Effect on anaerobic digestion performance of corn stover by freezing–thawing with ammonia pretreatment☆

Hairong Yuan ,Yanyan Lan ,Jialin Zhu ,Akiber Chufo Wachemo ,3,Xiujin Li,*,Liang Yu

1 Centre for Resource and Environmental Research,Beijing University of Chemical Technology,Beijing 100029,China

2 China National General Machinery Engineering Corporation,Beijing 100050,China

3 Department of Water Supply and Environmental Engineering,Arba Minch University,P.O.Box 21,Arba Minch,Ethiopia

4 Department of Biological Systems Engineering,Washington State University,Pullman,WA 99164,USA

Keywords:Freezing–thawing Ammonia pretreatment Anaerobic digestion Corn stover

ABSTRACT In order to enhance the biomethane production from corn stover,choosing effective pretreatment is one of the necessary steps before starting anaerobic digestion(AD).This study was aimed to analyze the effectiveness of freezing–thawing with ammonia pretreatment on substance degradation and AD performance of corn stover.Three ammonia concentrations(2%,4%,and 6%)with two different moisture contents(50%and 70%)were used to pretreat the corn stover at two temperatures(-20 °C and 20 °C).The result showed that an optimum pretreatmentcondition for corn stover was atthe temperature of-20°C,moisture contentof70%and ammonia concentration of 2%.Under the optimum pretreatment condition,the maximum biomethane yield reached 261 ml·(g VS)-1,which was 41.08%higher than that of the untreated.Under different pretreatment conditions,the highest loss of lignin at-20°C with 2%ammonia concentration was 63.36%compared with the untreated.The buffer capacity of AD system was also improved after the freezing–thawing with ammonia pretreatment.Therefore,the freezing–thawing with ammonia pretreatment can be used to improve AD performance for corn stover.This study provides further insight for exploring an efficient freezing–thawing with ammonia pretreatment strategy to enhance AD performance for the practical application.

1.Introduction

According to the BP Statistical Review of World Energy published in June 2017,fossil fuels are still the priority of the world's energy consumption structure.China is one of the world's largest energy consuming countries.In 2016,the energy consumption in China accounted for 23%ofglobalenergy consumption and contributed 27%to globalenergy demand[1].To reduce the reliance on unsustainable fossil fuels and to get relieved from the concern over global climate change,countries have to look for sustainable and renewable energy resources to enable partial or full replacement of using fossil fuels[2].Among different renewable energy technologies,anaerobic digestion(AD)is an efficient way of converting wastes,such as crop straws produced biogas[3].Moreover,biogas can be injected into the naturalgas grid or used forproduction of vehicle fuel via upgrading to increase the energy options[4].

Lignocellulosic biomass is a carbon-neutral source of energy.In recent years,it has become an increasingly important renewable raw material for biogas production in the process of anaerobic digestion[5].According to the 2016 China Statistical Yearbook,about 831.18 million tons of the crop residuals were produced in 2015,which was 2.12%higher than the data of 2014[6].However,the complex structures of lignocellulosic biomass limit the biodegradability of the crop residuals.To improve AD efficiency and enhance the degradability,it is necessary to break the lignin network and reduce the crystallinity of cellulose and polymerization of the components of lignocelluloses via suitable pretreatments[7].

Chemical pretreatment is one of the most promising approaches to improve the biodegradability of cellulose by removing lignin networks with hemicelluloses and to decrease the degree of polymerization and crystallinity of the lignocellulosic component[8].Most chemical pretreatment processes are implemented from ambient temperature(20 °C)to high temperature(220 °C)[9].Ammonia pretreatment is one of the chemical pretreatment methods[10]that can hydrolyze the polymers of cellulose and hemicellulose at 25–27 °C and also enhance the nitrogen content of crop residues for example corn stover,and straws during AD[11].Zhang and Zhang[12]reported that biomethane yield of rice straw pretreated using 2%ammonia at 110°C temperature increased by 17.5%compared to that of the untreated one.Li et al.[13]and Yuan etal.[14]found that 4%ammonia and 70%moisture content were the optimal conditions to treat corn stover and rice straw at(30±2)°C during biogas production process.

However,researchers found that sodium hydroxide with urea can hydrolyze cellulose better than sodium hydroxide alone at low temperature[15].Zhao et al.[16]obtained up to 70%glucose yield from spruce at low temperature(-15°C)pretreatment using the combination of 7%NaOHand 12%urea solution compared to 13%glucose yield fromuntreated spruce.Isogai and Atalla[17]developed an optimal pretreatment condition for cellulose by using 8.5 wt%NaOH solution,then freezing it at-20°C and holding it at that temperature until it became a solid frozen mass.Khor etal.[5]found thatthe pretreatmentat-18°C resulted in a 20.7%increase in anaerobic biomethane production,which was the maximum value in the low temperature calcium hydroxide pretreatment of maize straw,grass and sprout stem at the same lime loading.The biomethane yield of birch and spruce was improved by 56%and 600%with NaOH/thiourea(7/5.5 wt%)pretreatment at-15 °C,respectively[18–19].Therefore,pretreatment at low temperature may play an effective role to enhance the lignocellulosic fiber solubility in NaOH/urea solution[5].

More than 70%of the global land area(including 14%permafrost and 56%seasonally frozen soil)and 75%of the total land area of China(including 21.7%permafrost and 53.5%seasonally frozen soil)are covered by permafrost and seasonally frozen ground[20].These seasonally frozen regions experience significantfreezing and thawing cycles,especially in winter seasons.Freezing–thawing cycles affect the structural,physical,and mechanical properties of the concrete[21]and soil[22].In the same way,the freezing/thawing method could be used to breakdown cell walls to enhance the accessibility of their polysaccharides to the hydrolyzing enzymes.In order to produce ethanol from biomass,wheat straw was pretreated on the natural freezing–thawing condition by Yu et al.[23].Yu et al.found that freezing–thawing pretreatment can increase the enzymatic hydrolysis rate of wheat straw by 57.5%.On the other hand,the maximum theoretical ethanol yield of 90.87%was obtained with freezing–thawing treated Juncus maritimus[24].However,the freezing–thawing pretreatment method has rarely been tested for AD of crop straws.

The possible advantages of using freezing–thawing pretreatment are as follows:(1)itis applicable in seasonally low temperature regions without extra cooling or heating,(2)it reduces the use of excess chemicals and toxic reagents(no toxic products and no wastes are released)and(3)it doesn't require further equipment.This will provide extensive opportunities for the application of the low-temperature pretreatment technology for many countries where the seasonal temperature is very low.Considering that strong alkalization pretreatment cannot concurrently provide the required nitrogen for the AD of straw[13].Additionally,strong corrosion and pollution put NaOHatdisadvantage in the industrial application.The objectives of this study were:(1)to investigate the effect of freezing–thawing with ammonia pretreatment and moisture content on AD and(2)to look for the optimum pretreatment condition of corn stover.

2.Experimental

2.1.Raw material and inoculum

Corn stoverwas collected from ShunyiDistrict,Beijing.Itwas dried by natural air and ground to a desired size of 20-mesh by a hammer mill(FE130,Tianjin,China),then stored in the laboratory.The sludge wascollected from Xiaohongmen Wastewater Treatment Plant,Beijing,China.The main characteristics ofcorn stover and inoculumare listed in Table 1.

2.2.Experimental methods

During the pretreatmentprocess,25 wt%ammonia was used as pretreatment reagent.The ground corn stover was placed in a sealed bag,and then the air was purged from the bags.The amount of ammonia was added as 2 wt%,4 wt%,and 6 wt%of the dry mass of corn stover in respective plastic bags.The moisture contents of pretreatment were adjusted to 50%and 70%[14],meantime,the control group was used without ammonia pretreatment.The bags were immediately sealed and kneaded to fully mix the corn stover with the ammonia and water.One group of corn stover with ammonia was frozen in the refrigerator at-(20± 1)°C for 7 days.Another group of corn stover with ammonia was stored in a constant temperature incubator which was set at(20± 1)°C for 7 days.The pH values are measured every day.The pretreatment process was deemed complete when the pH of this system showed no obvious change.Pretreatment time was determined according to pH[13].

Table 1 Characteristics of raw materials and inoculum①

Batch AD was carried out in this study.The design of the anaerobic digester used in this experiment has a 1 L total volume(working volume was 0.8 L),a 1 L gas collecting bottle,and a 1 L beaker.According to pretreatmentresult,one group of corn stover with ammonia was frozen in the refrigerator at-(20±1)°C for 5 days and then immediately thawed at(20±1)°C temperature.Another group of corn stover with ammonia was stored in a constanttemperature incubatorwhich was set at(20±1)°C for 5 days.The pretreated and untreated corn stover was placed in each ready digester.The organic loading rate(OLR)of every digester was set at 65 g TS·L-1.Then the amount of inoculum added in each digester was 15 g MLSS(mixed liquid suspended solids)·L-1[12].The remaining volume of the digester was filled by tap water to maintain the same working volume of 0.8 L.Finally,the bottles were incubated ata(35±1)°C water bath for55 days.The daily biogasproduction and biomethane contentwere measured for further analysis.Allthe pretreatmentsand ADexperimentsforeach condition were in triplicate.

2.3.Analytical methods

The Hitachi S-4700,Japan scanning electron microscope(SEM)was used to observe the surface morphology of corn stover under the differentpretreatmentconditions.The total solid(TS),volatile solid(VS),and total alkalinity(TA)were determined by the APHA standard methods[25].The pH value was measured using a pH meter(CHN868,Thermo Orion,America).TC was analyzed by an elemental analyzer(Vario EL/micro cube elemental analyzer,Germany).TN was determined using the total Kjeldahl nitrogen analyzer(Model KDN-2C,Shanghai).The NH3-N was analyzed by a Kjeldahl analyzer(KT-260,Foss,Suzhou,China).The contents of cellulose,hemicellulose and lignin were measured using the extraction unit according to the procedures proposed by Van Soest et al.[26].VFAs were analyzed by a gas chromatograph(GC-2014,Shimadzu,Japan)equipped with a flame ionization detector(FID)and DBWAX123-7032 column.

The H2,N2,CH4and CO2contents ofbiogas were detected using a gas chromatograph(SP2100,Beifen,China)with a TDX-01 column and a thermal conductivity detector(TCD)and a standard curve made of known standard gases(H29.99%,N25.02%,CH449.98%,CO235.01%).The operational temperatures at the injection port,column oven,and detector were 150 °C,140 °C,and 150 °C,respectively.Argon was used as the carrier gas.Daily biogas production was recorded by water displacement method and daily biomethane content.

Fig.1.Changes of pH value with different pretreatment conditions.

2.4.Data analyses

The SGompertz,Slogistic1,and Fitzhugh(Chapman)[27–29]model equations were used to fit the CBP data curves in relative research.The formulas are showed in Eqs.(1),(2),and(3).

where P(t)is the cumulative CH4production(ml CH4)at time t;Pmis the maximum CH4potential(ml CH4)at the end of incubation time;t is time(d);Rmis the CH4production rate(ml CH4·d-1);λ is the lag phase(d);e is the base of natural logarithms,e.2.71828;k is the rate constant;and m is the shape factor.Pm,Rm,k,m and λ of the parameters were estimated by Nonlinear Curve Fit program in OriginPro 8.6.Excel 2010,SPSS 19.0,and OriginPro 8.6,AutoCAD were used fordata analysis and drawing,respectively.

3.Results and Discussion

3.1.Pretreatment

3.1.1.Pretreatment time

Fig.2.SEM micrographs of corn stover with 2%ammonia at 70%moisture content and untreated.

Table 2 Changes of lignocellulose compositions at different pretreatments

During the pretreatment process,pH is an important indicator to show the effect of pretreatment[30–31].The pretreatment time was determined based on the change in pH.For 50%and 70%moisture contents,the changes in pH values of different ammonia concentrations(2%,4%,and 6%)for the pretreatments of corn stover showed the similar trend at both-20 °C and 20 °C.For example,Fig.1 shows the trend of pH changes in the 50%moisture content sample.From Fig.1,the changes of pH values were higher at 20 °C than-20 °C temperature.In this pretreatment process,the pH values for corn stover pretreated with 2%,4%and 6%ammonia concentrations at-20°C were found to change since the first day(pH=9.90–10.30)and remained down on the fifth day(pH=8.94–9.52)(Fig.1A).While the pH values for corn stover pretreated by the corresponding conditions at 20 °C were found to change starting from the first day(pH=9.89–10.36)and remained down on the fifth day(pH=8.38–8.94)(Fig.1B).However,there was no clear change observed after the 6th day.The pH value of the control test remained constant at pH 8.10.Therefore, five days of pretreatment was used in this study.Moreover,the pretreatment experiment results also revealed that five days of pretreatment achieved the maximum lignin structure destruction which gave a good opportunity for anaerobic microbes to utilize the substrate[13].

3.1.2.Changes in physical structure and main compositions

In order to observe the changes of corn stover's physical structure before and after pretreatment,the SEM micrographs are shown in Fig.2.The structure morphology of the pretreated corn stover was fractured and deformed,as a result the internal structure was exposed at different temperatures using 2%ammonia and 70%moisture content.The complete and smooth surface of corn stover was destroyed by 20°C pretreatment.Some loose parts of the structure were dissolved and detached(Fig.2B).The morphological structure of corn stover was more seriously damaged and obvious structural exfoliation appeared by-20°Cpretreatment(Fig.2A).On the other hand,the surface of untreated corn stover was more complete and smooth,where the structure was aligned and tight without damage(Fig.2C).

Fig.3.The DBP of different pretreatment conditions.

Fig.4.The CBP of corm stover with different pretreatment conditions.

The compositions of lignin,cellulose and hemicellulose in corn stover before and after pretreatment are listed in Table 2.The cellulose,hemicellulose and lignin contentofraw corn stoverwas 38.63%,30.44%,and 8.68%,respectively.However,after pretreatment the components of cellulose,hemicellulose and lignin of corn stover were decreased by 2.30%–9.83%,8.85%–24.07%,and 39.04%–63.37%,respectively.After ammonia pretreatment the lignin content dropped from 8.68%to 3.18%.Compared with the untreated corn stover,the highest loss of lignin was 63.37%at-20°C with 2%ammonia pretreatment,which was signi ficantly higher than that of NaOH pretreated corn stover(19.1%)by Zheng[32].The lignin removalrate of corn stover with freezing–thawing pretreatment was 2.65 times higher than that of30°C pretreatment[14].This indicated that the freezing–thawing with ammonia pretreatment can remove the lignin networks from complex structures of lignocellulose,and improve the ADperformance.This is because underthe temperature around-20°C(frozen state),the plasma membrane of plant cells undergoes a rupture by the formation of a large amount of intracellular ice.When cells are exposed to high temperature(thawed state),the intracellular frozen cells are subjected to further ultrastructural changes during the thawing process resulting in serious cell damage[33].

3.2.Biomethane production

3.2.1.The daily biomethane production

The daily biomethane production(DBP)of pretreated sample showed three or four peaks during the entire period of batch experiment(Fig.3).The DBP showed higher production rate from the 15th to 40th days.However,the production rate rapidly dropped from the 5th to 15th days below 100 ml.The decrement in DMP could be related to the system acidification which drops the pH of the digester below optimum level.The first and second peaks of DBP appeared on the 2nd–4th and 19th–26th days,respectively,while the third peak of DMP appeared after the 22nd–30th days.Among these peaks,the 4%ammonia pretreatment of corn stover coupled with 70%moisture content at-20°C reached the first maximum daily biomethane yield of 1412 ml on the 19th day,which was 145.57%higher than that of 575 ml for the untreated corn stover.

Table 3 The parameters of system stability

Table 4 Parameters of CBP using different models

3.2.2.The cumulative biomethane production

The cumulative biomethane production(CBP)did notchange within the 5th to 20th days for 50%moisture contents and within the 5th to 15th days for 70%moisture contents(Fig.4).The CBP rapidly increased from the 20th to 30th days,and then the CBP gradually slowed down after day 31 of the digestion time.For samples with 70%moisture content,the CBP of 2%,4%,and 6%ammonia pretreatment corn stover was 261 ml·(g VS)-1,249 ml·(g VS)-1,and 221 ml·(g VS)-1at-20 °C,which were 15.49%–41.08%,14.75%–34.59%,and 15.24%–19.46%,respectively,higher than those of 20°C and untreated group.Besides,the CBPs of the group with 70%moisture content samples at-20°C were 10.67%–13.97%higher than those of the 50%moisture content group with the corresponding ammonia pretreatment.The highest CBP was 261 ml·(g VS)-1at 70%moisture content,2%ammonia and-20°C temperature.This result was 6.75%higher than the previous research on CBP[14]and 98.84%higher than that of the previous research using lime pretreated grass at-18°C[5].This result further suggested that the freezing–thawing with ammonia pretreatment has great potential to enhance biomethane yield.

3.3.Evaluation of the system stability

The system stability parameters such as pH,NH3-N,and TA are very important for maintaining stable and sustainable performance during anaerobic digestion process.The parameters for stability of the anaerobic system are presented in Table 3.Allthe initialpHvalues were keptat 8.0.The final pH was in between 7.25 and 7.48.This indicates that pH values were not beyond the range 6.5–7.8 and were normal,which was found to be suitable for the growth of methanogens[31].In this study,the amount of NH3-N and TA of the digesters was in the range of 733.6–2415.0 mg·L-1and 4480–7725 mg CaCO3·L-1,respectively.When the NH3-N is less than 1500 mg·L-1,the activity of methanogenic bacteria could improve and as a result the alkalinity could be above 2000 mg·L-1to maintain the buffer capacity of the substrate[34,8].The result showed that the ammonia concentration of 2%ammonia pretreatmentcorn stover was 1313.2–1457.4 mg·L-1and belonged to normal range.But ammonia concentrations of 4%–6%ammonia pretreatment corn stover were 1640.8–2331.0 mg·L-1and caused inhibition ofmethanogenesis in the ADsystem.The ratio ofVFAs/TAcan be used to evaluate the digester stability according to Callaghan et al.[35].If the ratio of VFAs/TA is more than 0.4,the digester should be unstable,however,in this study the ratio of VFAs/TA was 0.0044–0.0167,indicating anaerobic systems were stable.Therefore,for the 2%ammonia pretreatment group,the system stability was at optimum stage because there was no negative effectobserved from the process parameters(pH,NH3-N,TA,etc.).

Fig.5.The CBP fitted curves of corm stover by different pretreatment conditions.

Table 5 Kinetic parameters of biomethane production with modified SGompertz equation

3.4.Model valuation and kinetic parameters of biomethane production analyses

The SGompertz,Slogistic1,and Fitzhugh(Chapman)models(Eqs.(1),(2),(3))were used to fit the experimental data of the CBP.The fitted parameters of CBP using different models were listed in Table 4.According to the previous researches[36–38],root mean square error(RMSE)and the Akaike information criterion(AIC)play significant roles in characterizing and evaluating the quality ofthe model,as shown in Eqs.(4)and(5)

where Piis the predicted CH4production per gram VS,ml CH4·(g VS)-1,Eiis the actual CH4production per gram VS,ml CH4·(g VS)-1,n is the number of the test,and N is the number of formula.

The correlation coefficient R2was close to 0.9906–0.9921,0.9927–0.9934,and 0.9919–0.9895 for SGompertz,Slogistic1,and Fitzhugh(Chapman)(Table 4),respectively.Due to very close correlation coef ficient of different models,the RMSE and AIC were further used to evaluate the quality of different models.Models are thus ranked from the lowestto the highest RMSE and AIC scores,and the lowest is considered the ‘true’(best)model[39].As is given in Table 4,the quality of models under different pretreatment conditions was quantitatively judged as follows:SGompertz＞Fitzhugh(Chapman)＞Slogistic1.Therefore,the SGompertz equation was used to fit the CBP data.

The CBP curves werefitted by the SGompertz equation Eq.(1)for further investigation(Fig.5).The modeling results were listed in Table 5 with R2(0.9740–0.9986).In addition,the lag-phase time(λ)is another important indicator.The value of λ was in range of 10.47–20.19 in different pretreatment conditions(Table 5).For-20°C(Fig.5A),the CBPs of 50%moisture content with the 2%,4%,and 6%ammonia concentration were 23.78%,21.62%,and 7.57%higher than thatof the untreated,respectively.And the CBPs of the corn stovers with 70%moisture content and 2%,4%,6%ammonia concentration were 41.08%,34.59%,and 19.46%higher than that of the untreated,respectively.

4.Conclusions

AD performance of corn stover was investigated at freezing and thawing with different ammonia concentrations.The freezing–thawing with ammonia pretreatment was proved to be the best effect.The optimum pretreatment condition was-20°C,2%ammonia and 70%moisture content.Under the optimum pretreatment condition,the biomethane yield was 261 ml·(g VS)-1,which was 41.08%higher than that of the untreated.The highest loss of lignin was 63.36%compared with the untreated corn stover.The buffer capacity of the AD system was improved.The best fitting CMP curves were obtained from the SGompertz model.Therefore,the freezing–thawing with ammonia pretreatment strategy is recommended for the practical application because of its environmentally friendly characteristics and can be applied at seasonally frozen regions in winter reducing energy cost.