Performance of zeolite and trace elements on biogas production from alkaline hydrogen peroxide pretreated sweet sorghum bagasse slurry

In order to alleviate the inhibition of sodium ions and phenols on methanogenesis by anaerobic digestion of sweet sorghum bagasse pretreated with alkaline hydrogen peroxide, zeolite and trace elements (TEs) were added to sweet sorghum bagasse slurry (PSSBS) for biogas production. The in�uence of zeolite dosage and TE on the anaerobic digestion performance was revealed from the perspective of biogas production, as well as the cellulase and dehydrogenase activities. The results showed that zeolite and TE increased methane production and shorten the lag time. The maximum methane yield of 274.5 mL/gVS from PSSBS + 5 g/L zeolite + 1 mL TE could be obtained and 58.4% higher than that of untreated sweet sorghum bagasse. Addition of zeolite and TE was bene�cial to increase the activities of cellulase and dehydrogenase of the digestate. This work would provide a theoretical reference for the resource utilization of sweet sorghum bagasse for clean industrial application in future.


Introduction
Sweet sorghum is one kind of non-food crop, whose stem juice is rich in fermentable sugars such as sucrose, fructose and glucose, and often used in the production of fuel ethanol.The sweet sorghum bagasse (SSB) after squeezing contains not only the soluble sugars mentioned above, but also cellulose, hemicellulose and lignin, among which cellulose and hemicellulose can be hydrolyzed by cellulase or acid to glucose, xylose, arabinose and other monosaccharides, which can be used for ethanol production or anaerobic fermentation to produce biogas and other clean energy.Sweet sorghum bagasse contains approximate 15% of lignin, which generates cross-linked structure with cellulose and hemicellulose, hinding the hydrolysis of cellulose and hemicellulose by cellulase.Therefore, to improve the enzymatic hydrolysis e ciency of sweet sorghum bagasse, physical, chemical, biological or a combination of these methods are often required [1][2][3][4].The main purpose of these pretreatment methods is to increase the availability of enzymes to cellulose during the hydrolysis of cellulose, so as to improve the e ciency of enzymatic hydrolysis of lignocullulosic materials.
The Alkaline hydrogen peroxide (AHP) is a very effective chemical pretreatment method for lignocellulose because it can remove most of the acid-insoluble lignin from the bagasse by alkali lignin dissolution, while most of the cellulose and hemicellulose are retained [5,6].Using the improved AHP pretreatment method, Cao et al can greatly increase the hydrolysis e ciency of cellulose over 70% and the total sugar yield over 95% [5].After AHP pretreatment, the enzymatic hydrolysis e ciency of solid residue can be greatly improved, and it is easy to be converted into biofuel [7].The pretreatment e uent was not used for anaerobic digestion, mainly because the e uent contained high concentration of alkali metal ions, which had a strong inhibitory effect on microorganisms [8].It has been reported that if the concentration of Na + reached 10-11g/L, it would have a strong toxic effect on the methanogenesis process [9].In addition, phenolic substances and carboxylic acids generated in the process of lignin dissolution by alkaline also inhibited the biogas production process [10].These inhibition groups promoted the loss of bio lm and can affect its selectivity, slowed cell growth and lead to further sugar assimilation [11].The washing of bagasse will lead to the loss of soluble sugar and increase the amount of waste liquid production, which will also increase the use of enzyme catalyst and increase the cost [12].In fact, the e uent contains abundant fermentable substances such as free sugars and organic acids, which produced during the pretreatment process and can be converted into clean energy via anerobic digestion.Therefore, it is necessary to study the methane production potential of pretreatment sweet sorghum bagasse slurry (PSSBS), including pretreated bagassse and pretreatment e uent, not only to save energy, but also to solve potential environmental pollution problems.
In order to solve this problem, some researchers added zeolite into the digestion system to reduce the inbihion of sodium iont.Zeolite is a mineral material with large surface area, rough surface and perfect adsorption capacity.It can absorb metal ions and its porous structure has a strong agglomeration effect on microorganisms [13].Research shows that zeolite has a good adsorption effect on ammonia nitrogen of anaerobic digestion liquid of urban organic waste, and the unique microporous structure of zeolite itself is conducive to the growth and reproduction of microorganisms, so it can be used as an ideal biological carrier [14].In a study on anaerobic digestion of wastewater, it was also found that the addition of mineral materials could shorten the lag time of fermentation and increase methane production by 32%-117% [15].Some TE (Fe, Ni, Cu, Zn, Mn, Mo, etc.) play a vital role in the synthesis of coenzyme and enzyme activity in the process of biogas production by anaerobic digestion microorganisms.The material metabolism and energy metabolism of methanogens require the participation of TE, such as Fe, which is involved in the synthesis of cytochrome and cell oxidase in methanogens.It is also an electron carrier for intracellular redox reactions [16].The amount of TE required by methanogenic bacteria growth is rare, but the lack of TE can lead to the decrease of biological activity, and then affect the operation effect and stability of the whole anaerobic reactor [17].The importance of TE to anaerobic fermentation is mainly re ected in that they often appear in the enzyme system of anaerobic digestion microorganisms as coenzymes, cogroups and cofactors, and play an important role in regulating the methanogenesis stage of anaerobic fermentation.It is a feasible method to enhance the e ciency of anaerobic digestion by adding TE to enhance the activity of microorganisms.
Therefore, to alleviate the inhibition effect of phenolic substances and the sodium ions on methanogenesis process, and improve the methanogenesis potential of sweet sorghum bagasse, the PSSBS were used for biogas production with zeolite and TE addition.The optimal dosage of zeolite was determined and the effects of zeolite and TE on dehydrogenase and cellulase activities were also studied.
The results could provide reference for the industrial application of alkali pretreatment technology for sweet sorghum stalk residue.

Feedstocks and the pretreatment process
Sweet sorghum was taken from the planting base of Shunan Village, Fengxian District, Shanghai.The stalks were pressed and the bagasse was obtained.After natural drying, the bagasse was ground by a lab-scale crusher and passed through a 40-mesh sieve before storage.Zeolite with particle size of 0.5 ~ 1mm was purchased from Zhengzhou Henuo lter Material Co., LTD., Henan Province.The inoculum was the digestate from semi-continues stir tank reactor of anaerobic digestion of kitchen waste in our laboratory.Sweet sorghum bagasse was soaked in 2% of NaOH solution (solid-liquid ratio: 1:10) and kept in an autoclave at 121℃ for 60 min.After being cooled to room temperature, 5% of hydrogen peroxide was added to autoclaved SSB and kept in dark for 24h.In order to analyze the properties of the solid and liquid parts after pretreatment, the PSSBS obtained after pretreatment was ltered with G3 core funnel for the solid and liquid separation.The basic characteristics of SSB, PSSB, inoculum and pretreatment e uent before and after treatment are shown in Table 1.

Batched methane production assays
The SSB or PSSBS and inoculum were added into a 500mL glass reactor with a working volume of 400mL according to the VS ratio of substrate to inoculum of 1:2.The reactors were placed in a constant temperature water bath shaker under 36 ± 0.5 ℃and 80 rpm of the oscillation rate.The experimental design was shown in Table 2.The reactors were ushed with high-purity nitrogen for 5 minutes to maintain the anaerobic environment before the biogas production.The biogas yield was recorded by drainage method every day and the methane content in the biogas produced was measured by gas chromatography and the biogas volumes were calculated to standard temperature and pressure (273.15K, 101.325 kPa).The experimental device is shown in Fig. 1.Four parallel samples were set in each group, two of which were used for the analysis of biogas yield and methane content, and the others were used for the analysis of pH value, alkalinity, cellulase and dehydrogenase content of digestate by regularly sampling, and the results were averaged.

Analytical methods and the kinetic model
The cellulose, hemicellulose and lignin contents of SSB were determined by the Van Soest's method [18].The content of CH 4 and CO 2 in the biogas were determined by a gas chromatograph(Agilent 6820) equipped with a thermal conductivity detector and the He as the carrier gas [19], the methane production was calculated by multiplying biogas production by the volume fraction of methane in biogas.The methane production of each experimental group was obtained by deducting the methane production of inoculum.The TS, VS, pH and alkalinity of the digestate were measured according to the standard methods [20].The chemical oxygen demand (COD) and total phenolic compounds (TPC) in pretreatment e uent were determined by potassium dichromate oxidation method and Folin-Ciocalteu reagent method, respectively [21,20].The cellulase activity was determined were measured according to the reference [22].The cellulase in the digestate hydrolyzed sodium carboxymethyl cellulose (CMC-Na) at 1 hour to produce 1 mg of reducing sugar was de ned as a unit of enzyme activity.Dehydrogenase activity was determined by triphenyltetrazole chloride (TTC) method [23].TTC was reacted with the sample at 37 ℃ for 2h, and 1 mL of the sample was used to generate 1µg triphenylmethyl (TPF) in 1 hour, which was de ned as a unit of enzyme activity.
The results of methane production were tted with modi ed Gompertz model as shown below [24].
Where B is the experimental methane production, mL CH 4 /g VS; P is the methane production potential, mL CH 4 /g VS; t is the fermentation time, d; R m is the maximum methane production rate, mL CH 4 /g VS /d; λ is the lag-phase time, d; e is the constant (2.718282).

Results and discussion
3.1 The methane production from sweet sorghum bagasse with zeolite and trace elements addition Figure 2 shows the variation of accumulative methane production from sweet sorghum bagasse with time before and after pretreatment.As can be seen from Fig. 2 (a), the methane production from PSSBS increased by 6.0% compared with that of the control (SSB), while the accumulative methane yield increased to varying degrees after the addition of zeolite with different amount.The accumulative methane yield increased rst and then decreased with the addition amounts increase of zeolite.When the dosage of zeolite was 5g/L, the accumulative methane production reached 242mL/gVS, which increased by 39.6% compared with the control.Under the same conditions, after adding 1 mL TE, the accumulative methane production of all experimental groups was increased with different levels (Fig. 2 (b)), indicating that TE could promote the process of methane production, which was consistent with the results of Markus [25].After adding TE, the methane production of the PSSBS increased by 16.8% compared with the control, and increased by 32.7% compared with the experimental group neither adding zeolite nor adding TE.It could be concluded that the substantially enhancement of TE on methane production was related to the important regulation of TE on the process of anaerobic fermentation methane production [25].
As can be seen from Fi.2(b) that the accumulative methane production of PSSBS with 5g/L zeolite and 1 mL TE was the maximum (274.5 mL/gVS), which was increased by 58.4% when compared with the control.Notably, both zeolite and TE were bene t to improve methane production.This might be related to the ion exchange characteristics and adhesion of zeolite.Ca 2+ and Mg 2+ contained in the crystal structure of zeolite can be used as an ion exchanger to eliminate ammonia inhibition in the process of anaerobic digestion, and alleviate the accumulation of volatile acid [26].As a result, the biogas production was enhanced.
The importance of TE to anaerobic digestion was mainly re ected in that they often appear in the enzyme system of anaerobic digestion microorganisms as coenzymes, cogroups and cofactors, and played an important role in regulating the methanogenesis stage of anaerobic digestion [27].Fe was involved in the synthesis of cytochrome and cell oxidase in methanogens, and was also the electron carrier of reached 75%~80% [25].Because different substrates and anaerobic digestion systems required different TE, the amount of TE for biogas production needed to be further optimized.The modi ed Gompertz model was adopted for Gompertz nonlinear tting of the accumulative methane production in Fig. 2, and the tting results were shown in Table 3.As can be seen from Table 3, the variation of cumulative methane production over time in all experimental groups could be tted with the modi ed Gompertz (R2 ≧ 0.996).In the experimental group supplemented with zeolite (whether include TE or not), the maximum methane production potential increased rst and then decreased with the increase of zeolite addition dosage.The methane production potential of PSSB + 5 g/L zeolite + TE group was the largest, the R m value was higher, and the lag time was relatively short.The reasons might be the synergistic action of zeolite and TE, as the enrichment carrier of microorganisms, improved the concentration of microbial community.The presence of minerals alleviated the acid inhibition that might exist in anaerobic digestion system and enhanced its methanogenic capacity.However, excessive zeolite addition dosage might weaken the effect of TE on enzyme activies, and also cause excessive osmotic pressure in the system, thus reducing the methane production [15].The supplement of TE could participate in the synthesis of many enzymes, coenzymes and cofactors in anaerobic fermentation, and regulated the enzymatic reaction in the process of methanogenesis [30,31].

pH and alkalinity
3 (a) Fig. 3 (b) showed the changes of pH value of digestate during the process of biogas production.Figure 3 (c) and Fig. 3 (d) showed the variation of alkalinity of digestate during the process of biogas production.As can be seen from Fig. 3 (a) and Fig. 3 (b), the pH values decreased in the rst 3 days, and then gradually increased, until stabilized after 21 days.This variation tendency was consistent with Zhang's results [32].The pH values of the experimental groups with zeolite addition were higher than that of the control groups, but there was no signi cant correlation between pH value and zeolite addition dosage.At the initial stage of the biogas production from SSB/PSSBS, cellulose and hemicellulose in raw materials were hydrolyzed and generated small molecular acids, such as acetic acid and propionic acid, under the action of microorganisms.The accumulation of these acids would consume HCO 3 − , thus the pH value was reduced.The addition of zeolite is bene cial to regulate the acidi cation process in the reaction system, but methanogens not only consume fatty acids, but also produce HCO 3 − , which makes the pH value of the system rise subsequently [33].In addition, with the continuous degradation of macromolecules in the biogas production process, the degradation rate of macromolecules would slow down, and methanogens would also convert VFA to CH 4 and CO 2 , which gradually stabilized the pH value of the system.
In the process of anaerobic digestion, alkalinity was an important index to measure the buffer capacity of the reaction system and an important parameter to reveal whether the reactors were running stably.The bicarbonate ion (HCO 3 − ) was the main source of buffering capacity to maintain the system's pH in the range of 6.5 ~ 7.6 [34].As shown in Fig. 3(c) and Fig. 3 (d), from the beginning of the experiment to the 7th day, the alkalinity gradually decreased.At this stage, the VFA produced in the process of hydrolytic acidi cation was neutralized by HCO 3 − in the system, resulting in a decrease in alkalinity.Then the alkalinity began to rise and tended to be stable, and relatively stable alkalinity was conducive to maintaining the stability of the system [35].As can be seen in Fig. 3(c) and Fig. 3 (d), the alkalinity of the control rose the slowest, while the experimental groups supplemented with zeolite had a relative strong buffer capacity and could maintain a high alkalinity, thus reducing the adverse effects of the disturbance of system pH value on methanogenic microorganisms.

The and dehydrogenase activities in the digestate
Figure 4 the effect of zeolite addition dosage and TE on cellulase and dehydrogenase activities of the digestate.Cellulose is an important component of plant anaerobic fermentation substrate.Cellulase plays a very important role in the degradation of cellulose and hemicellulose during anaerobic digestion [36].Therefore, the activity of cellulase would re ect the degradation of lignocellulose in anaerobic digestion to a certain extent.According to Fig. 4 (a), the addition of zeolite and TE effectively improved the cellulase activities of the digestate, the cellulase activity of each experimental group reached a peak on the 7th day, in which the cellulase activity of PSSBS + 5g/L zeolite + TE was the highest (2.63U/g), which was increased by 27.7% compared with PSSBS + 5g/L zeolite and by 76.5% compared with the control.According to Fig. 3(a) and Fig. 3(b), the pH values on the 3rd and 7th were relatively low, it was not di cult to surmise acidi cation occurred in the system at the initial stage of gas production, which was conducive to the hydrolysis of cellulose and hemicellulose [37].Higher cellulase activity achieved faster hydrolysis rate and provided more substrates for methanogens, which was consistent with Yong et al. 's research results [38].
Dehydrogenase, as an important REDOX enzyme, will activate hydrogen ions of oxidized organic matter and transfer them to speci c hydrogen receptors.Dehydrogenase activity level is an important index of the average activity of microbial metabolic active population in anaerobic digestion [39].According to Fig. 4 (c) and Fig. 4 (d), the activities of dehydrogenase in digestate decreased to the lowest value in 3 ~ 7 days, which might be related to the decrease of pH value in the system, and acidi cation resulted in the decrease of dehydrogenase activities.Subsequently, dehydrogenase activities increased and peaked on the 21th day, which was similar to the variable trends of accumulative methane production.According to Fig. 4 (d), on the day of the 14th and 21st, the dehydrogenase activities achieved the highest.The experimental group of PSSBS + 5g/L zeolite + TE had the highest dehydrogenase activity, reaching 66.06µg/mL/h, increased by 32.6% compared with the control.Excessive zeolite addition dosage might result in high osmotic pressure during the biogas production, which lead to the decrease of dehydrogenase activity [40].

4.
Zeolite and TE effectively increased methane production, shortened the lag time and maintained the stability of the digestion system.Addition of 5g/L zeolite and 1 mL of TE was the optimal strategy for biogas production from PSSBS.Additions of Zeolite and TE were bene cial to increase the activities of cellulase and dehydrogenase.Adding zeolite promoted the cellulase activity, but the cellulase activity decreases when zeolite and TE coexist.The schematic diagram of biogas production intracellular REDOX reactions[28].Ni 2+ and Co 2+ were the active central component of the main enzymes involved in anaerobic digestion.Adding proper amount of Ni 2+ and Co 2+ could increase the activity of enzyme methyl-coenzyme reductase and coenzyme F 430 , and improve the biogas production[29].The results from Zhang et al showed that adding Fe(100 mg/L), Co(1 mg/L), Mo(5 mg/L) and Ni(5 mg/L) could increase the methanogenesis rate of kitchen waste anaerobic digestion by 35%[28].Ortner et al. showed that adding 11.4 mg/L Ni, 25.4 mg/L Co and 4.8 mg/L Mo could make the methane yield of anaerobic digestion of slaughterhouse waste reach 250 ~ 275 m 3 • t − 1 COD.The removal rate of COD

Table 1
The basic properties of SSB, PSSB, inoculum and pretreatment e uent a: acid soluble lignin; b: acid insoluble lignin; c: total phenolic content; NA: not analyzed.

Table 3
Kinetic tting results of accumulative methane production