Enhanced straw fermentation process based on microbial electrolysis cell coupled anaerobic digestion

Xinyu Yan,Bobo Wang,Hongxia Liang,Jie Yang,Jie Zhao,Fabrice Ndayisenga,Hongxun Zhang,2,Zhisheng Yu,2,*,Zhi Qian,*

1 College of Resources and Environment,University of Chinese Academy of Sciences,Beijing 100049,China

2 Research Center for Eco-Environmental Sciences-Institute of Microbiology,Chinese Academy of Sciences-University of Chinese Academy of Sciences(RCEES-IMCAS-UCAS)Joint-Lab of Microbial Technology for Environmental Science,Beijing 100085,China

Keywords:Microbial electrolysis cell (MEC)Methane Straw Fermentation Bioenergy

ABSTRACT The low quality and yield of methane severely hinder the industrial application of straw biogas fermentation,and no effective solution has been found so far.In this study,a novel method was developed when a microbial electrolysis cell(MEC)was coupled with normal anaerobic fermentation to enhance methane yield and purity.The fermentation process achieved a methane purity of more than 85%,which is considerably higher than that of previously published reports.With microbial stimulation and an electric current,the degradation of fibers has been greatly enhanced.The MEC system substantially improved the yield and purity of biogas,bringing a new path to the synthesis of methane by carbon dioxide and hydrogen ions in solution under electron irradiation.Electrochemical index analysis showed extra methane synthesis,due to the external circuit electron transfer.The results of the gas chromatography and solid degradation rate showed that the carbon source of extra methane was CO2 produced during normal fermentation and additional volatile solid degradation.These results show that the MEC considerably enhanced the quality and yield of methane in the straw fermentation process,providing insights into normal anaerobic fermentation.

1.Introduction

The emission of carbon dioxide from fossil fuel burning and other anthropogenic activities has substantially increased the average global temperature and greenhouse effect [1].Carbon dioxide,as the dominant component of greenhouse gases,has attracted increasing attention as a feedstock for the synthesis of various chemicals and fuels[2,3],with methane as one of the target products [4].The production of methane via anaerobic digestion is a biological technology that can achieve clean energy using organic waste as feedstock.It operates under mild reaction conditions,low energy consumption,and low secondary pollution [5].However,the common method for producing methane through anaerobic digestion has many disadvantages.The speed and efficiency of anaerobic digestion are slow[6],and do not provide satisfactory performance when treating low-strength wastewater [7].Moreover,the purity of methane has been unsatisfactory,while the carbon dioxide consumption in the anaerobic digestion gas has only been 30%–50% [8].

Bioelectrochemical systems (BES) are a promising strategy for overcoming these barriers during anaerobic digestion[9].BES have been employed in wastewater treatment [10,11],energy production [12,13],and heavy metal ion recycling [14],and have been advantageous owing to the high efficiency,easy operation,low energy input,and environmental friendliness [15].Microbial electrolysis cells (MECs),as an important component of BES,aim to provide chemicals through bio-catalyzed reactions supported by electric energy [16,17].New-generation integrated MECs have recently become a hot topic in the field of sustainable production of energy and chemicals [18,19].The main purpose is to decompose waste [20] and simultaneously generate valuable chemicals[21].Moreover,several studies have focused on wastewater treatment and obtaining methane as a byproduct [6,22].

Methane gas is producedviathree mechanisms [23].Produced by acetate decomposition (1),however,the intrinsic kinetics of acetate oxidation limit methane generation,rather than mass transfer [24];hydrogen synthesis (2);and electrons,hydrogen ion,and carbon dioxide (3) [22].

Mechanisms (1) and (2) also exist during normal anaerobic digestion,whereas mechanism (3) is unique to the MEC system.The performance of these mechanisms depends on the potential of the cathode.Functional microorganisms can directly obtain electrons from the cathode surface and reduce carbon dioxide to generate methane.When the cathode potential is less than -650 mVvsthe standard hydrogen electrode,the production of methane is mainly governed by the hydrogen and electron pathways [24].

Despite the advances in MEC technology,there is a scarcity of research exploring the use of macromolecule waste such as cellulose and lignin,low purity of methane,acidified fermentation broth during biogas fermentation,high-cost requirements,and low yield,which are still the most serious impediments to the development of biogas fermentation.In this study,we combined MEC with anaerobic digestion and used straw to produce methane.Straw is rich in cellulose and can be decomposed by cellulosedegrading bacteria and used by methanogens to produce methane.The pectin in straw can be decomposed into methanol,which can also be used to produce methane.Through a comparative analysis of total methane production and purity,and the electronic and energy balance,we propose a new efficient method for straw fermentation.

2.Materials and Methods

2.1.Straw preparation

Straw was collected from Wei County,Xingtai City,Hebei Province,washed with water,ground,and passed through a sieve(diameter of hole is 0.147 mm).The straw was then dried at 50°C.The total solid (TS) and the volatile solid (VS) contents of straw were 93.71% and 92.63%,respectively.

2.2.Reactor design

The reactors were built using plexiglass to avoid short circuits,and magnetic stirring was used to homogenize the feedstock.The inner diameter and height were set to 100 and 150 mm,respectively.To prevent the foam from blocking the air outlet,the liquid loading coefficient was set to 0.85.The sampling port was set at a height of 20 mm from the bottom of the reactor.The cover had two symmetrical holes to set the anode and cathode and set 30 mm apart to avoid a short circuit caused by direct contact between the anode and cathode.A tubular structure was set to release the gas collected in the gas bag for chromatography analysis.The gas bag and outlet tube were connected using two hoses coupled with a three-way valve (Fig.1).

2.3.Electrodes preparation

The used anode was carbon brush (length of the brush was 7.5 cm,length of brush holder was 15 cm,the brush diameter was 5 cm),and the brush holder was made of stainless steel(0.08% C,2% Mn,0.045% P,0.03% S,1% Si,18% Cr,and 9% Ni).The anode was then placed in the middle of the reactor.Carbon cloth was used as the cathode (0.5 mm×10 mm×35 mm) and seamed using a stainless steel(diameter of the hole is 8 mm).The cathode is adjacent to the inner wall of the reactor (Fig.2).

Fig.1.Reactor design in 3D (units in mm).

2.4.Inoculum selection

Activated sludge was used as the inoculum[20,25].Before inoculation,deoxyribonucleic acid(DNA)extraction,polymerase chain reaction (PCR),and agarose gel electrophoresis were performed to detect the presence of methanogens.The TS and VS values of the sludge were 5.54% and 3.02%,respectively.

2.4.1.DNA extraction

Activated sludge was chosen from five different sewage treatment plants:(a) Datong,(b) Taiyuan,(c) Shunyi,(d) University of Chinese Academy of Sciences(UCAS)Yanqi campus,and(e)Datong development zone sewage treatment plants.All activated sludge samples were collected in plastic bottles and refrigerated at 4°C.Genomic DNA from the collected activated sludge was extracted using the MO BIO Power Soil DNA kit(MO BIO Laboratories,Carlsbad,CA,USA),according to the manufacturer’s instructions.A 2 ml sample of activated sludge was centrifuged at 10,000 r?min-1.The precipitate was then used for DNA extraction.After DNA extraction,the samples were tested using NanoDrop 2000 UV–Vis spectrophotometer (Thermo Fisher Scientific,Waltham,MA,USA)(Table 1).

Table 1 DNA concentration in the product (≥10)

Fig.2.Electrodes and cover in 3D.The carbon brush anode (inside) and stainless steel mesh sewed on carbon cloth (outside);the carbon brush handle is bent to make sure it is placed in the middle of the reactor.The cathode is suspended by four copper wires.

2.4.2.PCR amplification and agarose gel electrophoresis

The samples were used for PCR amplification using the following universal primers for methanogens [26]: MLf:GGTGGTGTMGGATTCACACARTAYGCWACAGC and MLr:TTCATTGCRTAGTTWGGRTAGTT.Further details are shown in Figs.S2 and S3 in Supplementary Material .

2.5.Methane production and purity measurements

Methane production was calculated from the gas in the gas bag,and the purity was detected by gas chromatography at 25°C.The gas was mixed in the air bag with a 50 ml syringe after the baseline in the chromatogram was stable(about 30 min),then,a 200 μl gas sample was injected with a 250 μl syringe from the inlet.The gas content was then compared with that of the standard curve.

2.6.Fiber component measurements

The fiber component was measured using the van Soest method[27].The straw was boiled with neutral detergent,and the insoluble residue was neutral detergent fiber,mainly hemicellulose,cellulose,lignin,and silicate.The residue was treated with acid detergent,and the remaining residue was treated with acid detergent fiber,which includes cellulose,lignin,and silicate.The residues of the acid detergent fiber after 72% sulfuric acid treatment were lignin and silicate.Finally,the residue was ashed and the component that escaped during the ashing process was lignin.

2.6.1.Determination of neutral detergent fiber content (NDF)

One gram of the sample was accurately weighed in a crucible and dried to a constant mass.Next,100 ml of neutral detergent(3%C12H25SO4Na)and 3–5 drops ofn-octanol were added at room temperature.The mixture was heated to boiling and then refluxed for 1 h,filtered with boiling water,washed three times,and rinsed twice with cold acetone.The sample was heated in an oven at 105°C for 8 h,then placed in a desiccator and cooled to room temperature.The NDF (4) was calculated as follows:

wherem0is the mass of the crucible(g),m1is the mass of the crucible and the sample after drying(g),andmis the mass of the sample (g).

2.6.2.Determination of acid detergent fiber content (ADF)

One gram of the sample was accurately weighed in a crucible and dried to a constant mass.Subsequently,100 ml of acid detergent(2%CTAB)and 3–5 drops ofn-octanol were added to the crucible at room temperature.The mixture was heated to boiling and then refluxed for 1 h,filtered with boiling water,washed three times,and rinsed twice with cold acetone.The sample was heatedin an oven at 105°C for 8 h,then placed in a desiccator and cooled to room temperature.The ADF (5) was calculated as follows:

wherem0is the mass of the crucible(g),m1is the mass of the crucible and the sample after drying(g),andmis the mass of the sample (g).

2.6.3.Determination of acid-washed lignin content (ADL)

One gram of the sample was accurately weighed in a crucible and dried to a constant mass.Subsequently,100 ml of acid detergent and 3–5 drops ofn-octanol were added to the crucible at room temperature.The mixture was heated to boiling and then refluxed for 1 h,filtered with boiling water,washed three times,and rinsed twice with cold acetone.Finally,25 ml of 72% sulfuric acid was added to the mixture and left for 2 h at room temperature,before stirring for 1 h.After the cold extraction was completed,the extract was filtered and washed three times with boiling water.The sample was heated in an oven set at 105°C for 8 h,then was placed in a desiccator and cooled to room temperature.The ADL (6) content was calculated as follows:

wherem0is the mass of the crucible(g),m1is the mass of the crucible and the sample after drying(g),andmis the mass of the sample (g).

The cellulose (7) and hemicellulose (8) contents were then calculated as follows:

2.7.TS and VS determination

The samples were placed in a crucible,dried to a constant mass at 102°C,placed in a muffle oven and burned at 600°C for 2 h before weighing.

wherem0is the mass of the crucible(g),m1is the mass of the crucible and the sample after drying(g),m2is the mass of the crucible and the sample after burning(g),andVis the volume of the sample(ml).

2.8.Energy efficiency determination

The energy recovery rate (11)–(15) was used to measure the efficiency of the energy input and output in the system.The energy input was electric energy,while the energy output was the methane produced during fermentation (regardless of the energy in the straw) (see Supplementary Material s).

where η is the energy recovery rate,WEis the electric energy provided by the external power (J),WPis the electric energy obtained from the gas produced(J),Iis the electric current(A),Eis the external voltage (V),tis the reaction period(s),Qis the electric quantity(C),nis the amount of extra methane(mol)produced in the experimental groups,ΔHis the heat produced by burning 1 mol of methane,890.37 kJ?mol-1.

2.9.Fermentation parameters

All the reactors were operated at room temperature without illumination.The stirring speed of the reactors was set at 100 r?min-1and both the control and experimental groups contained cathode,anode,and reference electrodes (Ag/AgCl).The circuit of the control group was opened during the experiment;for the experimental group,the circuit was closed and the applied voltage was 1 V.

3.Results and Discussion

3.1.Total methane production

To enhance the methane production in the MEC coupled system,we added archaea growth medium [28] (Table S2,S3,and S4) to make the microorganisms metabolically more active.Then,we ran the reactor in batch operation for 58 d and recorded the cumulative methane production (Fig.3).

During the first four days,both the experimental and control groups produce almost no methane because the inoculated granular sludge is still adapting to the reactor environment.On day 8,the experimental groups greatly increase the methane production,whereas the control group only show a small increase on day 10.Therefore,we confirm that the MEC anode is enriched with electroactive microorganisms during fermentation[29].Then,the substrate is oxidized to provide the electrons that are transferred to the cathode through the external circuit and generated hydrogen using hydrogen ions on the surface of the cathode,thereby promoting the growth of hydrophilic methanogens.Meanwhile,the growth cycle of hydrotropic methanogens is shorter and more easily enriched compared to other types of methanogens [23].

Fig.3.Total methane production during fermentation period.

The methane production rate in the MEC-coupled reactors remain stable on day 14 and 19 for the control groups.During the stable period,the maximum daily production of experimental groups reaches 96 ml/day on day 15,whereas,for the control group,the maximum daily production reaches 63 ml/day on day 41.This difference is also observed at the end of the fermentation period.In the experimental groups,methane production stops by day 48,whereas for the control groups,it is by day 54.During the experimental period,the cumulative methane production in the control and experimental groups are (1199.97±19.56) ml(2122.77±7.75) ml,respectively,which corresponds to a(76.96±3.50)% promotion.The degradation of VS and TS show similar results (Fig.4).

The electrochemical system,a new method of methane production,converts organic matter to electrons and electrons to hydrogen,thereby promoting methanogenesis [30].Compared with normal anaerobic fermentation,in which methanogens utilize acetic acid salt to produce methane,the rate at which electrons are transferred to the cathode under an applied voltage to synthesize hydrogen gas is much higher than the rate of electron transport within the microbial community [31].Furthermore,the process by which methanogens form methane from hydrogen is easier than that used to produce methane from acetic acid salt[32].In MEC reactors,acetate provides electrons for the anodic substrate,and electrons are transferred to the cathode to generate hydrogen,which is subsequently utilized by methanogens to produce methane [33].Therefore,the methane production rate of the MEC reactors was much higher than that of the control group.

These data showed that the MEC-coupled system significantly promoted methane production compared to that by normal straw fermentation.The cumulative methane yield curves of the experimental groups were more similar and smoother than those of the control group.This means that the MEC system can also enhance fermentation stability.

3.2.Methane purity analysis

In addition to methane production,methane purity remains an important indicator of biogas fermentation.To investigate the efficiency of the MEC coupled system,we recorded the purity of methane during daily gas production (Fig.5).

Fig.4.Total solid (TS) and volatile solid (VS) degradation in the experimental and control groups after fermentation.

Fig.5.Daily methane purity during the fermentation period.Purity measured in the control and experiment groups.MEC,microbial electrolysis cell;AD,acid detergent.

During the whole process of anaerobic digestion,the methane content increased in the early phase,stabilized from days 14 to 38,and decreased from day 39 until the last day.The methane content was the same as the methane production during fermentation;however,the content did not follow the same trend during the mid-late phase.The methane content did not decrease with decreasing methane production.Although the substrate concentration was gradually reduced as fermentation progressed,the available substances of the acid-producing bacteria were reduced,and the dominant metabolically active microorganisms were still enriched with methanogens,which were responsible for methane production [34].Hence,the methane content of the system remained stable.During the entire period,as in the MEC coupled reactors,the methane purity was approximately (87±5)% during the stabilization period,whereas that in the traditional reactors was approximately (54±5)%.The methane content in the MECcoupled reactors also increased faster than that in the control reactors.This is because hydrogenotrophic methanogens propagate faster than acetic acid nutrient methanogens,and H2and CO2as by-products in traditional anaerobic fermentation are also methanogenic substrates in MEC coupling systems compared with traditional anaerobic fermentation processes.Consequently,the purity and yield of methane in the MEC coupling system was higher than that using traditional fermentation,which showed that the incorporation of MEC significantly improved the biogas composition of straw anaerobic fermentation.

3.3.Analysis of straw fiber components

Straw fibers were used as the carbon source during the fermentation period.The major components of straw fiber are cellulose,hemicellulose,and lignin.Hence,the degree of degradation indicates the degree of feedstock utilization.The difference in fiber degradation proved that the MEC coupled system not only utilized CO2and hydrogen ions to synthesize methane,but also promoted the decomposition rate of macromolecule fibers (Fig.6).

Fig.6.Fiber degradation during fermentation.Cellulose,hemicellulose,and lignin degradation after 60 days of fermentation.

At the end of the fermentation,the cellulose,hemicellulose,and lignin contents in the straw showed a significant downward trend compared to those before fermentation.The degradation rates of cellulose,hemicellulose,and lignin in the control groups were 10.60%/9.85%,6.80%/3.43%,and 3.22%/7.14%respectively,while the degradation rates of cellulose,hemicellulose,and lignin in the experimental groups were 20.05%/ 22.36%,24.44%/ 22.86%,and 36.89%/ 35.49% respectively.This phenomenon might have been caused by the enrichment of some bacteria,which can reduce the energy barrier to electron transport between cells and provide an extra driving force for electron transport in the cell[25].Moreover,the extra current broke the stable electrode structure of the benzene ring in the lignin and facilitated its degradation [35].These results indicate the remarkable role of MEC in enhancing the degradation of fibers,thereby increasing methane production.

3.4.Analysis of the electrochemical index

In the microbial electrolysis cell,the generated electrons were due to the oxidation of the substrate.The electrons reached the cathodeviathe circuit driven by the applied voltage,synthesized hydrogen gas from protons,and finally,was used by hydrophilic methanogens to synthesize methane.Therefore,the degradation rate of anodic organic matter influences the magnitude of the current.We then recorded the daily current and investigated the electron and energy efficiencies (S1 in Supplementary Material ).

The three different fitting situations represented the three different stages of the fermentation process (Fig.7).In the prophase,there were fewer methanogens,and electrons were partially utilized;meanwhile,little volatile acid production represented high resistance.Therefore,the fitting yield was slightly higher than the actual yield in the MEC-coupled reactor.With the accumulation of volatile acids and enrichment of methanogens during the metaphase,the fitting yield was similar but slightly lower than the actual yield,and a slightly lower yield resulted in the current not only providing the electrons for the synthesis of methane,but also promoting the degradation of macromolecular substances such as lignin.In the anaphase,with the consumption of substrates and accumulation of secondary metabolites during the fermentation process,the reaction was gradually terminated,and the electrons could not be used by bacteria such asGeobacter,resulting in a fitting yield much higher than the actual yield.In addition to electron efficiency,energy is an important index for assessing a method (Table 2).

Table 2 Energy balance

Fig.7.Electrons balance in the MEC coupled reactor.One mole extra methane production need eight moles electrons;all the electrons are provided by an external circuit.Through daily current,electron transport is calculated to obtain the theoretical extra methane production in the MEC system.

On the 38th day,the methane production in the experimental groups was declining,and energy efficiency reached 141.74%,which means that all the energy input can be made up by extra methane production,while 41.74% more energy can be achieved after incorporating electromethanogenesis.After the 53rd day,both the experimental and control groups stopped producing methane,with an energy efficiency of only 87.42%.Therefore,we suggest that the decline phase should be eliminated by applying a continuous fermentation mode to produce MECs with energy benefits.

3.5.Outlook

Overall,we report on a new and efficient approach to promote straw biogas fermentation and evaluate the purity and yield of methane and electrochemical indices in the process.Under an external voltage of 1.5 V,the yield and purity of methane were both significantly improved,owing to the additional methane production from the added MEC system.The external voltage also facilitates the transfer of extracellular and intracellular electrons,accelerating the microbial feedstock degradation.In contrast to traditional straw fermentation research,which focuses on the pretreatment of the feedstock,and the evaluation of water-soluble feedstock in MEC systems,this method directly uses straw in a coupled reactor and promotes biogas fermentation by breaking the covalent ring structure of benzene ring compounds in macromolecular substances,thus promoting the microbial degradation of the feedstock.Compared to existing methods,this method achieved high purity and production of methane;moreover,an additional net benefit of energy was realized.

Although this strategy has been successful,some basic and technical problems still need to be resolved.During the entire fermentation process,it is not known the mechanism by which microorganisms interact to increase methane production and purity.In previous studies,due to the simple feedstock and small molecules of feedstock,the dominant microorganisms were mainly methanogens such as Methanotrichaceae [34,36],Methanobacterium [37–39],Methanobrevibacter[40–42],and from the bacteria-assisted electron transfer,for example,Geobacter[43–45].These microorganisms have relatively simple functions and synergy.However,for macromolecular feedstocks,a variety of microbial communities are required to cooperate.During the fermentation process,the dominant microbial community was constantly altered with continuous changes in the main nutrient components.The synergy of multiple microorganisms and change in dominant microorganisms during the fermentation process also need to be studied further.In addition,encrustation in the electrodes is one of the obstacles affecting the application of the MEC system in the biogas fermentation process.Insights into these questions will assist in a better understanding of the mechanisms and MEC application in the biogas fermentation process.

4.Conclusions

In the present study,the MEC system was added to a simple anaerobic fermentation tank and compared with an open control group.The highest TS and VS degradation rates of the MECcoupled experimental groups reached 41.26%,which was 29.9%higher than the highest value obtained by the control group,and the highest VS gas production was 116.18 ml?(g VS)-1,which was 76.8%higher than the highest value obtained by the control group.The purity of methane in the MEC-coupled experimental group was (87±5)%,while that in the control group was only (54±5)%.Simultaneously,the start-up period of the MEC-coupled experimental group was considerably shorter than that of the control group.

The electrochemical index of the MEC coupling system was analyzed at the end of the comparison.The overall change in current showed a rapid increase to the peak value and a slight decrease thereafter until reaching a stable value.Moreover,the amount of methane synthesized by the theoretical amount of electron transfer and that of methane in the control group were substantially equivalent to the methane production of the experimental group MEC-coupled reactor before the end of the fermentation,which proves that most electron transfer was used to synthesize methane,while the degradation of VS and TS demonstrated that the other part of the electron transfer was most used to break the structure of the macromolecule feedstock.As for the energy balance,the energy efficiency reached 141.74%before the fermentation entered the decline phase,and an electric energy efficiency of 87.42% for the entire fermentation process.Therefore,continuous fermentation is more suitable than batch fermentation for MEC coupling.

In summary,the MEC system can significantly promote the traditional anaerobic fermentation,while the application of MEC can improve the utilization rate of the substrate,shorten the time required for fermentation,and increase the yield and purity of methane.At the same time,through the analysis of the energy balance in the reaction process,the MEC will have a better effect on continuous fermentation.The excellent performance of the proposed MEC coupling systems has promising prospects in the field of solid waste fermentation.

Acknowledgements

This work was supported by the National Key Research and Development Program of China (2018YFD0800403),the National Natural Science Foundation of China (No.21978287) and the Fundamental Research Funds for the Central Universities (No.292021000194).

Supplementary Material

Supplementary data to this article can be found online at https://doi.org/10.1016/j.cjche.2021.05.020.