increase the release of free ammonia when nitrogen was rich. Meanwhile, as a consequence of the toxic effect of ammonia on the anaerobic bacteria, large amount of volatile fatty acids greatly influenced the biogas production of anaerobic fermentation would be observed [Niu et al., 2011]. Then, different ratios of corncob and PM reflected different characteristics of biogas production in this study. However, methanogenic and acidogenic microorganisms in anaerobic inoculum behaved particularly active when they contacted with the anaerobic substrates. Then the biogas successfully obtained from the anaerobic system. Fig. 2 illustrates the daily biogas production from different ratios of corncob by anaerobic fermentation within 29 days. At the beginning of the experiments, the control reached a biogas production peak of about 2400ml with the biogas production rate of about 38.30mL/g·VS·d, then the gas production dropped rapidly to below 400ml. While daily biogas production of the ratios reached a peak of about 900, 1667, 2233, 1667 and 2900ml, with the biogas production rate of about 3.04, 7.65, 12.81, 15.32 and 36.98mL/g· VS·d, respectively. Thereafter, biogas production of the ratio 2:3 started to decrease rapidly than others, while the biogas production of other experiments played slightly decline at first few days. The biogas 7

production of the control declined until the end of the experiment. Meanwhile, the biogas production of the ratio 5:2 maintained at the level of 500ml. And the biogas production curve of the ratio 1:1 and 2:3 fell in a straight line until the biogas production declined to about 500ml. However, the biogas production of the ratio 2:1and 3:2 maintained at a level of about 1500 mL for 8 days and then the biogas production of them reduced to about 400ml at the end of the experiment. Daily methane content from different ratios of corncob by anaerobic fermentation within 29 days was shown in Fig. 3. Methane production of all the experiments performed almost the same production regular by its specific content in biogas. The methane contents were rapidly rising when the anaerobic fermentation began. The methane contents of the ratios maintained at the level about 60% until the end of the reaction. However, different ratios of corncob and PM lead to different biogas production after anaerobic fermentation. In order to find out the proper ratio between corncob and PM from different ratios for anaerobic digestion, analysis of the gas production result is essential. Fig. 4 describes the biogas and methane production of different ratios of corncob during 29 days anaerobic fermentation. Biogas and methane production performed different in gas yield and gas production rate as showed in Fig. 3. Comparing with the gas production rates, the Control showed a good gas production rate (based on anaerobic sludge and PM) and it reflected that the methanogenic microbials in anaerobic system were quite active. While biogas and methane production rates in ratios of 5:2, 2:1, 3:2, 1:1 and 2:3 (based on corncob) respectively were about 234 and 140 mL/g·VS, 55 and 31 mL/g· VS, 165 and 104 mL/g· VS, 216 and 134 mL/g· VS, 243 and 153 mL/g· VS, 374 and 226 mL/g·VS. Obviously, the ratio 2:3 was the highest in the aspect of biogas and methane production rate. It proved that the ratio 2:3 made the largest utilization of corncob. But the ratios contained different contents of corncob; the gas yields relative to 8

the gas production rate were different after the anaerobic digestion. Comparing with the gas yields in Fig. 4, the control produced the lowest biogas and methane, while total biogas and methane yields in ratios of 5:2, 2:1, 3:2, 1:1 and 2:3 were about 14.66 and 8.76L, 16.25 and 9.12L, 36.03 and 22.71L, 37.57 and 23.38L, 26.48 and 16.62L, 29.34 and 17.74L, respectively. Obviously, the ratio 3:2 presented the highest total biogas and methane yield. However, comparing with the ratio 3:2 and the ratio 2:3, the ratio 2:3 degraded with 90g corncob made better use of corncob than the ratio 3:2 with 200 g corncob, but the ratio 3:2 produced more biogas and methane than the ratio of 2:3 in the same working volume, the ratio 3:2 provided greater economic value. In this study, the ratio 3:2 was chose to be the proper ratio for the next experiment.

3.2. Amplification of the ratio of 3:2 for biogas production by corncob After the fermentations of different ratios between corncob and PM described in 3.1, 400 g corncob and about 30% inoculum of the ratio 3:2 between corncob and PM were added into glass bottles with working volume 4L for anaerobic digestion. The fermentation conditions were controlled as 2.2 described above. The experiment continued for 29 days. After the anaerobic fermentation of the amplification of the ratio 3:2, 86.70L of total biogas and 63.15L of total methane were produced, respectively. The average methane content was about 61.15%. Fig. 5 described the biogas and methane production rate after the amplification of the anaerobic fermentation of the ratio 3:2. It reflected the anaerobic fermentation was successful with the gas production trend by the daily biogas and methane production rate. Daily biogas and methane production rate showed the gas production was increased first and then decreased to a stable level, then increased and decreased until no effective gas for measure produced. The modeled parameters using the Gompertz equation for methane production 9

potential are shown in Fig. 6 and Table 2. It was observed that methane production rate stabilized after 2.23 days lag period. The methane production rate was 6.04 mLCH4/ g VS d. The maximum methane production potential reached 170.59 mLCH4 /g VS. Meanwhile, the time spent on the methane production yields reached the final 80% was 22 days. The accumulation of biogas production rate and methane production rate respectively reached 220.71 and 139.37 mL/g VSadded. Meanwhile, it was reported that biogas residue contained a large amount of carbon source, nitrogen source and other micronutrients after anaerobic digestion which could be further utilized [Sieling et al., 2013, Zirkler et al., 2014]. In this study, after corncob was used as renewable waste feedstock for methane production by anaerobic digestion, biogas residue was then served as fodder for grease production as extra energy recycling by BSFL.

3.3. Grease production from biogas residue by BSFL Lignocelluloses were partially digested to the monomeric sugars by pretreatment and converted into biogas by anaerobic digestion [Petersson et al., 2007], and it still retained substrates in biogas residue, such as a small amount of residual sugar from lignocellulose. Then the BSFL was contributed to energy recycling by feeding on biogas residue. In this study, 500 BSFL (0.0016g, individual weight) were inoculated into the biogas residue which was obtained from the fermentation of corncob described above in 3.2. After 8 days conversion, about 62.24g BSFL (0.1245g, individual weight) were obtained and 14.35g of dried biomass was obtained from 500 BSFL grown on 500g mixed feedstock of biogas residue, the dryed BSFL biomass was placed into a filter bag and soaked in petroleum ether (200 ml) for 48 h twice at room temperature. Resulted leach liquor was combined. Crude larval grease was then obtained by evaporating petroleum ether with a rotary evaporator which was reported in Li et al. [Zheng et al., 2012]. It yielded about 10

3.34g of BSFL grease. Thereafter, grease accounted for about 23.28% of the insect biomass as the comparison with three kinds of manures described in Table 3. Biogas residue is the second kind of waste after biogas production by anaerobic fermentation, it proved that biogas residue could be utilized for grease accumulation by BSFL.

3.4. Biodiesel production from BSFL grease A two step approach with acid-catalyzed esterification and sequential alkaline-catalyzed transesterification was used for biodiesel production. Briefly, as a pretreatment to convert free fatty acids in the crude grease into biodiesel, acid-catalyzed esterification was conducted at the following conditions: 1% H2SO4 as the catalyst, 75℃, methanol to grease ratio(8:1), and reaction for 1 h, to decrease the acid value of the crude grease. Resulted mixture was poured into a funnel for separation. The upper layer containing crude grease and biodiesel was further transferred into a new reactor for alkaline-catalyzed transesterification at the following conditions: methanol to grease ratio of 6:1, 0.8% NaOH (w/w) as the catalyst, 65℃, and reaction time of 30 min. After separation with a funnel, the biodiesel layer was distilled at 80℃ to remove residual methanol. About 3.17g of biodiesel was extracted from 3.34g BSFL grease with the extraction yield of 94.91%.

3.5. Change of Lignocelluloses s during two-step bioprocess Lignocelluloses reduction in the whole biomass utilization of corncob could objectively response the life activities of microorganisms. In this study, two main biological treatments of anaerobic digestion and biomass accumulation of insects were combined for corncob utilization, and it brought economic products of biogas and grease. As a feedback, it degraded a large number of lignocellulose of corncob. Fig. 6 shows the 11

changes of the lignocelluloses content in two-process biological treatment. The anaerobic digestion can achieve a special effect for the degradation of lignocellulose. From corncob to biogas residue by anaerobic digestion with 400g corncob of the ratio of 3:2, the cellulose, hemicellulose and lignin content in corncob decreased respectively from about 34.53%, 34.59% and 17.33% to about 27.67%, 25.59% and 13.59% in biogas residue. About 6.86% of cellulose, 9% of hemicellulose and 3.74% of lignin were degraded and utilized for biogas production by metabolism of microorganisms. Obviously, it confirmed that the degradation of hemicellulose was easier than cellulose and lignin, and cellulose was more likely to be digested than lignin in anaerobic digestion [Sawatdeenarunat et al., 2014]. Interestingly, from biogas residue to conversion residue, without the extra aid of cellulase for the biogas residue，the content of lignin was reduced from 13.59% to 11.54% in the residue after converted by BSFL. About 2.05% of lignin was removed in the biogas residue and utilized for biomass accumulation of BSFL. However, it was reported that BSFL could hardly use the lignin component in bran for feed [Zheng et al., 2012], but it reduced lignin more than that ratio by adding BSFL into biogas residue in this study. Meanwhile, there are many special bacteria with the ability of the degradation of lignin existing in our life [Brown and Chang, 2014]. So it could be hypothesized that the lignin degrading bacteria might survive in the conversion and it needed further research to prove.

3.6. Potential economic analysis for conversion According to the results of the experiments, possible economic analysis for the future application is made. If the output of biodiesel is 1 tons, about 1.05 tons BSFL grease should be produced by about 157.73 tons of biogas residue and 157.73 million of BSFL. To meet the need of biogas residue, about 126.18 tons of corncob and 353.17 tons of PM should be supplied. It is difficult to find the accurate data of corncob yield 12

from corn and pig manure yield from pig in the open literature. As a general rule of thumb, corncob accounts for about 1/8 of total weight of corn and every pig produces about 2.5kg feces every day. Meanwhile, data from FAO (food and agricultural organization) showed that corn yield in 2013 was about 60161.58 kg/hectares. In total, at least 16.78 hectares of acreage for planting corn and 387 pigs in farm can meet the output needs of producing 1 tons biodiesel during a year. But there are various errors may exist in expansion application that makes the reasoning data inaccurate. However, this research is conducted to study the feasibility of producing biogas and biodiesel by corncob, PM and BSFL, analysis on the costs from the possible application in future which involves a lot of practical issues need further research, such as problem of expansion application of energy production, distribution of raw materials and problem of transportation means and routes, especially the shortest path problem for transportation which can utilize the method of setting up complex mathematical model and using software of Matlab or Lingo to analyze. Meanwhile, insect protein can also be obtained from BSFL which can be another source of economic except grease [Li et al., 2011a].

4. Conclusions 86.70L biogas was obtained from 400g corncob with the accumulation of biogas yield of 220.71 mL/g VSadded by anaerobic digestion under the fermentation rate of 3:2 between corncob and PM. 3.17g biodiesel accounted for 94.91% was extracted from 3.34g grease after inoculating BSFL in the biogas residue for 8 days. Generally, 100g corncob could produce 20.43L biogas and 0.79g biodiesel. This study proves the corncob wastes can be further converted to high grade bioenergy of biogas and biodiesel by biotechnology methods, and lignocelluloses might have the opportunity to be further utilized by feeding special kind of insect. 13

Acknowledgements This work was supported in part by grants from the Fundamental Research Funds for the Central Universities (No2013PY061) and the National Fundamental Research Funds (No21172083). The authors gratefully acknowledge the financial support from National Science Foundation of China (No51376078). Thanks to Professor Jeffery K. Tomberli from Texas A&M University for providing black soldier fly and the method for culturing the insect.

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Figure Captions

Fig.1 Biogas and biodiesel production from corncob and BSFL

Fig. 2 Daily biogas production from different ratios of corncob

Fig. 3 Daily methane content from different ratios of corncob

Fig. 4 Biogas and methane production from different ratios of corncob

Fig. 5 Biogas and methane production rate of the ratio 3:2 under the amplification experiment

Fig. 6 Gompertz model of cumulative methane production of the ratio 3:2

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Fig.1. Biogas and biodiesel were produced from corncob by anaerobic fermentation and BSFL.

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30

Time (day)

Fig. 2. Daily biogas production from different ratios of corncob.

20

75

Methane content(%)

70 65 60 55 50 45

Control 5:2 1:1 2:1 3:2 2:3

40 35 30 25 0

3

6

9

12

15

18

21

24

27

Time(day)

Fig. 3. Daily methane content from different ratios of corncob.

21

30

Gas yield(ml)

35000

350

30000

300

25000

250

20000

200

15000

150

10000

100

5000

50

.

400

Gas production rate(ml/g vs)

40000

0

0 Control

5:2

2:1

3:2

1:1

2:3

Biogas production rate methane production rate

Biogas yield Methane yield

Fig. 4. Biogas and methane production from different ratios of corncob.

22

15

200 10

150

100 5

50

0

0

0

3

6

9

12 15 18 Time（day）

Cumulative biogas production rate Cumulative methane production rate

21

24

27

Daily gas production rate（mL/g.vs)

Cumulative gas production rate（mL/g.vsadded）

250

30

Daily biogas production rate Daily methane production rate

Fig. 5. Biogas and methane production rate of the ratio 3:2 under the amplification experiment.

23

Comulative CH4 production rate(ml/g VS)

150

120

90

60

30

CH4 production data Gompertz model

0 0

3

6

9

12

15

18

21

24

27

30

Time(d)

Fig. 6. Gompertz model of cumulative methane production of the ratio 3:2.

24

Table 1 Characteristics of anaerobic sludge, pig manure and corncob. Characteristics pH

Anaerobic sludge

Pig manure

Corncob

7.80

NA

NA

Total solids (%w)

8.72±0.92

26.38±0.77

89.01±1.29

Volatile solids (%w)

5.24±0.18

20.82±0.85

87.41±0.95

Hemicellulose (%)

NA

NA

34.59±1.31

Cellulose (%)

NA

NA

34.53±0.71

Lignin (%)

NA

NA

17.33±1.45

Note: NA (no analysis), w (wet base)

25

Table 2 Model parameters of modified Gompertz and experimental methane production rates during the ratio of 3:2. Substrate

3:2

P (mLCH4 /g VS)

170.59

λ (days)

Rmax (mLCH4/ g VS d)

6.04

2.23

R2

Methane production rate (mL CH4/g VSadded)

0.99

t80 is the time when accumulative methane production rate reach the 80% of final production rate.

26

139.37

t80 (d)

22

Table 3 Grease yield of BSFL fed on biogas residue in comparison with three kinds of manures.

Grease yield (%)

Biogas residue

Cattle manure[Li et al., 2011]

Pig manure[Li et al., 2011]

Chicken manure[Li et al., 2011]

23.28

29.90

29.10

30.10

27

Table 4 Change of lignocelluloses during biological treatments. Cellulose (%)

Hemicellulose (%)

Lignin (%)

Control

34.53±0.71

34.59±1.31

17.33±1.45

Anaerobic fermentation

27.67±1.43

25.59±0.92

13.59±0.31

BSFL bioconversion

26.71±1.32

25.31±0.91

11.54±0.48

28

Highlights

Biogas and biodiesel were obtained from corncob by anaerobic fermentation and BSFL.

29