Effects of temperature and organic loading rate on the performance and microbial community of anaerobic co-digestion of waste activated sludge and food waste

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    College of Environmental Science and Engineering, Hunan University, Changsha 410082, PR Chinaollution Control, Hunan University, Ministry of Education, Changsha 410082, PR China

    stigatedy werest endut stabilfected

    tank reactors. Results showed that the performance of co-digestion system

    walls are physical barriers that do not permit intracellular organicsto be easily biodegraded through digestion (Toreci et al., 2011). Inprevious studies, pretreatments such as mechanical (Nah et al.,2000), microwave (Toreci et al., 2011), alkaline (Li et al., 2012)

    release the intra-als were greatly

    h residuesto overco

    hazardous compounds, promote synergistic effects of microorgan-isms and enhance biogas yields (Wan et al., 2011). Food waste(FW), with its high organic contents and excellent biodegradability,was regarded as an appropriate substrate that can be treated by AD(Zhang et al., 2007). Furthermore, plenty of attention has been paiddue to its huge production from daily life in China. Nevertheless,some literatures pointed out that the digestion of FW alone maylead to the accumulation of abundant volatile fatty acids (VFA)

    Corresponding author at: College of Environmental Science and Engineering,Hunan University, Changsha 410082, PR China. Tel./fax: +86 0731 8882 2829.

    E-mail address: yzh@hnu.edu.cn (Z. Yang).

    Chemosphere xxx (2014) xxxxxx

    Contents lists availab


    evicess, as waste activated sludge (WAS) is mainly composed ofmicrobial cells within extracellular polymeric substances, and cell

    low digestibility in several studies (Habiba et al., 2009; Silvestreet al., 2011). Meanwhile, proper co-digestion could dilute potentialAnaerobic digestion (AD) is widely applied for sludge stabiliza-tion to reduce the sludge volume, generate methane gas, and yielda nutrient-rich nal product (Appels et al., 2011). However, its ef-ciency is largely limited due to the relatively slow hydrolysis pro-

    ciency of AD by disrupting sludge membranes tocellular nutrients, but extra energy or chemicconsumed simultaneously.

    Co-digestion of sludge with other organic-ricto be an attractive method which has been usedhttp://dx.doi.org/10.1016/j.chemosphere.2014.01.0180045-6535/ 2014 Elsevier Ltd. All rights reserved.

    Please cite this article in press as: Gou, C., et al. Effects of temperature and organic loading rate on the performance and microbial community of anco-digestion of waste activated sludge and food waste. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.01.018seemsme itsReceived in revised form 12 January 2014Accepted 15 January 2014Available online xxxx

    Keywords:Anaerobic co-digestionWaste activated sludgeFood wasteTemperatureOrganic loading rateMicrobial community

    was distinctly inuenced by temperature and organic loading rate (OLR) in terms of gas production rate(GPR), methane yield, volatile solids (VS) removal efciency and the system stability. The highest GPR at55 C was 1.6 and 1.3 times higher than that at 35 and 45 C with the OLR of 1 g VS L1 d1, and the cor-responding average CH4 yields were 0.40, 0.26 and 0.30 L CH4 g

    1 VSadded, respectively. The thermophilicsystem exhibited the best load bearing capacity at extremely high OLR of 7 g VS L1 d1, while the mes-ophilic system showed the best process stability at low OLRs (< 5 g VS L1 d1). Temperature had a moreremarkable effect on the richness and diversity of microbial populations than the OLR.

    2014 Elsevier Ltd. All rights reserved.

    1. Introduction and ultrasonic (Xu et al., 2011) were reported to improve the ef-Article history:Received 17 August 2013

    Anaerobic co-digestion ofusing continuously stirredKey Laboratory of Environmental Biology and P

    h i g h l i g h t s

    Co-digestion of WAS and FW was inve The CH4 yield and VS removal efcienc The thermophilic system had the highe The mesophilic system showed the bes The microbial community was more af

    a r t i c l e i n f oat different temperatures and OLRs.decreased as OLR gradually increased.rable OLR of 7 g VS L1 d1.ity at low OLR (< 5 g VS L1 d1).by temperature than the OLR.

    a b s t r a c t

    waste activated sludge and food waste was investigated semi-continuouslyChengliu Gou, Zhaohui Yang , Jing Huang, Huiling Wang, Haiyin Xu, Like WangTechnical Note

    Effects of temperature and organic loadinand microbial community of anaerobic csludge and food waste

    journal homepage: www.elsrate on the performancedigestion of waste activated

    le at ScienceDirect


    er.com/locate /chemosphereaerobic

  • greatly disturbed by the OLR (Luste and Luostarinen, 2010). Themajor problem is that with an extremely high OLR, the rate of

    working volume was lled with inoculums, and the co-substrateswere introduced into the reactors as the starting material. The sys-

    supernatant was ltrated through a 0.45-lm membrane lter

    Standard Methods (APHA, 2005).

    2.4. Microbial community analysis

    Samples were taken at steady state of each temperature undervarious OLRs for microbial community analysis. Total genomicDNA was extracted from 0.2 g digestion samples (wet weight)according to the method described by Yang et al. (2007). DNAwas dissolved in 200 lL of 50 TAE (2 M Tris, 1 M Acetate, 0.1 MNa2EDTA2H2O) buffer and 4 lL of DNA was used for agarose gelelectrophoresis.

    The PCR amplication was performed on an iCycler IQ5 Ther-mocycler (Bio-Rad, USA). The primer set GC341F and 534R wereused for amplication. DGGE was carried out by using the Dcode

    herhydrolysis/acidogenesis could be higher than methanogenesis,and the high concentration of VFA accumulated from hydrolysis/acidogenesis can eventually lead to an irreversible acidication(Nagao et al., 2012). Nevertheless, previous studies mainly focusedon the methane production and volatile solids (VS) removal ef-ciency of co-digestion system in a tolerable range of OLR (Heoet al., 2004; Kim et al., 2004, 2006), the information on maximumfeasible loading rate is still lacking. Therefore, it is interesting toinvestigate the critical value by increasing the OLR stepwisethrough long-term experience.

    Temperature, another important environmental factor, directlyaffects the dynamic situation of microorganisms. Earlier studiesmainly concentrated on improving the efciency of co-digestionprocess at a certain operating temperature, such as mesophilic(Dai et al., 2013), thermophilic (Kim et al., 2011), and even underhyperthermophilic condition (Lee et al., 2009). It seems that moredata were demanded to compare the co-digestion performances atdifferent temperatures applying the similar OLR, especially usingcontinuously stirred tank reactors (CSTRs) in which all of the reac-tions (hydrolysis, acidogenesis, and methanogenesis) happenedsimultaneously. In addition, although the microbial communityhas been analyzed in the acidogenic fermenter for anaerobic co-digestion of kitchen garbage and sewage sludge (Lee et al., 2009),the information concerning the effects of the gradient temperatureand OLR on microbial community structures in CSTRs is currentlyinsufcient.

    Consequently, the objective of this study was to evaluate theefciency and stability of anaerobic co-digestion of WAS and FWunder a wide range of OLRs at different temperatures in CSTRs.Meanwhile, the microbial community involved in anaerobicco-digestion process was also investigated by means of 16S rDNApolymerase chain reaction (PCR) and denaturing gradient gelelectrophoresis (DGGE).

    2. Methods

    2.1. Preparation of feed stocks and inoculums

    WAS used in this study was obtained from the secondary clari-er of the second municipal wastewater treatment plant in Chang-sha, China. Fresh sludge was concentrated by sedimentation for 6 hbefore being used. FW was collected continuously over 5 consecu-tive working days from a refectory in Hunan University, Changsha,China. To facilitate the digestion effectively, the FWwas crushed toa mean size of 35 mm by an electrical food grinder. Samples werestored at 4 C for no longer than one week.

    WAS and FW were mixed with a total solids (TS) ratio of 2:1,especially at high organic loading rate (OLR), which could inhibitthe methanogenesis and even destabilize the anaerobic process(El-Mashad et al., 2008; Nagao et al., 2012). These ndings led tothe investigation of its co-digestion with WAS as an alternative.Moreover, co-digestion of WAS and FW could be a strategic andcross-sectorial solution to deliver benecial synergies for the waterindustry and FW management authorities (Iacovidou et al., 2012).

    Efciency and process stability are proved to be the criteria forthe performance of AD (Lv et al., 2010). Most recently, impacts ofmixing ratios and hydraulic retention time (HRT) on the perfor-mance of co-digestion of WAS and FW have been discussed (Heoet al., 2004; Kim et al., 2007; Lee et al., 2009). In fact, the equilib-rium and productivity of the fermentation process can also be

    2 C. Gou et al. / Chemospwhich was demonstrated to have the best stability and efciencyin our preliminary experiments, and the value was approximatelyequal to the optimal mixture suggested by Kim et al. (2007). The

    Please cite this article in press as: Gou, C., et al. Effects of temperature and orgaco-digestion of waste activated sludge and food waste. Chemosphere (2014), h(Whatmann, USA). TS, VS, total VFA (TVFA), alkalinity, total nitro-gen, ammonium, and SCOD were determined according to thetems were ushed with N2 for 3 min to create an anaerobic envi-ronment before sealing. Then the feeding and withdrawing wereconducted once a day according to the needed OLR. Starting-upOLR was 1 g VS L1 d1 and the corresponding HRT was 33 d. TheOLR was then increased to next step when the system reached asteady state. Corresponding operation process is shown in Table 2.

    2.3. Analytical methods

    Biogas volume was daily measured with water displacement,and methane content was analyzed by a gas chromatograph (GC2010 Shimadzu) equipped with a thermal conductivity detectorand a 2 m 3 mm stainless-steel column packed with Porapak Q(80/100 mesh). Samples from the reactors were immediately cen-trifuged at 5000 rpm for further analyses. For the analysis of solu-ble chemical oxygen demand (SCOD) and ammonium, thedetailed characteristics of these substrates are summarized inTable 1.

    2.2. Semi-continuous anaerobic co-digestion systems

    Three series of lab-scale CSTRs were installed with a workingvolume of 2.0 L. The temperatures were controlled at 35 2,45 2 and 55 2 C by three water baths, and were referred toas R1, R2 and R3, respectively. Mixing was performed intermit-tently by magnetic stirrers at uniform speed of 200 rpm beforeand after the new substrate was added. About 60% of digesterTable 1Characteristics of feed stocks and inoculums.

    Characteristics Unit Inoculums WAS FW Co-substrate

    TS g L1 20 25 150 45VS % of TS 58 63 90 72pH 7.8 7.2 5.6 6.8SCOD mg L1 2324 2165 7260 14838C/N 5.8 7.2 34 13VFA mg L1 860 467 842 748Alkalinity mg L1 as

    CaCO3212 143 453 3662

    Ammonium mg L1 221 163 113 160

    e xxx (2014) xxxxxxUniversal Detection System in accordance with the manufacturersinstructions (Bio-Rad, USA). The detailed running procedures forPCR and DGGE were operated as described by Zhang et al. (2011).

    nic loading rate on the performance and microbial community of anaerobicttp://dx.doi.org/10.1016/j.chemosphere.2014.01.018

  • 3. Results and discussion

    3.1. Effect on gas production rate (GPR)

    A semi-continuous operation was conducted to verify the per-formances of R1 (35 C), R2 (45 C) and R3 (55 C) throughout160, 178 and 188 d, respectively. A comparison of GPRs of the threesystems is shown in Fig. 1. It illustrated that GPR apparently in-creased with increasing OLR before the extreme OLR was reached.

    The rst 30 d served as a start-up stage for each reactor at anOLR of 1 g VS L1 d1. As seen from Fig. 1, GPRs in R1, R2 and R3 in-creased gradually and then became stable around day 7, 14 and 12,

    Obviously, at the same OLR, average GPR of R3 was about 1.6and 1.3 times higher than those in R1 and R2, respectively. Despitethe same tendency in the case of R2 and R3, the maximum avail-able OLRs were achieved at 6 and 7 g VS L1 d1. These data indi-cated that thermophilic methanogens performed more effectivelyand had a better bearing capacity for high OLR than those at theother two lower temperatures, which was consistent with previousstudies that reported the superior performance of the thermophilicprocesses (Bolzonella et al., 2012; Cavinato et al., 2013).

    3.2. Effect on CH4 yield and VS removal efciency

    Table 2Operational conditions of semi-continuous anaerobic co-digestion CSTR systems.

    Days 130 3160 6190 91120 121150 151170 171180 181188

    OLR (g VS L1 d1) 1 0.3 2 0.3 3 0.3 4 0.3 5 0.3 6 0.3 7 0.3 8 0.3HRT (d) 33.3 16.7 11.1 8.3 6.7 5.6 4.8 4.2Flow rate (L d1) 0.06 0.12 0.18 0.24 0.30 0.36 0.42 0.48FW (g) 4.2 0.1 8.4 0.1 12.6 0.1 16.8 0.1 21.0 0.1 25.2 0.1 29.4 0.1 33.6 0.1

    C. Gou et al. / Chemosphere xxx (2014) xxxxxx 3with average values of 0.35, 0.43 and 0.55 L L1 d1, respectively.The relatively low GPR during the rst several days was most likelydue to the predominant acidogenic activity, and the subsequentsteady value indicated that a delicate balance was achieved be-tween the rates of hydrolysis/acidogenesis and methanogenesis.

    In mesophilic digestion system, GPR went up to approximately1.3 L L1 d1 with OLR stepwise increased to 4 g VS L1 d1. The en-hanced GPR was mainly ascribed to the increased biodegradablematerials in the feedstock. Thereafter, a signicant pH drop to6.3 was noticed with an OLR of 5 g VS L1 d1 on day 121. In orderto maintain a steady state, moderate NaHCO3 (1 M) was added toalleviate pH inhibition. Under that condition, R1 could operatefavorably at 5 g VS L1 d1 despite with a relatively low GPR of1.2 L L1 d1. As OLR further increased to 6 g VS L1 d1 on day151, however, GPR decreased drastically close to zero with pHdropping to 4.9, and an irreversible acidication occurred evenwith a large amount of alkalinity being added into the system.Based on these results, it was possible to predict that the maxi-mum endurable OLR was 5 g VS L1 d1 for mesophilic co-diges-tion of WAS and FW in this study. For R2, the highest averageGPR of 1.6 L L1 d1 was achieved when the reactor operated atan OLR of 5 g VS L1 d1, while for R3, the corresponding valueswere 2.1 L L1 d1 at 6 g VS L1 d1.Fig. 1. Variation of GPR according to different temperatures an

    Please cite this article in press as: Gou, C., et al. Effects of temperature and organco-digestion of waste activated sludge and food waste. Chemosphere (2014), hFig. 2 compares the CH4 yields and VS removal efciencies ob-tained from the three systems. As shown in Fig. 2a, it was clear thatthe CH4 yield decreased with increasing OLR especially for thethermophilic system, which was reduced by approximately 43%as OLR increased from 1 to 6 g VS L1 d1. While for R1, only asmall decline that varying from 0.26 to 0.23 L CH4 g1 VSadded(14 g VS L1 d1) was found. Although a stable GPR was achievedat the maximum OLR for each system, the CH4...


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