Bioresource Technology 230 (2017) 24–32

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Bioresource Technology

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Batch and semi-continuous anaerobic co-digestion of goose manure withalkali solubilized wheat straw: A case of carbon to nitrogen ratio andorganic loading rate regression optimization

http://dx.doi.org/10.1016/j.biortech.2017.01.0250960-8524/� 2017 Elsevier Ltd. All rights reserved.

⇑ Corresponding author.E-mail address: wmding@njau.edu.cn (W. Ding).

Muhammad Hassan a, Weimin Ding a,⇑, Muhammad Umar b, Ghulam Rasool c

aCollege of Engineering, Nanjing Agricultural University, Nanjing, Jiangsu Province 210031, ChinabDepartment of Food Engineering, University of Agriculture, Faisalabad 38000, PakistancCollege of Water Conservancy and Hydropower Engineering, Hohai University, Nanjing 210098, China

h i g h l i g h t s

� Development of C/N and OLR regression optimization models.� Methane enhancement up to 96.10% due to C/N optimization.� 20–30 is the optimum C/N range for AD of goose manure and wheat straw.� 71.19% lignin removal due to NaOH solubilization of the wheat straw.� Optimum OLR of 3 g.VS/L.d at C/N of 25 during CSTR experimentations.

a r t i c l e i n f o

Article history:Received 17 November 2016Received in revised form 6 January 2017Accepted 11 January 2017Available online 13 January 2017

Keywords:Organic loading managementC/N optimizationNaOH solubilizationMethane enhancement

a b s t r a c t

The present study focused on carbon to nitrogen ratio (C/N) and organic loading rate (OLR) optimizationof goose manure (GM) and wheat straw (WS). Dealing the anaerobic digestion of poultry manure onindustrial scale; the question of optimum C/N (mixing ratio) and OLR (daily feeding concentration) havesignificant importance still lack in literature. Therefore, Batch and CSTR co-digestion experiments of theGM and WS were carried out at mesophilic condition. The alkali (NaOH) solubilization pretreatment forthe WS had greatly enhanced its anaerobic digestibility. The highest methane production was evaluatedbetween the C/N of 20–30 during Batch experimentation while for CSTRs; the second applied OLR of (3 g.VS/L.d) was proved as the optimum with maximum methane production capability of 254.65 ml/g.VS forreactor B at C/N of 25. The C/N and OLR regression optimization models were developed for their com-mercial scale usefulness.

� 2017 Elsevier Ltd. All rights reserved.

1. Introduction

Due to high rate of industrialization and commercialization inthe developing countries, a swift improvement in the rural devel-opment was found predominant during the last decade. Thesedevelopments results in high demand of energy and food require-ments that ultimately boosts the poultry sector among rural side.In China especially, goose is considered a favorite protein sourceand 2.5 million tones of goose production by food and agricultureorganization (FAO) were reported in 2010, which contributes about2.6% of the world poultry meat production (FAO, 2012). Enhancedpoultry industry also emerged as a major source of environmentaleutrophication that seriously deteriorates the atmosphere, soil

structure and groundwater contamination due to direct applica-tion of goose manure within the fields (Abouelenien et al., 2010).Likewise, wheat straw is also an abundant biomass resource foundin China with 125.6 million tons production and most of it is burntstanding in fields or for direct burning in kitchen that results inaddition of green house gas emissions (GHG) (Hassan et al.,2016b; Li et al., 2015b).

Anaerobic digestion (AD) also emerged as a promisingtreatment option to reduce the risk of environmental pollutionby volumetric reduction of the poultry manure. Anaerobic diges-tion is a biochemical conversion of volatile solids of the substrateinto useful gas termed as methane or generally biogas consistingof (50–70)% methane (Zhen et al., 2016). Biogas productionthrough anaerobic digestion process has become Chinese nationalstrategy and goal in order to improve the socio-economic situation

M. Hassan et al. / Bioresource Technology 230 (2017) 24–32 25

of the farmers, promotion of rural construction and clean ruralenvironment. (Li et al., 2015b). Anaerobic digestion is well devel-oped for the livestock manures but for the lignocellulosic biomass,still reluctance was observed on industrial scale due to their highlignin content that controls the hydrolysis step of the AD, that’swhy pretreatment was found prerequisite prior to AD of the ligno-cellulosic biomasses (Hassan et al., 2016a; Kumar et al., 2013).

The intrinsic property of the lignocellulosic straw reveals itshigh C/N ratio, floatation in the reactor, high lignin content and cel-lulosic crystallinity that creates critical situation to the methano-gens. Different researches were carried out to reduce the ligninand cellulosic crystallinity to enhance the fermentation stabilityof the lignocellulosic biomass like pretreatment and co-digestion(Li et al., 2015b). On the other side, livestock manure consistingof high nitrogen content with elevated recalcitrant fiber that limitstheir anaerobic biodegradability during the AD process (Gronrooset al., 2005). Therefore, C/N optimization was found essential andco-digestion of poultry manure with agricultural biomass has pro-vided a pivotal approach to regulate the nutrient balance duringthe anaerobic digestion process with enhanced methane produc-tion (Comino et al., 2010). Dealing the anaerobic digestion insemi-continuous stirring tank reactor (CSTR), the question of opti-mum organic loading rate (OLR) has significant importance. Espe-cially, dealing with the livestock manure, those posses highcontents of total ammonium nitrogen (TAN), their higher OLRmay results with elevated concentrations of TAN within the CSTRthat can possibly inhibit the anaerobic digestion process. There-fore, determining the influence of OLR on the anaerobic digestionperformance stability of co-digestion has a significant importancein order to optimize the OLR and evaluate the appropriate opera-tional conditions for a commercial scale CSTR.

Numerous studies were reported about the co-digestion of live-stock manure and agricultural straw. Wang et al. reported a largenumber of experiments on co-digestion of wheat straw with dairymanure and chicken manure, corn stover with chicken manure anddairy manure and rice straw with pig manure, chicken manure anddairy manure (Wang et al., 2012a, 2013). Another study conductedon co-digestion of goat manure with rice straw, wheat straw andcorn stover (Zhang et al., 2013). Wu et al. conducted co-digestionby using swine manure with corn stover, wheat straw and oat straw(Wu et al., 2010). The significant methane enhancement wasreported by all the above stated researches due to co-digestion ofthe animal manures and agricultural straw. Dong Li et al. have con-ducted co-digestion of rice straw and pig manure about the organicloading optimization at OLRs of 3.0, 3.6, 4.2, 4.8, 6.0, 8.0, and 12.0 g.VS/(L.d) and specific biogas production of 413 ml/g.VS was achievedat different OLR of (3–8) g.VS/(L.d) (Li et al., 2015b). Another similar

Table 1Chemical characterization of the goose manure (GM), pretreated and untreated wheat str

Parameters Units GM WS untr

TS % 29.94 ± 3.1 98.2 ± 0VS % 69.1 ± 3.3 95.9 ± 0TN % 2.5 ± 0.2 0.85 ± 0TP % 1.1 ± 0.2 0.41 ± 0TAN mg/L 1835.3 ± 56 –FAN mg/L 92.2 ± 9.8 –TOC % 38.5 ± 1.2 52.1 ± 0OM % 66.3 ± 2.1 89.9 ± 0C/N – 15.3 ± 0.7 61.4 ± 0Cellulose % – 39.3 ± 0Hemi-cellulose % – 21.3 ± 0Lignin % – 10.1 ± 0pH – 7.7 ± 0.0 –CODs mg/L 7564 ± 89 –TVFAs mg/L – –Protein % 15.1 ± 0.5 –

study conducted by the same author about the co-digestion and OLRoptimization of rice straw and cow manure at the same OLRsreported above with maximum average specific biogas productionof 383.5 ml/g.VS at OLR of 6 g.VS/(L.d) (Li et al., 2015a).

The present research focused on the co-digestion of goose man-ure and pretreated wheat straw to optimize the proper C/N andOLR. The Batch experiment were carried out to evaluate optimumC/N ratio and C/N regression model while semi-continuous exper-iments were adopted to develop OLR regression optimizationmodel. The effects of pretreatment on the chemical compositionof the wheat straw were also determined. The anaerobic digestionprocess performance parameters like total ammonia (TAN), freeammonia (FA), total volatile fatty acids (TVFAs), ethanol produc-tion, soluble chemical oxygen demand (CODs) and pH werestrongly monitored during the whole AD process.

2. Material and methods

2.1. Experimental material collection and preparation

Fresh goose manure (GM) was collected from Changzhou GooseProduction Farm, Nanjing, Jiangsu Province, China. The goose man-ure was stored in refrigerator at 2 �C for Batch and downstreamCSTR daily feedings. The wheat straw (WS) was collected fromJiangpu agricultural research station. It was first dried in the ovenat 105 �C for 24 h. Afterwards it was chopped and grinded to lessthan 1 mm size. This grindedWS was utilized for pretreatment fur-ther and stored in refrigerator. The chemical composition of theGM, untreated and pretreated WS and seed sludge were deter-mined and the results were presented in Table 1. The inoculumwas collected from a biogas plant in working condition located atPukou district, Nanjing, China. That biogas plant was (UASB)upflow anaerobic sludge blanket type working at mesophilic con-ditions and equipped with horizontal stirrers. As the seed sludgewas fully digested from that biogas plant, that’s why it was keptin anaerobic conditions for further two weeks for activation pur-pose. The sludge was feed with 2 g of glucose/L.day to improvethe methanogenic consortium within the sludge (Hassan et al.,2016a). Afterwards, it was strained through 1 mm polyester screento remove all the foreign substance before it was used as sludge inBatch and CSTR experimentation (see Table 2).

2.2. Batch and CSTR experimental setup and pretreatment of wheatstraw

In order to reduce the lignin and cellulosic crystallinity andenhance the anaerobic biodegradability of theWS, thermochemical

aw (WS) and seed sludge (mean values ± standard deviation).

eated WS Pre-treated with 7.5% NaOH Seed sludge

.1 99.2 ± 0.4 2.4 ± 0.9

.5 97.4 ± 0.6 75.8 ± 1.6

.1 0.82 ± 0.3 3.3 ± 0.4

.5 – 2.2 ± 0.3– 1683 ± 45– 65.3 ± 4.2

.1 43.4 ± 0.1 22.4 ± 1.2

.2 74.9 ± 0.1 38.6 ± 0.7

.4 53.0 ± 0.8 6.9 ± 0.4

.9 40.4 ± 0.7 –

.2 16.7 ± 0.5 –

.2 5.9 ± 0.3 –– 7.5 ± 0.1– 6304 ± 369– 283.8 ± 0.0– –

Table 2Literature review about the co-digestion of the different agricultural wastes to enhance methane production.

Feed stock Composition or C/N Type Methane enhancement References

CM: DM:WS C/N-27.2 Batch 46% Wang et al. (2012a)GM:CS C/N-21.2 Batch 2.11 folds Zhang et al. (2013)SM:CS C/N-20 Batch 16 folds Wu et al. (2010)OW:WAS C/N-27.6 ASBR 43.8% Bouallagui et al. (2009)CM:CS 1:3 Batch 14% Li et al. (2013)CM:SS 0.30:0.70 Batch 50% Borowski and Weatherley (2013)MA:FW 0.2:0.8 Batch 4.99 folds Zhen et al. (2016)CM:VPW 0.25:0.75 Batch 41% Molinuevo-Salces et al. (2013)CM:CS C/N-20 CSTR 121% Li et al. (2014)CM:AWS 3:1 Batch 93% Abouelenien et al. (2014))GM:WS C/N-20 Batch 96.1% Present studyGM:WS C/N-25 CSTR 41.47% Present study

Chicken manure (CM), Dairy manure (DM), Wheat straw (WS), Goat manure (GM), Corn stalks (CS), Swine manure (SM), Organic waste (OW), Waste activated sludge (WAS),Sewage sludge (SS), Micro algae (MA), Food waste (FW), Vegetable processing waste (VPW), Coconut, cassava waste and coffee grounds (AWS), Goose manure (GM).

26 M. Hassan et al. / Bioresource Technology 230 (2017) 24–32

pretreatment with 7.5% NaOH was carried out. All the pretreat-ment conditions and details were derived from the previousresearch (Hassan et al., 2016a). The pretreatment effects on theintrinsic characteristics of the wheat straw were evaluated andresults were presented in Table 1. Batch experiments were carriedout in 1 L laboratory fabricated glass reactors in triplicates. All theanaerobic digestion experiments were carried out at mesophilicconditions. In order to optimize the C/N ratio, four different com-positions were made (C1, C2, C3 and C4) having C/N ratio of35:1, 30:1, 25:1 and 20:1 respectively. To determine the methaneproduction capability and comparison with different compositions,the mono anaerobic digestion of pretreated WS, untreated WS andGM were also carried out while untreated WS was termed as con-trol during the experiment. The batch experiments were carriedout for about 40 days until the negligible biogas production wereobserved. The CSTRs were fabricated having 10 L working volumecapability. Detailed design of the CSTRs was shown in Fig. 1 witheach component labeled. At the head of the each reactor an electricmotor was installed for stirring purpose and a gear box was alsoattached with the motor to provide variable stirring speeds. Themotor was further connected with numeric time controller switch

1

1

2

3

4

5

6

8

9 10

11

12

7

1: Hot water supply pipes, 2: Digestate, 3: Hot water jacket, inlet, 8: Electric stirrer motor, 9: Gear box, 10: Biogas pipe, 1saturated water, 14: Gas holder, 15: Water recharging port, 1

Fig. 1. Schematic mechanism for semi-co

to maintain automatic working of the stirrer. The stirring speedwas 150 rpm and stirring frequency was 5 min every hour. TwoCSTR, termed as Reactors A and B were used in the present studyhaving C/N ratio of 20 and 25 respectively. Three different organicloading rates were applied in each reactor; 1.5 g-VS/L.day, 3 g-VS/L.day and 4.5 g-VS/L.day with their corresponding calculated totalsolids (TS) concentration of 4%, 6% and 8% respectively; thereforeconsequently termed as Phase-I, Phase-II and Phase-III. Thehydraulic retention time (HRT) was kept 10 days with furtherone week as to achieve pseudo-state steady stabilization after eachapplied OLR. The daily feeding and slurry removal was carried outwhile process biochemistry parameters like TAN, FA, TVFAs, etha-nol, pH and CODs were measured at each three days interval whilebiogas was measured and analyzed daily to determine methanecontents.

2.3. Analytical procedures

The total solids (TS), volatile solids (VS), total nitrogen (TN),total phosphorous (TP), total organic carbon (TOC), organic matter(OM) and soluble chemical oxygen demand (CODs) of the GM, WS

13

14

15

16

4: Stirring blades, 5: Valve, 6: Exhaust pipe, 7: Feeding 1: Biogas sampling port, 12: Slurry outlet, 13: Brine

6: Graduated cylinder

ntinuous stirring tank reactor (CSTR).

M. Hassan et al. / Bioresource Technology 230 (2017) 24–32 27

and sludge were determined according to standard method pro-posed by (APHA) American Public Health Administration (APHA,2006). Methane contents were evaluated by gas chromatography(GC, 7820A, USA). The thermal conductivity detector (TCD) of theGC was kept at 250 �C while Helium (He) was used as carrier gasand 50 ml biogas sample was injected for biogas compositiondetermination. The pH values were determined by a digital pHmeter (Mettler-Toledo, FE20 K-Switzerland,). For ethanol and totalvolatile fatty acid (TVFAs) concentration measurement in thedigestate, GC-Shimadzu (GC2014, Shimadzu, Japan) was usedequipped with thermal conductivity detector (TCD) of specification(DA-Stabilwax, 30 m � 0.53 mm � 1 lm) was used. During experi-ment, the total ammonia nitrogen (TAN) concentration were mea-sured by (Lianhua Technological Company, Limited, PR China)while free ammonia nitrogen (FAN) was calculated by using theEq. (1) (Hansen et al., 1998; Nie et al., 2015).

FAN ¼ TAN 1þ 10�pH

10� 0:09018þ2729:92Tkð Þ

" #�1

ð1Þ

where, FAN was the concentration of the free ammonia in mg/L, andTAN was the total ammonia nitrogen concentration in mg/L and Tkwas the temperature measured in Kelvin. The chemical oxygendemand, CODs removal were calculated by the equation proposedby (Hassan et al., 2016a) while VS removal (%) was estimated byEq. (3) (Nie et al., 2015).

CODs change % ¼ CODi� CODfCODi

� 100 ð2Þ

While, i and f were the initial and final CODs values during theanaerobic digestion process in each phase of experiment.

% VS removal ¼ 1� VSdigestate � ð100� VSfeedÞVSfeed � ð100� VSdigestateÞ

� �� 100 ð3Þ

2.4. Statistical analysis

To find out the compositional changes after the pretreatment ofwheat straw, their respective methane production and CSTRsmethane production during different organic loadings, analysis ofvariance (ANOVA) has been utilized by using Analytical Software(Statistix 8.1, Tallahassee, USA). The complete randomized design(CRD) was used with significant level (P < 0.05) and to develop acontrast between treatment means, LSD pair-wise comparison testwas used. To improve the visibility and clarity of the experimentaldata, OrigionPro 8.1 (Originlab, USA) was utilized.

3. Results and discussion

3.1. Pretreatment after effects on the chemical composition of wheatstraw

During anaerobic digestion of the lignocellulosic biomass,hydrolysis is considered as the rate determining step (Fu et al.,2015). Different pretreatment methods have been reported by dif-ferent researchers that have significant effects on reduction of thelignin and cellulosic crystallinity, enzymatic digestibility anddegree of polymerization. The main objective of all these pretreat-ment was to enhance the anaerobic efficiency of the lignocellulosicbiomass (Hassan et al., 2016a). In the present study, thermochem-ical pretreatment namely NaOH solubilization was applied for theWS and significant results were achieved as presented in Table 1.Overall, 27.54% hemicellulose solubilization, 71.19% lignin removaland 15.85% reduction in C/N ratio were calculated. The higherlignin removal as compared with the other researches could be jus-tified that the thermochemical pretreatments are considered more

effective in terms of chemical compositional changes and as com-pared with mechanical and biological pretreatments. The alkalinepretreatments produced significant swelling in the hemi-ligninmatrix that enhanced the easier microbial approach during anaer-obic digestion process. This phenomena increase the hemi-ligninsolubilization, redistributed the lignin, reduced cellulose crys-tallinity, dislocation and modification of lignocellulosic tissues.These entire phenomenon have directly associated with the ligninremoval of WS (Hendriks and Zeeman, 2009; Silverstein et al.,2007; Zhu et al., 2010). Zong et al. conducted pretreatment of cornstover with NaOH with (17.8–29.3)% hemicellulose solubilizationand (32.9–42)% lignin removal were achieved. In another study,10% NaOH pretreatment were applied to rice straw while 54%and 45% hemicellulose and lignin removal were evaluated respec-tively (He et al., 2009). Alkaline pretreatments are considered morefavorite in cleaving the complex lignin carbohydrate linkages,increased porosity and surface area, swelling of the structure,reduced crystallinity and lignin matrix destruction (Kumar et al.,2015; Zheng et al., 2014). The thermal-NaOH had improved thedestruction of hemi-lignin structure that leads to higher ligninremoval, hemicellulosic solubilization and anaerobic digestibility(Sambusiti et al., 2013). All the results of the current NaOH solubi-lization for WS were found in accordance with the above refer-enced literature.

3.2. Batch anaerobic co-digestion of goose manure and wheat strawand C/N regression optimization

Carbon to nitrogen ration (C/N) is considered as one of the mostimportant and governing parameter during the AD process. Theoptimum C/N ratio is responsible for the regulation of nutrient bal-ance to the methanogens within the reactor. The optimum value ofthe C/N ratio was reported by many researchers and the excellentanaerobic performance was achieved between C/N of 20 to 30(Wang et al., 2012a). The low C/N ratio of the substrate would pos-sibly inhibit the anaerobic digestion process due to the abundanceof free and residual ammonia like in case of poultry while high C/Nratio will results in higher nitrogen consumption rate during thestartup of AD process by methanogens which would possibly leadsthe lignocellulosic biomass to poor anaerobic digestibility (Hassanet al., 2016b). To enhance the methane production; C/N optimiza-tion were found quite perquisite prior to anaerobic digestion. Inthe current study, two approaches were applied: i) pretreatmentof the wheat straw that have reduced the C/N ratio of the WS to15.80% ii) co-digestion of the pretreated wheat straw with goosemanure that significantly (P < 0.05) improved the methane produc-tion from all the compositions. Four different compositions of thepretreated WS and goose manure were prepared having C/N ratioof (35:1, 30:1, 25:1 and 20:1) consequently termed as (C1, C2, C3and C4) respectively. Mono-digestion of the goose manure, pre-treated and untreated WS was also carried out in order to makea comparison between different treatments.

The daily and cumulative methane production profiles weredepicted in Fig. 2(a) and (b) respectively while methane enhance-ment capability of different compositions and respective C/N opti-mization is presented in Fig. 4(a). The C/N regression optimizationmodel was also developed and presented as Eq. (4). The highestcumulative methane production of 408.16 ml/g.VS were producedby the treatment C4 followed by the treatment C2, C3 and C1 with387.09 ml/g.VS, 380.42 ml/g.VS and 365,04 ml/g.VS respectively.The treatment C4 were proved as the optimum composition havingC/N ratio of 20 with 96.10% methane enhancement capability ascompare with the untreated WS having C/N ratio of 61.35. Gener-ally speaking, the maximummethane enhancement of 86–96% wasobserved within the C/N range of 20–30 that also had justified thestudy conducted by (Wang et al., 2012a). The NaOH pretreatment

Fig. 2. Daily and cumulative methane production of batch co-digestion of goose manure and wheat straw along with the digestion period.

Fig. 3. Daily methane production of the both CSTRs.

Fig. 4. (a) C/N optimization and respective methane enhancement capability of different compositions in batch anaerobic digestion while (b) CODs and VS removal capabilityof different organic loading rates in both CSTRs.

28 M. Hassan et al. / Bioresource Technology 230 (2017) 24–32

of the WS was found quite significant with 55% enhanced methanegeneration as compared with the control. The methane productioncapability of the goose manure was found quite close to thepretreatedWSwith 56.39% enhanced as compared with the controlwhile the untreated WS had produced only 208.14 ml/g.VSmethane. The maximum daily methane production of 24 ml/g.VSwas observed by the treatment C2 and the least methane

production of 11.75 ml/g.VS was determined obviously for the con-trol on the 5th and 3rd day of the experiment. Generally speaking,during first two weeks of the anaerobic digestion period normallyall peak methane production was observed for all the treatments ascould be seen in Fig. 2(a). At the startup of the third anaerobicdigestion period, the daily methane production peaks were provedhaving declining behavior with almost daily methane production

M. Hassan et al. / Bioresource Technology 230 (2017) 24–32 29

rate of 10 ml/g.VS to 14 ml/g.VS. After fourth week of the digestionperiod, again sudden declining behavior of the daily methaneproduction was predominant with almost less than 5 ml/g.VS dailymethane production. It was observed that almost 90% methanewas produced during the first four weeks of the anaerobic diges-tion process and this phenomenon was found more significant incase of the pretreated WS that also had proved the role andeffectiveness of the pretreatment. On the basis of the cumulativemethane production of the different treatments and their respec-tive C/N value, a regression model was developed that will be help-ful to determine the relationship among the two parameters atP < 0.05.

Y ¼ 443:08� 2:94X ð4Þwhere, Y is the specific methane production in ml/g.VS, X is the C/Nratio, 443.08 is the intercept while 2.94 is the slope of line having R2

of 0.96.Different studies were present in the literature focusing on the

importance of the C/N optimization. Wang et al. studied the co-digestion of wheat straw with chicken manure and dairy manureby surface response methodology and 240 ml/g.VS methane wasproduced at C/N ratio of 27.2 (Wang et al., 2012a).In one study,Batch anaerobic co-digestion of the chicken manure was carriedout with the vegetable waste with 41% enhanced methane gener-ation (Molinuevo-Salces et al., 2013). Another study, sewagesludge was co-digested with chicken manure and 50% methaneenhancement were evaluated (Borowski and Weatherley, 2013).In another study, corn stover was co-digested with chicken manurewhile 218.8 ml/g.VS methane production were calculated at cornstover: chicken manure of 3:1 respectively (Li et al., 2013). Aboue-lenien et al. studied the co-digestion of chicken manure with threedifferent crop residues and 50–93% methane enhancement wereevaluated as compared with the control (mono-digestion ofchicken manure) (Abouelenien et al., 2014). All of these researchedprovided a clear agreement with the results of the current study.

However, anaerobic co-digestion and C/N optimization wasfound quite complex phenomenon that not only depend on thespecific C/N ratio but also on the asynchronism, synergetic effectsof the substrate and their interaction together on microbial colo-nial growth. Furthermore, more comprehensive research isrequired to find out the synergetic effects of the co-digestion.

3.3. Semi-continuous anaerobic co-digestion of goose manure andwheat straw and OLR regression optimization

To deal with the industrial scale poultry manure based biogasplants, the question of organic loading rate have significant impor-tance. The higher applied OLR may speed up the treatment of poul-try waste but also had increased the risks of anaerobic processinhibition due to higher residual nitrogen contents of the poultrymanure. Therefore, determination of an optimum OLR is veryimportant to reduce the maintenance and operational cost of thecommercial scale anaerobic reactors (CSTR). Based on the resultsof the C/N optimization from Batch experiments, Reactor A(C/N = 20) and B (C/N = 25) were setup for the CSTRs co-digestionperformance evaluation. Three different OLRs were applied,consequently termed as Phase-I, Phase-II and Phase-III having 1.5(g.VS/L.d), 3.0 (g.VS/L.d), 4.5 (g.VS/L.d) respectively. The dailymethane production chart was presented as Fig. 3.

Generally speaking, the methane production has increased withthe increasing OLR but this increasing trend was found very sharpas the reactors enters from Phase-I to Phase-II. But as the bothreactors enter from Phase-II to Phase-III almost negligible methaneenhancement was observed. This phenomenon could be attributedwith the total ammonia and TVFAs accumulation due to higherorganic loading rate. The maximum daily methane production of

3127 ml and 3074 ml was recorded for the Reactors A and B onthe 14th and 2nd day of the experiment respectively duringPhase-I. Moreover, in the second Phase-II, 8242 ml and 8756 mlmethane was produced on the 29th and 31th day of the experi-ment for Reactors A and B respectively. In order to compare thespecific methane production, the Phase-II proved as the optimumwith maximum methane generation. The highest methanegeneration capability of 254.65 ml/g.VS was noticed for the ReactorB during Phase-II followed by the Reactor A within the same Phaseof 252.20 ml/g.VS. Overall, both reactors almost behave similarlyduring all Phases of the experiment proving Reactor B a little betterthan the Reactor A in terms of methane production. During thePhase-III of the experiment, the higher organic loading rateresulted in the accumulation of total ammonia and total volatilefatty acids, but overall no process inhibition was observed. Thedaily specific methane yield by CSTRs provided significantly lowerresults as compared with their respective Batch methane produc-tion. The phenomenon could be justified that in case of the Batch,complete anaerobic digestion was performed during 40 days untilalmost negligible methane production was recorded. But in case ofCSTR; the specific methane production were measured on daybasis that obviously should be lower than the Batch results(Aboudi et al., 2015). As the AD process proceeds, the methane con-tents were going to rise and reached to about 60% for both reactorsduring the third Phase of the experiment that proved the AD stabi-lization. On the basis of the methane production (ml/g.VS) duringdifferent Phase of the AD and applied OLR (g.VS/L.d) OLR regressionoptimization models were developed for the commercial scaledreactors at P < 0.05.

Y ¼ 202:78þ 3:38X ðReactorAÞ

Y ¼ 194:84þ 5:03X ðReactorBÞwhere, Y is the methane production in ml/g.VS, X is the OLR g.VS/(L.day) having R2 of 0.99 and 0.98 respectively for Reactors A andB respectively.

Different researches were carried out to find the optimum OLRfor different substrates. In a previous study, organic waste was co-digested with waste activated sludge in ASBR reactor, having OLRbetween 2.46 g.VS/(L.day) to 2.51 g.VS/(L.day), with 43.8%enhanced biogas production and 85.4% VS removal efficiency(Bouallagui et al., 2009). Another study concluded optimum rangeof OLR between 2.4 g.VS/(L.day) to 9.0 g.VS/(L.day) dealing with theco-digestion of primary sludge and fruit and vegetable fraction ofmunicipal solid waste (Gómez et al., 2006). Li et al. conductedco-digestion of chicken manure and corn stover at C/N of 20 inCSTR, and 223 ml/g.VS methane were achieved while with thesame parameters and substrate 281 ml/g.VS methane wererecorded for the Batch AD experiments (Li et al., 2014). All of theseresearches were found pertinent with the current study.

3.4. Anaerobic digestion stability performance of the CSTR reactors

The biochemistry of all the three different anaerobic digestionphases like TVFAs (total volatile fatty acids), ethanol production,pH value, TAN (total available ammonia), FA (free ammonia) andCODs (soluble chemical oxygen demand) were determined at eachthree days interval and the results were depicted in the Fig. 5.

Anaerobic digestion process stability is directly associated withthe total volatile acids production and ethanol production and theirstabilization that were produced during the hydrolysis step of theAD process. Hydrolysis of the GM and WS results in the productionof amino acids, long chain and short chain volatile fatty acids, etha-nol molecules and monomers during the acidogenesis step of theAD that leads to the raised concentration levels of the TVFAs. Thesmooth biogas and methane generation is directly associated with

Fig. 5. Different inter process parameters of semi-continuous anaerobic digestion like TVFAs, C2H5OH, pH, TAN, FAN and CODs in mg/L (a) and (b) for Reactor A while (c) and(d) for Reactor B respectively.

30 M. Hassan et al. / Bioresource Technology 230 (2017) 24–32

the conversion and stabilization of these (TVFAs) intermediateproducts into hydrogen, methane and carbon dioxide with the helpof methanogens duringmethanogenesis (Hansen et al., 1998;Wanget al., 2012a). In the current study, six prominent types of totalvolatile fatty acids were determined named as acetic acid (the mostabundant specie), propionic acid, butyrate (butyric acid),iso-butyric acid, valeric and iso-valeric acid concentrations weredetermined. Fig. 5(a) and (c) presented the TVFAs, ethanol and pHprofiles of the Reactors A and B respectively. Almost similar TVFAs,ethanol and pH values were evaluated for both reactors. The TVFAsand ethanol and having similar increasing trend as the AD processstartup. The TVFAs started from about 2000 mg/L while at the endof the AD TVFAs reached up to 4386 mg/L. The ethanol productionalso provided a similar fashion, starting from 18.76 mg/L and endwith 62.39 mg/L as could be seen in Fig. 5(a) and (c). The higherTVFAs and ethanol concentration could be attributed with thehigher OLRs applied during the second and third Phases of theexperiment but the phenomenon is also directly associated withthe consumption level of TVFAs and alcohol by the microbial com-munities and anaerobic digestion efficiency. A minor TVFAs accu-mulation was found due to its elevated concentration level andleast TVFAs stabilization and consumption by the methanogens,but overall AD system was proved significant for methane produc-tion. Furthermore, pH values behave differently, as they haveinverse relationship with TVFAs concentration (Hassan et al.,2016b), and this theory also had justified by the results of the pre-sent study. The pH value started from 7.43 while ends with 7.27during the whole AD period. As the TVFAs concentration goeshigher the pH value drops suddenly as could be observed byFig. 5(a) and (c) for Reactors A and B respectively.

During the hydrolysis step of the AD, hydrolysis of thepeptides, proteins, monomers and amino acids would results in

the production of ammonia and obviously the ammonia produc-tion rate was quite higher in case of poultry manure. This producedammonia has a significant role on the population of the methano-gens while their presence also increased the buffering capacity ofthe sludge and has significant importance in neutralization of theTVFAs. However, their higher concentration could inhibit theanaerobic digestion process (Li et al., 2015a). Due to the accumula-tion of the total ammonia, hydrogenotrophic and acetoclasticmethanogenic activities were seriously affected. As a consequence,inhibited methanogens could not be able to consume the producedvolatile fatty acids and they continue to rise that leads to higheraccumulation of the TVFAs. The accumulated TVFAs and totalammonia, both have negative synergetic effect on the methaneproduction. However, it is very difficult to define the optimumrange for the total ammonia and TVFAs concentration becausedifferent anaerobic digestion systems behave differently withrespect to their tolerance level. Poultry and livestock manure areconsidered having high contents of ammonia and indigestible pro-teins. Furthermore, as the AD process proceeds; these indigestibleproteins are further hydrolyzed and produce amino acids, peptidesthen ultimately results in accumulation of ammonia contents. Themost dangerous and undesired forms of the total ammonia is freeammonia (FAN) (Hansen et al., 1998) and its acceptable limit is100 mg/L to 150 mg/L, while its higher concentration could betoxic for the biological activities within the digester (Aboudiet al., 2015). The optimum limit for the TAN is different fordifferent AD systems depending upon the tolerance level of theAD and its averagely discussed value should be between(1500–3000) mg/L in order to achieve the smooth methane pro-duction while higher than the 3000 mg/L could possibly inhibitthe AD process (Abouelenien et al., 2010). Co-digestion is the bestknown and cheap technology in order to reduce the risk of the

M. Hassan et al. / Bioresource Technology 230 (2017) 24–32 31

ammonia inhibition because it can provide a wide range of thedesired C/N ratio by diluting the total ammonia content of thepoultry manure. The TAN and FAN results were described bythe Fig. 5(b) and (d) for Reactors A and B respectively. The TANvalue started 1490 mg/L while ending with 1860 mg/L and almostsimilar values were found for the both reactors. The maximum TANof 2385 mg/L was reported for Reactor A during third Phase of theexperiment. During second and mostly in third experimentalPhase, almost higher than 2000 mg/L of TAN were found and thiscould be explained due the higher OLR which also had provedthe minor TAN accumulation. But the overall AD cycle were foundunaffected because all TAN values were lied within the permissiblerange. The FAN value also had a similar pattern like TAN withmaximum production of 110.36 mg/L during the Phase-II for theReactor B. The starting and end value for both reactors were foundabout 50 mg/L while during the experiment higher value up to100 mg/L were found but overall AD process was found quitesmooth and un affected.

Soluble chemical oxygen demand (CODs) is considered as one ofthe most important parameter during the AD process. The CODs isbasically ameasure of the organic loading during the AD. Higher theCODs meant the presence higher volatile and consumable productsthat are suitable feedstock for themethanogens to consume. HigherCODs removal indicated the enhanced anaerobic digestibility of thesubstrate and ultimately higher methane production (Li et al.,2015a). The graphical representation of the CODs were found inFig. 5(b) and (d) for the Reactors A and B respectively. Generallyspeaking, during the Phase-I, highest CODs of 12,200 mg/L wererecorded for both reactors. Afterwards, at the end of Phase-I thisvalue reached to about 6900 mg/L while at the startup of thePhase-II again sudden rise in the CODs were found. The phenomenacould be explained the application of higher OLR that was carriedout. Similar increasing trend of the CODs were also observed duringthe startup of the Phase-III but lower CODs value about 8200 mg/Lwere calculated. The results of the anaerobic process stability werefound in accordance with the previous researches (Aboudi et al.,2015; Li et al., 2015a,b).

3.5. Anaerobic downstream characteristics of the CSTR digestate (VSand CODs removal)

Livestock manure and agricultural biomass are considered hav-ing high volatile solids. Anaerobic digestion is basically a microbialvolatile solids degradation process by which VS reduction andmethane generation are directly associated (Hassan et al.,2016b). Anaerobic co-digestion is also environmental friendlytechnology that leads to the reduced risk of the environmental pol-lution especially in the rural sector. The volatile solids of the goosemanure and wheat straw are consumed by the microbial commu-nities and being converted into useful gaseous products likemethane (Fang et al., 2014; Hassan et al., 2016a). Pretreatment ofthe WS and co-digestion with goose manure had increased thevolatile solids consumption by the microbial activities and the phe-nomenon was also proved by the enhanced methane generationfrom the present study. Volatile solids consumption and CODsremoval are also directly associated with each other. The higherCODs value meant the higher intra products (acetates, monomers,fatty acids, alcohol, amino acids, peptides) production and con-sumption during the anaerobic digestion process thus leads toenhanced methane production (Hassan et al., 2016b). The relativeVS and CODs consumption during different OLR stages of bothReactors (A and B) were presented in Fig. 4(b). The highest VSand CODs removal efficiencies of 59.26% 56.07% were providedby the Reactor A respectively during second phase of theexperiment that also confirmed their respective role in the highestmethane production during this stage as shown in Fig. 4(b).

Similarly, for the Reactor B, the highest VS and CODs removal of57.62% and 54.05% were also observed during the second appliedOLR respectively. During the first and third OLRs, almost the simi-lar values for the VS (higher than 50%) and CODs removal (higherthan 42%) were achieved as described by the Fig. 4(b). The resultsof the present study were found pertinent with the previous pub-lished researches (Hassan et al., 2016b; Wang et al., 2012a,b).

4. Conclusion

Therefore, co-digestion of the goose manure with pretreatedwheat straw provided an excellent approach in C/N optimization.The C/N optimization was concluded as prerequisite for the goosemanure prior to anaerobic digestion. The maximum methaneenhancement was concluded between C/N ratio of 20 –30 thatwas 86 –96% respectively proving C4 (C/N = 20) as the optimumduring Batch study while in case of CSTR the second applied OLR(3 g.VS/L.day) was found as the optimum in order to enhance themethane production. The OLR optimization was found necessaryto commercialize the AD process for goose manure.

Acknowledgements

This current research was sponsored by Jiangsu AgricultureScience and Technology Innovation Fund (CX(15)1008) and NationalScience and Technology Support Program (2013BAD08B04). Besidesthat; the principal author Muhammad Hassan is thankful to (HECPakistan) for providing him (Master leading to PhD) scholarshipunder scholarship program titled (HRDI-UESTPs/UETs Batch III).

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