Energy Conversion and Management 75 (2013) 21–24

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Energy Conversion and Management

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Biogas performance from co-digestion of Taihu algae and kitchen wastes

0196-8904/$ - see front matter Crown Copyright � 2013 Published by Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.enconman.2013.05.037

⇑ Corresponding author. Tel./fax: +86 510 85197091.E-mail address: wqruanjnedu@yahoo.com.cn (W.-Q. Ruan).

Ming-Xing Zhao, Wen-Quan Ruan ⇑School of Environment and Civil Engineering, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu Province 214122, China

a r t i c l e i n f o a b s t r a c t

Article history:Received 1 April 2013Accepted 27 May 2013

Keywords:Taihu algaeKitchen wastesBiogasNeutral protease enzymeDehydrogenase enzyme

Co-digestion of Taihu algae with high carbon content substrate can balance the nutrients in thefermentation process. In this study, optimal mixing ratio for co-digestion of Taihu algae and kitchenwastes were investigated in order to improve biogas production potential. The results indicated thatthe biogas yield reached 388.6 mL/gTS at C/N15:1 group, which was 1.29 and 1.18 times of algae andkitchen wastes only. The maximum concentration of VFA reached 4239 mg/L on 8th day in kitchenwastes group, which was 1.21 times of algae group. Neutral protease enzyme activity in algae groupreached maximum of 904.2 lg/(gTS h), while dehydrogenase enzyme at C/N 15:1 group reached maxi-mum of 3402.2 lgTF/(gTS h). The feasibility of adjusting the C/N with co-digestion of Taihu algae andkitchen wastes to increase biogas production was demonstrated. Remarkably, the C/N of 15:1 was foundto be the most appropriate ratio.

Crown Copyright � 2013 Published by Elsevier Ltd. All rights reserved.

1. Introduction

As the third largest freshwater lake in China, Taihu Lake suffersalgae blooming throughout the summer in recent years due to theeutrophication [1]. Salvaging of algae from polluted lake was anefficient way to retrieve nitrogen and phosphorus from lake, there-by reduce lake pollution. However, algae collected could amount tothousands of tons every day, and further disposal of skimmed algaewas necessary to avoid serious secondary environmental contami-nation [2].

Anaerobic digestion technology was considered to be a practicalway to deal with the skimmed algae, since the process did not re-quire advanced dewatering or further chemical extraction, more-over, it not only decrease the amount of waste, but also generatebiogas as sustainable energy [3,4]. The low C/N ratio of algae itselfwas a serious problem to the anaerobic fermentation, which led toabound total ammonia nitrogen release during the digestion pro-cess. Some excellent research had proved that co-digestion of algaewith high carbon content substrate could provide a good solution.Yen and Brune [5] indicated that adding waste paper in algaesludge increased the methane production rate, Zhong et al. [6]showed that co-digestion of blue algae and corn straw improvedmethane yield by 61.69% compared with algae digestion alone.

As a burden of environment issue in cities, the generation ofkitchen wastes has been growing extremely fast every year inChina [7]. Kitchen wastes can be a valuable alternative feedstockfor biogas production due to its carbohydrate-rich nature [8]. How-ever, the operation of anaerobic digester fed with kitchen wastes

was not very effective and stable due to the accumulation of vola-tile fatty acids (VFAs). Therefore, co-digestion of kitchen wasteswith other organic wastes, such as municipal sludge, agriculturalstraw and animal manure become more popular [9]. Co-digestionof Taihu algae and kitchen wastes can balance the fermentationnutrients during the digestion process, and this was a fresh at-tempt since there was sparse information about such mixed sub-strates degradation. Hydrolysis was a limiting step duringanaerobic digestion [10], but the important information about thisprocess can be obtained by investigating hydrolysis enzymaticactivities. Neutral protease was an important hydrolysis enzymeconcerned with the nitrogen cycle [11], while dehydrogenase en-zyme corresponded with the microbial dehydrogenation capabilityof organics, could indirectly indicator of microbial activity [12].However, little is known about the performance of such enzymesduring the co-digestion of Taihu algae and kitchen wastes.

The viability of co-digestion Taihu algae with kitchen wastes inbatch mode was indicated in this study. The optimum mixing ratioof two substrates for digestion was discussed. In addition, the per-formance of neutral protease enzyme and dehydrogenase enzymeduring fermentation process was also presented.

2. Materials and methods

2.1. Taihu algae and kitchen wastes

The wet algae was collected from Bogong Island, Taihu Lake,Wuxi, China, the dominant strain of algae was Microcystis spp.The kitchen wastes were collected from dining hall in JiangnanUniversity. Kitchen wastes were grinded after bones, crushed

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paper and caps, etc. were singled out. Characteristics of substrateswere shown in Table 1.

2.2. Inoculum

Anaerobic granular sludge was obtained from an anaerobic di-gester with 1000 m3 working volume at Xielian Thermal PowerPlant Ltd., Wuxi, China. The digester was operated of 35 �C at aHRT of 1 day. The TS and VS of the sludge were 7.20% and 73.62%(TS), respectively.

2.3. Experiment setup

The mixed substrate was made up of Taihu algae and kitchenwastes with total TS of 5 g. The volume of experiment bottle was500 mL. Bottle 1 (B1) and bottle 2 (B2) were operated with Taihu al-gae only and kitchen wastes only at a C/N ratios of 6.35:1 and24.08:1, respectively. Bottle 3 (B3), bottle 4 (B4) and bottle 5 (B5)were operated with mixed substrate at a C/N ratios of 10:1, 15:1and 20:1, respectively. Anaerobic granular sludge was inoculatedwith the rate of 1:1 (based on TS) to each reaction bottle. The head-space of the reaction bottles was purged with nitrogen for 2 min tomaintain an anaerobic environment, and then the bottles were agi-tated at 70 rpm and reaction temperature 55 ± 1 �C in a shakingwater bath. Biogas production was recorded by quantifying waterdisplacement. Each experiment was performed in triplicate.

2.4. Analytical methods

Volatile fatty acid was analyzed using an HPLC (Agilent 1100,USA) equipped with a UV detector at the wavelength of 210 nmand with a ZORBAX SB-A column (300.0 � 7.8 mm, Biorad, USA)at the column temperature of 30 �C. The mobile phase consistedof 0.5% acetonitrile and 99.5% KH2PO4 (0.02 mol/L), at a flow rateof 0.5 mL/min. Total nitrogen and protein were determined bythe Kjeldahl method. Carbohydrate was determined by the phenolsulfuric acid method [13]. Lipids were determined using the Soxh-let method [14], total carbon was monitored with a TOC analyzer(Elementar, Germany).

Neutral protease enzyme measurement was based on the pro-tein transition to amino acid [11]. Dehydrogenase enzyme activityassay was based on estimation of the triphenyltetrazolium chloride(TTC) reduction rate to triphenyl formazan [12].

The Modified Gompertz equation which used to describe theproduct formation in the biogas production process as follows[15]. P was the product formed at fermentation time, mL; Pmax

was the potential maximum product formed, mL; Rmax was the max-imum rate of product formed, mL/d; k was the lag time to exponen-tial product formed, d:

P ¼ Pmax exp � expRmax � e

Pmaxðk� tÞ þ 1

� �� �ð1Þ

3. Results and discussion

3.1. Biogas performance from co-digestion fermentation

The cumulative biogas of algae and kitchen waste fermentationwas plotted in Fig. 1. The biogas increased rapidly with time up to

Table 1The characteristics of Taihu algae and kitchen wastes.

Substrate Total solids (TS/%) Volatile solids (VS/%) Total carbon (mg/g TS)

Taihu algae 2.15 1.68 512.43Kitchen

wastes15.76 14.35 726.78

20 days, after which it remained stable in all bottles, and the wholeduration of biogas production lasted 26 days. Interestingly, therewas a second peak biogas during 12–20 days in B3, B4 and B5. Bio-gas yield of each group finally reached 1503.5, 1642.3, 1827.4,2003.7 and 1978.8 mL, respectively. Data indicated that the mixedsubstrate at a C/N ratio of 15:1 had a better performance thanother two groups (B3 and B5). Yen and Brune [5] suggested an opti-mum C/N ratio for algae and other substrate co-digestion was nec-essary. Zhong et al. [6] found adjusting the C/N increased 61.69%compared the algae only. These reports were in agreement withour research.

Biogas generation was accompanied by the degradation of or-ganic component in substrate. Compared with algae only group(B1), the benefit of adding kitchen wastes was evident (B3, B4and B5), this was attributed to the mixed substrate could not onlybalance the nutrients for anaerobic bacteria, but also enhance thebuffering capacity of the digester. Lay et al. [16] indicated that car-bohydrate was preferentially degraded than protein and lipids.Kitchen wastes in mixed substrate probably preferred degradationin the initial because of the higher concentration of carbohydrateitself, while algae degradation relatively delay attributed to the cellwalls and higher concentration of protein itself. That could explainwhy there were two peaks during the biogas generation process(Fig. 1).

The parameters of the fitted equation for the biogas were sum-marized in Table 2. The maximum correlation coefficient (R2) was0.9991 in B1, and it was all over 0.988 in other groups, suggestingthat the modified Gompertz equation was able to describe the bio-gas generation. As shown in Table 2, the Pm and Rm reached maxi-mum of 1943.2 mL and 197.2 mL/d in B4. Mixed substrate groupimproved the specific biogas yield than that of algae and kitchenwastes only, since it was 13.9–14.9 mL/(gTS d) in B3, B4 and B5,while it was only 11.6 mL/(gTS d), 12.6 mL/(gTS d) in B1 and B2,respectively. The delay time (k) was about 1–2 days, suggestingthe anaerobic sludge could adapt to the reaction system quickly,and generate biogas in a short time in each group. The biogas pro-duction rate achieved maximum of 388.6 mL/gTS in B4, which washigher than 86.5 and 60.1 mL/gTS of algae and kitchen wastesgroup, respectively. The biogas performance of algae group (B1)was higher than that the batch anaerobic digestion of Microcystisspp., with 166.8 mL/gTS [17]. But the maximum biogas yield ofco-digestion group was lower than that of algae and corn strawmixed fermentation [6], which was probably due to the differentdigestion temperature and the characteristic of adding substrate.

3.2. Metabolities development during reaction process

VFA is important metabolic products, as the most biogas is de-rived from VFA [18]. An accumulation of VFA can indicate factorsthat might be affecting the methanogenic bacteria [19]. The con-centration of VFA significantly increased in the first 8 days, but itdecreased afterward in all groups except B1 (Fig. 2). B3, B4 andB5 had a second raise during 14–16th days. The maximum concen-tration of VFA reached 4239 mg/L on 8th day in B2, which was 1.21times of B1. B3, B4 and B5 had a peak of 3721, 3803 and 4076 mg/Lon the 8th day, respectively. While the concentration of VFA ran-ged from 849 to 1309 mg/L at the end of reaction.

Total nitrogen (mg/g TS) Carbohydrate(TS�1)

Protein(TS�1)

Lipids(TS�1)

80.72 6.43 46.43 2.3230.18 52.32 18.45 10.65

Fig. 1. Variation of biogas generation from Taihu algae and kitchen wastes.

Table 2Parameters results using modified Gompertz equation for biogas generation.

Group Pm

(mL)Rm

(mL/d)

k(d)

Biogasproductionrate (mL/gTS)

Specificbiogas yield(mL/(gTS d))

Correlationcoefficient(R2)

B1 1510.4 142.6 2.1 302.1 11.6 0.9991B2 1632.4 164.0 1.4 328.5 12.6 0.9988B3 1800.3 155.4 0.8 360.1 13.9 0.9940B4 1943.2 197.2 0.9 388.6 14.9 0.9886B5 1916.0 184.4 0.9 383.2 14.7 0.9906

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VFA was accumulated at the beginning due to the organic mat-ter hydrolysis, after which it was continuously consumed with thereaction proceeds. The hydrolysis of kitchen wastes seemed fasterthan algae as B1 reached the peak on the 10th day, while it was onthe 8th day in B2. Mussgnug et al. [20] suggested that algae wasresistant to bacteria attack due to the strong cell walls. Becauseof slowly nutrients released in algae as reaction proceeded, thereaction system had a second peak of VFA in B3, B4 and B5 with3501, 3395 and 3198 mg/L on the 16th day, respectively.

The data indicated that the concentration of VFA was increasedwith the increasing of kitchen wastes in mixed substrate. A bal-anced C/N ratio in feedstock was likely to beneficial to the methan-

Fig. 2. Change of VFA during the biogas process.

ogen activity and resulted in VFA concentration decreased by moreVFA converted to methane. However, the maximum VFA concen-tration was lower than that in a digester with algae and waste pa-per as co-substrate, which reached 5220 mg/L [5]. Most of theremaining VFA in this study was analyzed to be propionate acidas agreed to the results of Zhong et al. [6].

3.3. Neutral protease enzyme characterization in the anaerobic system

The protease enzyme activity values could sensitively indicatethe changes occurred in the equilibrium of the protein degradationprocess [21]. It is suggested to use protease activity measurementsto monitor the hydrolysis reaction process [22]. Fig. 3 presents thechange of protease enzyme activity during the digestion process.All groups showed an increasing then decreasing trend. Each grouprapidly increased from initial about100 lg/(gTS h) to the maxi-mum value of 904.2, 753.4, 843.3, 833.4 and 829.3 lg/(gTS h),respectively. Noticeably, the time reached the peak was different,the fastest was on 4th day in B2, while it was on 8th day in B1.After that, the enzyme decreased quickly to 112.5–233.3 lg/(gTS h) at the end.

The activity of enzyme showed a slight increase at the begin-ning due to the hydrolysis of protein in substances. As the highconcentration of protein in Taihu algae, enzyme in B1 was signifi-cantly, the maximum value was 120.0% of B2. Enzymes excretionand transmission was more favorable in a liquid system than in adry system, making the maximum values of protease better thanthat reported by Lu et al. [23]. With the decreasing compositionof Taihu algae in mixed substrate, the value of protease enzyme de-clined, which was due to the reducing of total protein in substrate.

Previous studies had shown that protease enzyme can be af-fected by temperature [21], metal ions [24], reaction devices[25], et al. In our study, it was found that the composition of co-digestion substrate was also an important factor. Levente et al.[21] indicated protease activity was sensitive to the external envi-ronment change, which also detected in this study, since proteasevalue represented a large variation during the digestion process.

3.4. Dehydrogenase enzyme characterization in the anaerobic system

Dehydrogenase enzyme activity can be a good parameter forcharacterizing the microorganism activity [26,27]. The dehydroge-nase enzyme initially increased sharply in all groups, but it de-creased afterward. The activity of microorganism abound

Fig. 3. Variation in the enzymatic activity of neutral protease enzyme.

Fig. 4. Variation in the enzymatic activity of dehydrogenase enzyme.

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increased as the organic matters were hydrolyzed during the firsttime, B1 and B2 reached maximum of 2109.2 and 2892.3 lgTF/(gTS h), which was lower than the group of B3, B4 and B5 of3283.3–3402.2 lgTF/(gTS h) (Fig. 4). The peak time was on 12th,6th, 10th, 10th and 10th day, respectively. It was found that biogasaccumulation was in accordance with the trend of dehydrogenaseenzyme, since the high biogas output also occurred during the first10 days and subsequently weakened (Fig. 1). This observation re-vealed that the dehydrogenase enzyme might indicate the micro-bial activity, which was consistent with our previous findings inhydrogen production process [28]. The finally concentration ofdehydrogenase ranged from 400.2 to1098.0 lgTF/(gTS h).

It seemed that adding kitchen wastes activated the degradationmicroorganisms in mixed substrate group, as dehydrogenase activ-ity in B4 had a maximum value of 3402.2 lgTF/(gTS h), which was161.3% of B1. Because of the solid cell walls in algae, algae cannotbe broken and utilized by microorganisms in a short time, makingits peak value on 12th day. Zhong et al. [6] found anaerobicdigestion of algae with high carbon material could enhance biogasefficiency, the possible reason for explaining increased biogas yieldwas the improve of enzyme activities in the digestion systemthrough our study.

4. Conclusions

In this study, significant increase of biogas can be achieved byadjusting the C/N with co-digestion of Taihu algae and kitchenwastes. The group with C/N of 15:1 reached maximum biogas yieldof 2003.7 mL, which was 33.3% higher than Taihu algae only. Withthe increasing constituent of kitchen wastes in mixed substrate,the concentration of VFA was increased. The neutral protease anddehydrogenase enzyme represented first increased then decreasedtrend. Co-digestion mode promoted the enzyme activity duringfermentation process.

Acknowledgements

This research was supported by National Scientific andTechnological Support of China (2012BAC18B01-2); Scientific andTechnological Support of Jiangsu Province, China (BE2012615);and The Fundamental Research Funds for the Central Universities(JUSRP111A12).

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