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Anaerobic digestion of biorefinery lignin: Effect of different wet explosion pretreatment conditions
Journal Pre-proofs Anaerobic digestion of biorefinery lignin: effect of different Wet Explosion pretreatment conditions Muhammad Usman Khan, Birgitte Kiaer Ahring PII: DOI: Reference:
S0960-8524(19)31767-5 https://doi.org/10.1016/j.biortech.2019.122537 BITE 122537
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Bioresource Technology
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1 October 2019 27 November 2019 29 November 2019
Please cite this article as: Usman Khan, M., Kiaer Ahring, B., Anaerobic digestion of biorefinery lignin: effect of different Wet Explosion pretreatment conditions, Bioresource Technology (2019), doi: https://doi.org/10.1016/ j.biortech.2019.122537
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Anaerobic digestion of biorefinery lignin: effect of different Wet Explosion pretreatment conditions Muhammad Usman Khana, Birgitte Kiaer Ahringa,b* aBioproducts,
bGene
Sciences and Engineering Laboratory, Washington State University, Tri-Cities, Richland, WA-99354
and Linda Voiland School of Chemical Engineering and Bioengineering & Biological Systems Engineering, Washington State University, Pullman, WA-99163
Corresponding author Dr. Birgitte K. Ahring Bioproducts Sciences and Engineering Laboratory Washington State University, Tri-cities 2710 Crimson Way Richland, WA 99354 Tel.: 01-(509)-372-7682 Fax: 01-(509)-372-7690. E-mail address: [email protected]
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Abstract This study examine ways to make biorefinery lignin accessible for anaerobic digestion. The raw material was the residue after removing carbohydrates by Wet Explosion pretreatment at 190oC and 7.5% O2 followed by enzymatic hydrolysis. The residual solid was mainly composed of lignin and was the raw material for a second WEx pretreatment operated at 220oC with 4 % oxygen and variable concentrations of NaOH (0-2%). Lignin B was the residue after pretreated without NaOH, Lignin C was pretreated at 1% NaOH, and Lignin E at 2% NaOH. Anaerobic digestion was carried out on all lignin fractions (Lignin A, B, C and E) at thermophilic conditions (52oC) by mixing 70% of each lignin fractions with 30% clarified manure. The results showed that the lignin samples were demethoxylated as part of the biodegradation and that the highest severity pretreatment (with oxygen and 2% NaOH) resulted in the highest methane yield. Key Words: Biorefinery lignin, Anaerobic Digestion, Lignin degradation, Pretreatment
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Introduction Currently, 80% of the global energy demand is being fulfilled by fossil fuels (Guo et al., 2015). The global energy consumption was 5.5 × 1020 J in 2010, which is predicted to increase to 6.6 × 1020 J in 2020 and 8.6 × 1020 J in 2040 (Guo et al., 2015). The continuously increasing use of conventional fossil fuels not only increase the environmental pollution but is also causing an increase in global warming (Bahrami and Abbaszadeh, 2013). Production of biofuels and bioenergy from abundantly available plant biomass can assist in a reduction of the problems linked to fossil fuels consumption. The global annual production of plant biomass is 200 × 1011 tons/year, which can produce 2.2 × 1021 J of energy. This energy is 3-4 times higher than the current global energy demand (Guo et al., 2015). Lignocellulose is one of the most abundant and renewable biomass resources on the earth which makes it an ideal feedstock for production of biofuels and bioproducts. It is mainly composed of lignin, cellulose and hemicellulose. Lignin is the second most abundant natural polymer and the only abundant aromatic polymer in nature. In the plant cell wall lignin is associated with cellulose and hemicellulose and its percentage varies from 10-30% in the lignocellulosic biomass (Asgher et al., 2014). The paper and pulp industry alone further produces about 50 million tons of lignin but currently the lignin is only used in major amount for the production of low value products such as production of steam, binders or dispersants in cement and gypsum blends. The cost of production of high-value products like vanillin is still high and the product yield is low. Today, only 2% of the available lignin from paper and pulp industry is being used for other purposes than as a low value fuel (Gosselink et al., 2004) . Besides this amount the global cellulosic biofuels industry is expected to produce a huge amount of lignin ca. 225 million tons per year) (Jung et al., 2015; Sahoo et al.,2011). A sustainable and cost-effective method is needed to tackle the large amount of lignin, which would be produced from second generation biofuel production both for improving the economics of the biofuels as well as for reduction in the greenhouse gas emission. Until now the valorization and conversion of lignin residues have proven to be challenging within conventional paper and pulp industries and no viable solutions have been implemented for lignin from production of second generation biofuels(Vishtal and Kraslawski, 2011). One solution which has been investigated in larger detail is the conversion by pyrolysis to produce bio-oil and chemicals by heating the lignin in an oxygen-free environment at a temperature of about 220-400oC (Lu et al .,2019). The need for upgrading of the products from pyrolysis is, however, very energy intensive and costly and the yield of final product is generally low (Pham et al., 2015). Anaerobic digestion of lignin into useful methane could potentially be a useful and cheap solution with far higher conversion rates compared to pyrolysis. Anaerobic digestion of 3
lignin can also provide energy for operating the whole biorefinery as well as a raw material for producing hydrogen for further upgrading in the biorefinery (Sawatdeenarunat et al.,2015) Anaerobic digestion is a mature and reliable technology used widely for handling different types of organic waste and biomass materials with the production of bioenergy in the form of biogas(Chen et al., 2008). The complex organic compounds in the biomass raw material are converted to biogas by the concerted action of several groups of microorganism, performing all from hydrolysis of the polymers to acidogenesis, acetogenesis, and methanogenesis (Ali Shah et al., 2014). Methane (50-75%) and carbon dioxide (25 to 50%) are the main constituents of the biogas and their amounts vary by the composition of the feed stock and the operating conditions of the anaerobic process (Hosseini and Wahid, 2014). It is generally believed that only the cellulose and hemicellulose fraction of lignocellulosic materials is converted to biogas during anaerobic digestion, while the lignin fraction remains left behind. Still, lignin degradation has been observed in many anaerobic natural environments (Ko et al.,2009; DeAngelis et al.,2011; DeAngelis et al.,2013). Lignin degradation has, however, not been studied broadly in anaerobic digesters. Lignocellulosic materials such as straw and deep litter are increasing used as a feedstock for biogas plants. However, the limited hydrolysis of this substrate is a major challenge (Tsapekos et al., 2019). Different types of biomass pretreatments are increasingly being researched for increasing the hydrolysis of these materials to produce a higher methane yield (Yu et al.,2019). Overall, these pretreatments are found to increase the biogas yield (Khalid et al., 2019; Hashemi et al.,2019) but lignin degradation during anaerobic digestion was not observed or examined. Moreover, most of the pretreatments of lignocellulosic materials will include costly chemicals, which further results in changes to the biomass, which can produce hazardous waste, which needs to be managed carefully to avoid environmental pollution (Carrere et al.,2016; Paudel et al.,2017). Wet explosion (WEx) is a thermochemical pretreatment process of biomass with oxygen addition, where the wet oxidation process is combined with steam explosion. This pretreatment process has shown good performance for destruction of the structure of lignocellulosic materials along with good conversions during enzymatic hydrolysis(Biswas et al., 2012; Rana et al., 2012). Wet explosion pretreatment have many advantages for use as part of anaerobic digestion compared to other pretreatments due: (1) this pretreatment mainly attack the lignin structure, which is the major recalcitrant part of the biomass under anaerobic conditions, (2) During the pretreatment of the feedstock, there is no need for addition of any other chemicals to run the process except from oxygen and water, (3) After the pretreatment process, no recycling and recovery of added chemicals is required, (4) Energy requirement of the process is low because the oxygen used for the process generates heat during its reaction at 170oC or above (Rana et al.,2012). Wet explosion pretreatment is also cheaper compared to the other process because the pretreatment of lignocellulosic materials can be carried out at higher concentrations of dry 4
matter, resulting in a higher throughput through the system (Ahring and Munck, 2014). This pretreatment will, further, not use any hazardous chemicals reducing potential extra cost for treatment or disposal of any hazardous waste (Rana et al., 2012). The economy of the process is improved compared to dilute acid pretreatment and AFEX, which require a proper system for the recycling and recovery of sulfuric acid or ammonia (Sendich et al., 2008;Martin et al.,2019). The WOEx method of for pretreatment of lignocellulosic biomass was patented in 2014 (Ahring and Munck 2014) and was further developed into an optimized continuous process patented in 2016 (Merrill and Petersen 2016). Importantly, the WEx process has previously been found to make lignin accessible during anaerobic digestion (Ahring et al.,2015). The lignin degradation was explained in the light of the extensive methoxylations within the lignin molecule during WEx pretreatment (Rane et al 2015, Khan and Ahring 2019). In the current study we will examine the optimal conditions for WEx pretreatment to improve lignin degradation during anaerobic digestion including the effect of NaOH in addition to oxygen during pretreatment. 2. Methods 2.1. Feed Material and Inoculum Wheat straw and clarified manure were collected from Five D Farms Pasco Washington. The clarified manure is manure, which has been screw pressed to remove particular solids and five 2L anaerobic reactors were started on this material under thermophilic conditions with a hydraulic retention time of 20 days. The wheat straw was dried at ambient temperature for two weeks until the moisture contents were below 10%, after which it was grinded to reduce the particle size to 6 mm before it was pretreated. The wheat straw used for the experiment consists of 35.9% glucan, 23.9% xylan and 2.6% arabinan of the total weight (oven dry basis). Moreover, wheat straw also contains 2.4% acetyl group and 17.7 % total lignin (acid soluble and insoluble). Further, the concentration of cell wall extractives, that are soluble in water and ethanol, was determined to be 9.1% in this raw material. 2.2 Wet explosion pretreatment and enzymatic hydrolysis The down-sized wheat straw was pretreated using a pilot scale wet explosion pretreatment system. Wet explosion pretreatment of wheat straw was carried at optimized conditions (190oC, 7.5%O2, 25 min) for maximum sugar production at high solid concentration (25% DM) using a 100 L stainless steel pretreatment reactor. After pretreatment the material was enzymatically hydrolyzed without removal of liquid fraction using 15L Applikon Bioreactor with the addition of cellulolytic enzymes: Cellic®CTec2:27.8 mg protein/g glucan Cellic®HTec2;10% of CTec2 (Novozymes, USA), respectively. The enzymatic hydrolysis was done for 90 hrs at pH 5 at 50oC. The final biorefinery lignin stream (Lignin A) was separated using a combination of water 5
washing and solid liquid separation leading to a liquid fraction composed of high concentrations of sugars such as glucose and some xylose along with the solid Lignin A fraction mainly composed of lignin and some residual cellulose besides minor concentrations of hemicellulose. Lignin A was then further wet exploded at 220oC for 10 minutes including 4% O2 with different concentrations of alkali (0, 1 & 2 wt%) to examine the effect of further attack to the lignin’s aromatic structure. These lignin fractions were named as B, C and E, respectively, as shown in Fig.1 . All lignin fractions A-E were used as feed materials for anaerobic bioreactors to test the influence of the different pretreatment conditions on anaerobic digestion of the materials. 2.3. Experimental set-up and Feeding Strategy for the Anaerobic Bioreactors The anaerobic digestion of the biorefinery lignin was carried out using 2 L glass bioreactors operated semi-continuously using anaerobically digested clarified manure as an inoculum from an active laboratory bioreactor. The reactors were operated under thermophilic conditions (52oC) with a hydraulic retention time of 20 days and with one daily feeding at a fixed time. The four pretreated lignin fractions (A-E) were diluted by adding 30% clarified manure to the pretreated lignin samples to ensure that sufficient nutrients were present during the experiment. A bioreactor was further fed with 100% clarified manure to examine the biogas production from this raw material. Gas production and pH was registered daily. 2.4. Characterization of the Feed Stock Total solids (TS), Volatile solids and ash content of all types of lignin feed were analyzed using NREL standard methods (Sluiter et al., 2008). The feed material was taken in pre-dried crucibles and placed in a convection drying oven at a temperature of 105±3 oC for 24h. The dried samples were taken out from the oven after 24 hours and placed in a desiccator to allow them to cool to room temperature. The crucibles were weighed after 20 minutes of cooling and the weight was recorded. After weighing all the crucible containing dried samples were again placed in convection oven so that they can be dried to constant weight. After one hour the samples were removed again from the oven and weighed for the total solids. The ash contents of the material was determined by heating the crucibles at a temperature of 575±25 in a muffle furnace for 6 hours according to the method described by (Wychen and Laurens 2013). After 6 hours the samples were removed from the muffle furnace and placed in desiccator and allow them to cool to the room temperature. The weight of the cooled samples were recorded for volatile solids determination. 2.5 Compositional Analysis The feed and effluent samples were dried thoroughly for the determination of total lignin (acid soluble and Acid Insoluble) and total carbohydrates according to the NREL standard method described by Sluiter et al., 2008 The total carbohydrates (sum of glucose, xylose and arabinose) 6
and the Acid Insoluble lignin were measured after two-step hydrolysis, whereas the Acid soluble lignin was determined using a UV spectrophotometer (Jenway 6405 UV/Visible,NJ,USA) with a wavelength of 320nm. Monomers were analyzed by using an UltiMate® 3000 HPLC system (Dionex, Sunnyvale, CA) with an Aminex® 87H Column 250 × 4.6 mm (Bio-Rad, Hercules, CA) and a Shodex RI-101 refractive index detector at 60 °C using 4 mmol H2SO4 as an eluent with a flow rate of 0.6 mL/min. All the measurements of lignin and carbohydrates analysis were done in duplicates. 2.6 Gas Analysis Analysis of the biogas composition from all the bioreactors was done on a daily basis. The gas samples were taken from the gas valves provided in gas pipeline of each of the reactor using a syringe. The collected gas sample was then analyzed by using Universal Gas Analyzer, UGA series (Stanford Research Systems, Sunnyvale, CA). 2.7 Volatile Fatty Acid Analysis High performance liquid chromatography (HPLC) was used for the volatile fatty acid analysis of the feed and effluent samples. About 2ml of the sample was taken in an eppendorf tube and centrifuged for 10 min at 10,000 rpm. After centrifugation the supernatant was separated using a pipette and then it was diluted 2 times using 4mM sulfuric acid and the diluted samples were filtered through 0.2 µm filter using a 3 ml syringe. The filtered samples were analyzed using an UltiMate ® 3000 HPLC system (Dionex,Sunnyvale,CA) with an Aminex ® 87 H column 250×4.6 mm (Bio-Rad, Hercules,CA) and a Shodex RI-101 refrective index detector. The flow rate of the eluent (4mM Sulfuric acid) flowing through 87H column and the oven temperature were kept constant at 0.6 ml/min and 60oC respectively as done in the previous study (Murali et al., 2017). The total time for the VFA analysis of the feed and digested lignin and manure samples was 68 min. 2.8 pH Analysis The pH of the effluent from all the reactors was monitored regularly during the whole experiment. The pH was measured by Oakton pH-510 pH meter. The pH was measured immediately after removing samples from the reactors to avoid any changes of pH during storage. Between each measurement the pH electrode was washed with deionized water and then wiped with paper towel. 2.9 Elemental Analysis and Methoxyl groups Content The Carbon ©, Hydrogen (H), Sulfur (S) and Nitrogen (N) analysis of each of the lignin sample was carried out by using TRUSPEC-CHN® elemental analyzer (LECO, U.S.). The elemental analysis was done by using by using 0.15 g of the lignin sample. The percentage of oxygen was 7
determined by subtracting the percentage of C, H, N and S from 100%.The methoxyl groups in feed and effluent samples were found by using eq.1 as was determined by (Jablonsky et al.,2015). OCH3 = ―18.5769 + 4.0658(H) + 0.34543(O)
(1)
The demethoxylation (%) was determined by using eq.2 given below Demethoxylation (%) =
(OCH3)Feed ― (OCH3)Eff (OCH3)Feed
(2)
3. Results and discussion 3.1 Substrates Characteristics The compositional analysis of the different lignin feed materials (A-E) before anaerobic digestion and after digestion (effluent determinations) has been presented in Table.1. The table shows that about 3/5 of the organic matter in all the feed materials was composed of lignin in which only 3.1% is acid soluble in lignin A, 2.7% in lignin B, 2.6% in lignin C, and, 2.8% lignin E. In comparison, only 1/10 of the organic matter in manure was lignin in the clarified manure of which 20.3% was acid-soluble lignin, while the rest was acid insoluble. Acid insoluble is the higher molecular weight (HMW) lignin and its molecular weight is generally over 850kD. The anaerobic digestion of this type of lignin is generally difficult unless it has been modified to allow for microbial attack. Anaerobic microorganisms cannot produce the radicals and enzymes, which can attack large lignin molecules, but need pretreatment to make necessary modifications to the molecule such as methoxylations. When the lignin molecule is methoxylated, these points can be attacked by the microbial communities operating in the anaerobic bioreactor (Khan and Ahring,2019;Ko et al., 2009). The percentage of Acid insoluble lignin in Lignin A was found to be 55.9% (VS basis) while the further pretreated lignin B,C and E showed higher percentage of acid insoluble lignin accounting for 60.4, 60.7 and 62.4 on a volatile solid basis. The manure showed only 10.6% Acid insoluble lignin. The percentage of Acid soluble lignin was found to be almost similar in both primary and secondary wet exploded lignin in the range of 1.6-1.8 (% of VS) while the manure was found to have a somewhat higher 2.7% (% of VS) percentage of Acid soluble lignin as compared to the primary and secondary wet exploded lignin. The percentage of carbohydrates was found to be 42.3%, 37.9%, 37.7 % and 35.8% in lignin A, B, C and E, respectively, whereas the manure had 86.7% carbohydrates on a VS basis.
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The lignin to carbohydrate ratio in all five types of feed materials i.e. Lignin A, Lignin B, Lignin C, Lignin E and clarified manure was found to be 1.36, 1.63, 1.65, 1.79, and 0.15, respectively. The lignin to carbohydrate ratio by the compositional analysis of effluent from all the reactors i.e. Lignin A, Lignin B, Lignin C , Lignin E and Manure was observed to be 1.15,1.38, 1.26, 1.05 and 0.20 which showed that the lignin to carbohydrate ratio in the effluent was significantly reduced in all four types of lignin, while it was increased in the manure feed. These results show that lignin is being converted to methane in all types of lignin samples, whereas the carbohydrate is the main source being converted to methane in the manure feed. The results are comparable with a previous from the study of Ahring et al.,2015 where significant amount of wet exploded lignin was converted to methane as compared to non-pretreated lignin. Our results further showed that the maximum reduction in lignin to carbohydrate ratio was observed in Lignin E, which was secondary wet exploded with 2% NaOH. This was probably due to the fact that lower molecular weight lignin fragments is produced when higher concentrations of NaOH (2%) is added compared to lower concentrations of NaOH (1%) leading to a better bioconversion. The finding that lignin depolymerization is more efficient at higher NaOH concentrations has been previously described by Rodriguez et al.,2017. 3.2. Biogas Potential of the Feed Materials Methane yields from all five semi-continuous bioreactors (R1 to R5) has been presented in Fig.2 (moving average of 7 days). The reactors R1, R2, R3 and R4 were fed with Lignin A, Lignin B, Lignin C and Lignin E along with 30% clarified manure; R5 was only fed with clarified manure. During the whole experiment the methane production from all the reactors was measured on daily basis. During startup of the experiments, all of the five reactors were only fed with the clarified manure until stable and similar gas production was recorded from all of reactors. Afterwards, the reactors were fed with 70% lignin (A-E) and 30% clarified manure. Methane yield varied during the initial period of experiment (day1-22) from 5 to 94 ml/gVS/day with average values of 35.9±30, 37.8±32, 37.5±31, 38.7±33 and 40.1±32 ml/gVS/day in R1 to R5, respectively. After the gas production stabilized in all the reactors the average methane production from 23-26 days was found to be 93.7±1, 93.9±1, 93.9±0.6, 93.2±0.54 and 92.6±0.92 ml/gVS/day in R1 to R5, respectively. From 27th day Lignin A, Lignin B, Lignin C and Lignin E with 30% clarified manure and 100% clarified manure were used as a feed material in R1 to R5, respectively. The methane yield from the reactors started varying after addition of lignin. The methane yield from all the reactors which were fed by lignin stabilized after 64 days resulting in an average methane yield from R1 to R4 of 128.7±2, 146.4±2, 176.3±2.5,195.4±2.3 respectively, while the R 5 reactor with only clarified manure gave a methane yield of 94.8±2. All the reactors fed with different wet exploded lignin in addition to clarified manure produced higher methane yield compared to the reactor, which were only fed with clarified manure. There was significant differences (T-test) between Reactor 1 (Lignin A), Reactor 2 (lignin B), Reactor 3 (lignin C) and Reactor 4 (lignin E) yielded 36, 54.5, 86 and 106.2% more methane as 9
compared to Reactor 5, which only received clarified manure. The wet exploded lignin showed higher methane yield due to the fact that the small fractions of hemicellulose and parts of the lignin solubilized during the wet explosion pretreatment and could be converted along with some of the solid lignin (Khan and Ahring, 2019). Overall, the secondary wet explosion pretreatment was found to increase the methane yield and the lignin B produced 14% more methane as compared to the primary wet exploded lignin A. The alkali assisted wet exploded lignin showed even higher methane yield as compared to just wet explosion with oxygen at higher temperatures, and Lignin C and Lignin E produced 37 and 52% higher methane yield compared to the lignin A. Moreover the addition of base during the secondary wet explosion pretreatment was found to increase the methane yield and lignin C and E produced 21 and 33% more methane as compared to the lignin B, which was pretreated without any base. The higher concentration of alkali during pretreatment was found beneficial for increasing the methane yield and Lignin E with 2 % alkali added produced 11% more methane compared to the Lignin C, which had only 1% alkali added. The lignin depolymerization is more uniform and the molecular weight of the lignin fragments can be expected to be lower at 2% NaOH as compared to the 1% (Rodriguez et al., 2017), which could explain why the methane yield is higher under these conditions. 3.3 Biogas Composition Methane contents of the biogas in all the reactors has been presented in Fig.3. During the start of experiment the methane contents were found to be very low in all of the reactors. But with the passage of time and as the bacteria started acclimizing the methane contents increased of the biogas produced. After that the methane contents in all of the reactors were stabilized the average methane contents were found to be 59±1, 59±1, 59±1, 60±1, 59±1% in R1 to R5, respectively. After starting the feeding with lignin, the methane percentage started decreasing in all of the reactors, while the methane percentage remained constant in the reactor fed with clarified manure. The average methane contents from 28-95 days was found to be 62.6±1% in the R5 where as in R1,R2,R3 and R4 average methane contents were found to be 54±2,54±2,55±2 and 55±2%, respectively. Overall R5 showed 13.7% higher methane content as compared to R1 and R2 and 12% higher methane contents as compared to R3 and R4.The lower methane contents in the rectors which were fed by lignin can be explained by the higher carbon content as compared to the R5. 3.4 Biodegradation of Substrate The compositional analysis of the solids in the feed and effluent after digestion was determined to understand which part of the raw material carbohydrates or lignin acid-solvable or insoluble fractions, which was converted during anaerobic digestion. As shown in Fig. 4, the volatile solids (VS) destruction for all five feed material i.e. manure, Lignin A, Lignin B, Lignin C and Lignin E was found to be 22.1, 26.9, 37.6, 43.1 and 46.3%, respectively, showing that the wet explosion 10
pretreatment increased the degradation of feed material and that the destruction of VS increased with increased severity of the wet explosion pretreatment. The volatile solids conversion (46.3%) in Lignin E with 2% NaOH in addition to oxygen is 72.1% increased compared to Lignin A, 23.1% increased compared to Lignin B, and 7.4% increased compared to Lignin C. It is also clear from the figure that carbohydrates (23%) are the main portion of the feed material that was converted into biogas in manure during the anaerobic digestion process. In contrast, in all the tested lignin materials i.e. Lignin A, Lignin B, Lignin C and Lignin E, lignin was the major component that was converted to methane with conversion of 32.1, 41.6, 48.8 and 56.3% of the lignin fraction, respectively. It is obvious from these findings that high lignin conversions demand a more severe pretreatment compared to the standard pretreatment used for producing cellulosic sugars from lignocellulosic materials. It was previously found that the wet explosion pretreatment makes significant changes to the lignin structures leading to a higher degree of methoxylation of the lignin molecule (Rana et al.,2015; Ahring et al 2015). Recently, we further discussed the importance of methoxylation of the lignin for biodegradation (Khan and Ahring, 2019). The methoxy-groups was found to be the principal point of attack during microbial degradation of lignin and demethoxylation was the initial process followed by ring cleavage of the aromatic ring of the lignin molecule. It is obvious to assume that the secondary wet explosion further increase the methoxylation of the lignin molecule in excess of the parts, which were methoxylated during the primary wet explosion resulting in even higher methane yield compared to lignin A. The addition of NaOH is known to depolymerize lignin and reduce the molecular weight of the lignin fragments (Rodriguez et al.,2017). Previous studies on anaerobic digestion of smaller fragments of lignin showed that anaerobic bacteria are capable of utilizing the smaller fragments of lignin and convert them into biogas (Jayasinghe et al., 2011). 3.5 Correlation between lignin degradation and methane yield The correlation between methane yield and lignin degradation is shown in Fig.5. As can be seen the methane yield increased linearly with the amount of lignin degradation as previously found by Zhao et al 2014. The results shows that the pretreatment has improved the lignin degradation. The lignin is partly depolymerized during wet explosion pretreatment due to the breakage of the abundantly available beta-aryl ether bonds between the phenyl propanoid units (Biswas et al 2014). The phenolic and non-phenolic lignin fragments can be accessible for further degradation by anaerobic bacteria resulting in increased methane production during the AD process as shown by Yokoyama and Matsumoto, 2010. The primary goal of wet explosion pretreatment is to reduce the recalcitrance of lignocellulosic materials and another important goal is to make changes to the lignin structure, so that cellulose degradation can occur ether by the action of enzymes, or as in this study, due to anaerobic digestion (Khan and Ahring, 2019). During the wet explosion pretreatment of lignocellulosic materials, hemicellulose and parts of the lignin solubilizes while the cellulose fraction remain conserved with minimum structural changes. 11
3.6 Elemental analysis and Methoxyl contents The elemental analysis of the feed and effluent lignin samples is shown in Table 2. As can be seen, carbon is significantly consumed in all of the reactors receiving lignin samples, whereas the manure sample showed far lower carbon consumption. Moreover, the methoxyl group’s percentage was found to be higher in all of the pretreated lignin samples as compared to the clarified manure. This is in accordance with previous findings that the wet explosion pretreatment increases methoxylation of lignin (Rana et al., 2015).The methoxylation of lignin was further found to have increased significantly during the secondary wet explosion pretreatment. The highest degree of methoxylation was found in lignin E. The fact that the largest fraction of lignin has been converted (56.3%) in Lignin E can be explained by the increase of methoxyl groups within this lignin sample. Previously it has been found that the methoxyl groups in lignin can be increased with addition of NaOH (up to 5%)- while higher percentage of NaOH results in decrease of methoxyl groups (Hidayati et al.,2018). The demethoxylation of lignin during anaerobic digestion was also found to be highest for Lignin E (49.54%) as compared to the other lignin samples tested. The highest degree of demethoxylation found in lignin E correlate with the fact that demethoxylation is the primary way for attacking lignin by anaerobic bacteria (Khan and Ahring 2019). The correlation between lignin degradation and demethoxylation has been shown in Fig.5. As shown in the Figure, demethoxylation and lignin degradation are highly correlated in accordance with the general understanding of anaerobic lignin degradation (Wu and He,2013). 3.7 Volatile Fatty Acids Analysis of volatile fatty acids (VFA) was carried out weekly to assess the performance of all the reactors and the data are presented in Table 3. The analysis showed a considerable amount of fatty acids in the feed material but the effluent analysis showed a stable low concentration of VFA in the effluents of all bioreactors throughout the experiment. The VFA concentration for the pretreated lignin with 2% NaOH was found to be considerably higher as compared to the other lignin feed materials. The higher concentration of VFAs in this feed material was due to the higher solubilization during pretreatment with the highest addition of NaOH tested. The results of the VFA concentration in the feed and effluent are comparable with the previous study of Ahring et al.,2015. Conclusion This study demonstrates that biorefinery lignin can be a good substrate for biogas production. The wet explosion pretreatment can increase the biodegradability of the lignin especially when alkali is added in addition to oxygen. Increased severity of wet explosion pretreatment with base addition (2%) increased the biogas yield and lignin conversion (56.3%) during AD. Wet 12
explosion pretreatment resulted in an increase in the methoxylation of the lignin and a clear correlation was found between lignin degradation and degree of methoxylation. The highest degree of methoxylation was found for lignin E, which further was found to have the highest degradation and methane yield. Lignin E produced 106.2% more methane compared to clarified manure alone.
Acknowledgement. This work was supported by a grant from Easterday Farm to support research on Anaerobic Digestion and from The Government of Punjab in collaboration with University of Agriculture, Faisalabad, Pakistan to Muhammad Usman Khan for studies abroad.
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Tables: Table.1.Compositional Analysis of Feed and Effluent Samples Lignin A Feed
Lignin B
Lignin C
Lignin E
Manure
Effluent Feed
Effluent Feed
Effluent Feed
Effluent Feed
Effluent
Carbohydrates (% 42.3 of VS)
46.4
37.9
42
37.7
44.1
35.8
48.61
86.7
82.8
Acid Soluble 1.8 Lignin (% of VS)
1.4
1.7
1.2
1.6
1.1
1.8
1.09
2.7
1.6
Acid Insoluble 55.9 Lignin (% of VS)
52.2
60.4
56.8
60.7
54.8
62.4
50.3
10.6
15.6
Lignin: Carbohydrates
1.15
1.63
1.38
1.65
1.26
1.79
1.05
0.15
0.20
1.36
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Table.2. Elemental analysis and Methoxyl contents of Feed and Effluent samples
Clarified Manure Lignin A Lignin B Lignin C Lignin E
C(%)
H(%)
N(%)
O(%)
S(%)
-OCH3 (%)
Demethoxylation (%)
Feed Effluent
37.1 36.6
3.1 2.8
2.8 2.3
57 58.3
0 0
13.7 12.9
5.6
Feed Effluent Feed Effluent Feed Effluent Feed Effluent
40.5 37.6 41 38 41.5 38 42.4 38.2
5.9 4.7 6.3 4.3 6.8 3.4 6.9 3
1.6 1.1 2.1 1.3 2.1 1.2 1.5 1.3
52 56.5 50.5 56.3 49.5 57.3 49.1 57.5
0 0 0 0 0 0 0 0
23.3 20.0 24.6 18.3 26.3 15.1 26.7 13.4
18
14.22 25.58 42.51 49.54
Table.3. Volatile fatty acids analysis for the feed and effluent Samples
Acetic Acid (g/L) Propionic Acid(g/L) Isobutyric Acid (g/L) Butyric Acid (g/L) Isovaleric Acid (g/L) Valeric Acid (g/L) Isocaproic Acid (g/L) Hexanoic Acid(g/L) Heptanoic Acid(g/L) Formic Acid(g/L)
Manure Feed 1.6
Effluent n.a
Lignin A Feed Effluent 1.8 0.3
Lignin B Feed Effluent 3.4 0.193
Lignin C Feed Effluent 2.1 0.6
Lignin E Feed 3.4
Effluent 0.6
n.a
n.a
1.9
0.1
1.8
0.1
3.2
0.1
3.5
0.3
0.3
n.a
0.3
n.a
0.1
n.a
1
n.a
0.7
0.5
0.4
n.a
0.6
n.a
0.2
n.a
3.2
n.a
1.6
n.a
0.3
n.a
0.3
n.a.
0.1
n.a
0.9
n.a
0.6
n.a
n.a
n.a
0.1
n.a
n.a
n.a
0.7
n.a
0.2
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a.
n.a
0.3
n.a
n.a
n.a
0.1
n.a
n.a
n.a
0.4
n.a
0.4
n.a
n.a
n.a
n.a.
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a.
n.a
n.a
n.a
n.a
n.a
n.a
m.a
n.a
n.a
19
Figure Captions Fig.1 Wet Explosion pretreatment of lignin Fig.2. Effect of Wet Explosion pretreatment on methane yield Fig.3 Methane content of biogas Fig.4. Volatile solids, carbohydrate and lignin composition for the feed and effluent samples. The results are an average of 2 replicates. Fig.5 Correlation between lignin degradation, demthoxylation and methane yield
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To whom it may concern
The Authors hereby declare that the work presented herein is original and is not published anywhere. They have no conflict of interest. Sincerely, Birgitte K. Ahring, Ph.D. Battelle Distinguished Professor Bioproducts, Sciences and Engineering Laboratory, BSEL
Highlights
Wet Explosion Pretreatment of Biorefinery Lignin Increased methoxylation of lignin increased lignin degradation The addition of NaOH and O2 increased lignin degradation A total of 56.3% lignin was converted in to methane
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S0960-8524(19)31767-5 https://doi.org/10.1016/j.biortech.2019.122537 BITE 122537
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
1 October 2019 27 November 2019 29 November 2019
Please cite this article as: Usman Khan, M., Kiaer Ahring, B., Anaerobic digestion of biorefinery lignin: effect of different Wet Explosion pretreatment conditions, Bioresource Technology (2019), doi: https://doi.org/10.1016/ j.biortech.2019.122537
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Anaerobic digestion of biorefinery lignin: effect of different Wet Explosion pretreatment conditions Muhammad Usman Khana, Birgitte Kiaer Ahringa,b* aBioproducts,
bGene
Sciences and Engineering Laboratory, Washington State University, Tri-Cities, Richland, WA-99354
and Linda Voiland School of Chemical Engineering and Bioengineering & Biological Systems Engineering, Washington State University, Pullman, WA-99163
Corresponding author Dr. Birgitte K. Ahring Bioproducts Sciences and Engineering Laboratory Washington State University, Tri-cities 2710 Crimson Way Richland, WA 99354 Tel.: 01-(509)-372-7682 Fax: 01-(509)-372-7690. E-mail address: [email protected]
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Abstract This study examine ways to make biorefinery lignin accessible for anaerobic digestion. The raw material was the residue after removing carbohydrates by Wet Explosion pretreatment at 190oC and 7.5% O2 followed by enzymatic hydrolysis. The residual solid was mainly composed of lignin and was the raw material for a second WEx pretreatment operated at 220oC with 4 % oxygen and variable concentrations of NaOH (0-2%). Lignin B was the residue after pretreated without NaOH, Lignin C was pretreated at 1% NaOH, and Lignin E at 2% NaOH. Anaerobic digestion was carried out on all lignin fractions (Lignin A, B, C and E) at thermophilic conditions (52oC) by mixing 70% of each lignin fractions with 30% clarified manure. The results showed that the lignin samples were demethoxylated as part of the biodegradation and that the highest severity pretreatment (with oxygen and 2% NaOH) resulted in the highest methane yield. Key Words: Biorefinery lignin, Anaerobic Digestion, Lignin degradation, Pretreatment
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Introduction Currently, 80% of the global energy demand is being fulfilled by fossil fuels (Guo et al., 2015). The global energy consumption was 5.5 × 1020 J in 2010, which is predicted to increase to 6.6 × 1020 J in 2020 and 8.6 × 1020 J in 2040 (Guo et al., 2015). The continuously increasing use of conventional fossil fuels not only increase the environmental pollution but is also causing an increase in global warming (Bahrami and Abbaszadeh, 2013). Production of biofuels and bioenergy from abundantly available plant biomass can assist in a reduction of the problems linked to fossil fuels consumption. The global annual production of plant biomass is 200 × 1011 tons/year, which can produce 2.2 × 1021 J of energy. This energy is 3-4 times higher than the current global energy demand (Guo et al., 2015). Lignocellulose is one of the most abundant and renewable biomass resources on the earth which makes it an ideal feedstock for production of biofuels and bioproducts. It is mainly composed of lignin, cellulose and hemicellulose. Lignin is the second most abundant natural polymer and the only abundant aromatic polymer in nature. In the plant cell wall lignin is associated with cellulose and hemicellulose and its percentage varies from 10-30% in the lignocellulosic biomass (Asgher et al., 2014). The paper and pulp industry alone further produces about 50 million tons of lignin but currently the lignin is only used in major amount for the production of low value products such as production of steam, binders or dispersants in cement and gypsum blends. The cost of production of high-value products like vanillin is still high and the product yield is low. Today, only 2% of the available lignin from paper and pulp industry is being used for other purposes than as a low value fuel (Gosselink et al., 2004) . Besides this amount the global cellulosic biofuels industry is expected to produce a huge amount of lignin ca. 225 million tons per year) (Jung et al., 2015; Sahoo et al.,2011). A sustainable and cost-effective method is needed to tackle the large amount of lignin, which would be produced from second generation biofuel production both for improving the economics of the biofuels as well as for reduction in the greenhouse gas emission. Until now the valorization and conversion of lignin residues have proven to be challenging within conventional paper and pulp industries and no viable solutions have been implemented for lignin from production of second generation biofuels(Vishtal and Kraslawski, 2011). One solution which has been investigated in larger detail is the conversion by pyrolysis to produce bio-oil and chemicals by heating the lignin in an oxygen-free environment at a temperature of about 220-400oC (Lu et al .,2019). The need for upgrading of the products from pyrolysis is, however, very energy intensive and costly and the yield of final product is generally low (Pham et al., 2015). Anaerobic digestion of lignin into useful methane could potentially be a useful and cheap solution with far higher conversion rates compared to pyrolysis. Anaerobic digestion of 3
lignin can also provide energy for operating the whole biorefinery as well as a raw material for producing hydrogen for further upgrading in the biorefinery (Sawatdeenarunat et al.,2015) Anaerobic digestion is a mature and reliable technology used widely for handling different types of organic waste and biomass materials with the production of bioenergy in the form of biogas(Chen et al., 2008). The complex organic compounds in the biomass raw material are converted to biogas by the concerted action of several groups of microorganism, performing all from hydrolysis of the polymers to acidogenesis, acetogenesis, and methanogenesis (Ali Shah et al., 2014). Methane (50-75%) and carbon dioxide (25 to 50%) are the main constituents of the biogas and their amounts vary by the composition of the feed stock and the operating conditions of the anaerobic process (Hosseini and Wahid, 2014). It is generally believed that only the cellulose and hemicellulose fraction of lignocellulosic materials is converted to biogas during anaerobic digestion, while the lignin fraction remains left behind. Still, lignin degradation has been observed in many anaerobic natural environments (Ko et al.,2009; DeAngelis et al.,2011; DeAngelis et al.,2013). Lignin degradation has, however, not been studied broadly in anaerobic digesters. Lignocellulosic materials such as straw and deep litter are increasing used as a feedstock for biogas plants. However, the limited hydrolysis of this substrate is a major challenge (Tsapekos et al., 2019). Different types of biomass pretreatments are increasingly being researched for increasing the hydrolysis of these materials to produce a higher methane yield (Yu et al.,2019). Overall, these pretreatments are found to increase the biogas yield (Khalid et al., 2019; Hashemi et al.,2019) but lignin degradation during anaerobic digestion was not observed or examined. Moreover, most of the pretreatments of lignocellulosic materials will include costly chemicals, which further results in changes to the biomass, which can produce hazardous waste, which needs to be managed carefully to avoid environmental pollution (Carrere et al.,2016; Paudel et al.,2017). Wet explosion (WEx) is a thermochemical pretreatment process of biomass with oxygen addition, where the wet oxidation process is combined with steam explosion. This pretreatment process has shown good performance for destruction of the structure of lignocellulosic materials along with good conversions during enzymatic hydrolysis(Biswas et al., 2012; Rana et al., 2012). Wet explosion pretreatment have many advantages for use as part of anaerobic digestion compared to other pretreatments due: (1) this pretreatment mainly attack the lignin structure, which is the major recalcitrant part of the biomass under anaerobic conditions, (2) During the pretreatment of the feedstock, there is no need for addition of any other chemicals to run the process except from oxygen and water, (3) After the pretreatment process, no recycling and recovery of added chemicals is required, (4) Energy requirement of the process is low because the oxygen used for the process generates heat during its reaction at 170oC or above (Rana et al.,2012). Wet explosion pretreatment is also cheaper compared to the other process because the pretreatment of lignocellulosic materials can be carried out at higher concentrations of dry 4
matter, resulting in a higher throughput through the system (Ahring and Munck, 2014). This pretreatment will, further, not use any hazardous chemicals reducing potential extra cost for treatment or disposal of any hazardous waste (Rana et al., 2012). The economy of the process is improved compared to dilute acid pretreatment and AFEX, which require a proper system for the recycling and recovery of sulfuric acid or ammonia (Sendich et al., 2008;Martin et al.,2019). The WOEx method of for pretreatment of lignocellulosic biomass was patented in 2014 (Ahring and Munck 2014) and was further developed into an optimized continuous process patented in 2016 (Merrill and Petersen 2016). Importantly, the WEx process has previously been found to make lignin accessible during anaerobic digestion (Ahring et al.,2015). The lignin degradation was explained in the light of the extensive methoxylations within the lignin molecule during WEx pretreatment (Rane et al 2015, Khan and Ahring 2019). In the current study we will examine the optimal conditions for WEx pretreatment to improve lignin degradation during anaerobic digestion including the effect of NaOH in addition to oxygen during pretreatment. 2. Methods 2.1. Feed Material and Inoculum Wheat straw and clarified manure were collected from Five D Farms Pasco Washington. The clarified manure is manure, which has been screw pressed to remove particular solids and five 2L anaerobic reactors were started on this material under thermophilic conditions with a hydraulic retention time of 20 days. The wheat straw was dried at ambient temperature for two weeks until the moisture contents were below 10%, after which it was grinded to reduce the particle size to 6 mm before it was pretreated. The wheat straw used for the experiment consists of 35.9% glucan, 23.9% xylan and 2.6% arabinan of the total weight (oven dry basis). Moreover, wheat straw also contains 2.4% acetyl group and 17.7 % total lignin (acid soluble and insoluble). Further, the concentration of cell wall extractives, that are soluble in water and ethanol, was determined to be 9.1% in this raw material. 2.2 Wet explosion pretreatment and enzymatic hydrolysis The down-sized wheat straw was pretreated using a pilot scale wet explosion pretreatment system. Wet explosion pretreatment of wheat straw was carried at optimized conditions (190oC, 7.5%O2, 25 min) for maximum sugar production at high solid concentration (25% DM) using a 100 L stainless steel pretreatment reactor. After pretreatment the material was enzymatically hydrolyzed without removal of liquid fraction using 15L Applikon Bioreactor with the addition of cellulolytic enzymes: Cellic®CTec2:27.8 mg protein/g glucan Cellic®HTec2;10% of CTec2 (Novozymes, USA), respectively. The enzymatic hydrolysis was done for 90 hrs at pH 5 at 50oC. The final biorefinery lignin stream (Lignin A) was separated using a combination of water 5
washing and solid liquid separation leading to a liquid fraction composed of high concentrations of sugars such as glucose and some xylose along with the solid Lignin A fraction mainly composed of lignin and some residual cellulose besides minor concentrations of hemicellulose. Lignin A was then further wet exploded at 220oC for 10 minutes including 4% O2 with different concentrations of alkali (0, 1 & 2 wt%) to examine the effect of further attack to the lignin’s aromatic structure. These lignin fractions were named as B, C and E, respectively, as shown in Fig.1 . All lignin fractions A-E were used as feed materials for anaerobic bioreactors to test the influence of the different pretreatment conditions on anaerobic digestion of the materials. 2.3. Experimental set-up and Feeding Strategy for the Anaerobic Bioreactors The anaerobic digestion of the biorefinery lignin was carried out using 2 L glass bioreactors operated semi-continuously using anaerobically digested clarified manure as an inoculum from an active laboratory bioreactor. The reactors were operated under thermophilic conditions (52oC) with a hydraulic retention time of 20 days and with one daily feeding at a fixed time. The four pretreated lignin fractions (A-E) were diluted by adding 30% clarified manure to the pretreated lignin samples to ensure that sufficient nutrients were present during the experiment. A bioreactor was further fed with 100% clarified manure to examine the biogas production from this raw material. Gas production and pH was registered daily. 2.4. Characterization of the Feed Stock Total solids (TS), Volatile solids and ash content of all types of lignin feed were analyzed using NREL standard methods (Sluiter et al., 2008). The feed material was taken in pre-dried crucibles and placed in a convection drying oven at a temperature of 105±3 oC for 24h. The dried samples were taken out from the oven after 24 hours and placed in a desiccator to allow them to cool to room temperature. The crucibles were weighed after 20 minutes of cooling and the weight was recorded. After weighing all the crucible containing dried samples were again placed in convection oven so that they can be dried to constant weight. After one hour the samples were removed again from the oven and weighed for the total solids. The ash contents of the material was determined by heating the crucibles at a temperature of 575±25 in a muffle furnace for 6 hours according to the method described by (Wychen and Laurens 2013). After 6 hours the samples were removed from the muffle furnace and placed in desiccator and allow them to cool to the room temperature. The weight of the cooled samples were recorded for volatile solids determination. 2.5 Compositional Analysis The feed and effluent samples were dried thoroughly for the determination of total lignin (acid soluble and Acid Insoluble) and total carbohydrates according to the NREL standard method described by Sluiter et al., 2008 The total carbohydrates (sum of glucose, xylose and arabinose) 6
and the Acid Insoluble lignin were measured after two-step hydrolysis, whereas the Acid soluble lignin was determined using a UV spectrophotometer (Jenway 6405 UV/Visible,NJ,USA) with a wavelength of 320nm. Monomers were analyzed by using an UltiMate® 3000 HPLC system (Dionex, Sunnyvale, CA) with an Aminex® 87H Column 250 × 4.6 mm (Bio-Rad, Hercules, CA) and a Shodex RI-101 refractive index detector at 60 °C using 4 mmol H2SO4 as an eluent with a flow rate of 0.6 mL/min. All the measurements of lignin and carbohydrates analysis were done in duplicates. 2.6 Gas Analysis Analysis of the biogas composition from all the bioreactors was done on a daily basis. The gas samples were taken from the gas valves provided in gas pipeline of each of the reactor using a syringe. The collected gas sample was then analyzed by using Universal Gas Analyzer, UGA series (Stanford Research Systems, Sunnyvale, CA). 2.7 Volatile Fatty Acid Analysis High performance liquid chromatography (HPLC) was used for the volatile fatty acid analysis of the feed and effluent samples. About 2ml of the sample was taken in an eppendorf tube and centrifuged for 10 min at 10,000 rpm. After centrifugation the supernatant was separated using a pipette and then it was diluted 2 times using 4mM sulfuric acid and the diluted samples were filtered through 0.2 µm filter using a 3 ml syringe. The filtered samples were analyzed using an UltiMate ® 3000 HPLC system (Dionex,Sunnyvale,CA) with an Aminex ® 87 H column 250×4.6 mm (Bio-Rad, Hercules,CA) and a Shodex RI-101 refrective index detector. The flow rate of the eluent (4mM Sulfuric acid) flowing through 87H column and the oven temperature were kept constant at 0.6 ml/min and 60oC respectively as done in the previous study (Murali et al., 2017). The total time for the VFA analysis of the feed and digested lignin and manure samples was 68 min. 2.8 pH Analysis The pH of the effluent from all the reactors was monitored regularly during the whole experiment. The pH was measured by Oakton pH-510 pH meter. The pH was measured immediately after removing samples from the reactors to avoid any changes of pH during storage. Between each measurement the pH electrode was washed with deionized water and then wiped with paper towel. 2.9 Elemental Analysis and Methoxyl groups Content The Carbon ©, Hydrogen (H), Sulfur (S) and Nitrogen (N) analysis of each of the lignin sample was carried out by using TRUSPEC-CHN® elemental analyzer (LECO, U.S.). The elemental analysis was done by using by using 0.15 g of the lignin sample. The percentage of oxygen was 7
determined by subtracting the percentage of C, H, N and S from 100%.The methoxyl groups in feed and effluent samples were found by using eq.1 as was determined by (Jablonsky et al.,2015). OCH3 = ―18.5769 + 4.0658(H) + 0.34543(O)
(1)
The demethoxylation (%) was determined by using eq.2 given below Demethoxylation (%) =
(OCH3)Feed ― (OCH3)Eff (OCH3)Feed
(2)
3. Results and discussion 3.1 Substrates Characteristics The compositional analysis of the different lignin feed materials (A-E) before anaerobic digestion and after digestion (effluent determinations) has been presented in Table.1. The table shows that about 3/5 of the organic matter in all the feed materials was composed of lignin in which only 3.1% is acid soluble in lignin A, 2.7% in lignin B, 2.6% in lignin C, and, 2.8% lignin E. In comparison, only 1/10 of the organic matter in manure was lignin in the clarified manure of which 20.3% was acid-soluble lignin, while the rest was acid insoluble. Acid insoluble is the higher molecular weight (HMW) lignin and its molecular weight is generally over 850kD. The anaerobic digestion of this type of lignin is generally difficult unless it has been modified to allow for microbial attack. Anaerobic microorganisms cannot produce the radicals and enzymes, which can attack large lignin molecules, but need pretreatment to make necessary modifications to the molecule such as methoxylations. When the lignin molecule is methoxylated, these points can be attacked by the microbial communities operating in the anaerobic bioreactor (Khan and Ahring,2019;Ko et al., 2009). The percentage of Acid insoluble lignin in Lignin A was found to be 55.9% (VS basis) while the further pretreated lignin B,C and E showed higher percentage of acid insoluble lignin accounting for 60.4, 60.7 and 62.4 on a volatile solid basis. The manure showed only 10.6% Acid insoluble lignin. The percentage of Acid soluble lignin was found to be almost similar in both primary and secondary wet exploded lignin in the range of 1.6-1.8 (% of VS) while the manure was found to have a somewhat higher 2.7% (% of VS) percentage of Acid soluble lignin as compared to the primary and secondary wet exploded lignin. The percentage of carbohydrates was found to be 42.3%, 37.9%, 37.7 % and 35.8% in lignin A, B, C and E, respectively, whereas the manure had 86.7% carbohydrates on a VS basis.
8
The lignin to carbohydrate ratio in all five types of feed materials i.e. Lignin A, Lignin B, Lignin C, Lignin E and clarified manure was found to be 1.36, 1.63, 1.65, 1.79, and 0.15, respectively. The lignin to carbohydrate ratio by the compositional analysis of effluent from all the reactors i.e. Lignin A, Lignin B, Lignin C , Lignin E and Manure was observed to be 1.15,1.38, 1.26, 1.05 and 0.20 which showed that the lignin to carbohydrate ratio in the effluent was significantly reduced in all four types of lignin, while it was increased in the manure feed. These results show that lignin is being converted to methane in all types of lignin samples, whereas the carbohydrate is the main source being converted to methane in the manure feed. The results are comparable with a previous from the study of Ahring et al.,2015 where significant amount of wet exploded lignin was converted to methane as compared to non-pretreated lignin. Our results further showed that the maximum reduction in lignin to carbohydrate ratio was observed in Lignin E, which was secondary wet exploded with 2% NaOH. This was probably due to the fact that lower molecular weight lignin fragments is produced when higher concentrations of NaOH (2%) is added compared to lower concentrations of NaOH (1%) leading to a better bioconversion. The finding that lignin depolymerization is more efficient at higher NaOH concentrations has been previously described by Rodriguez et al.,2017. 3.2. Biogas Potential of the Feed Materials Methane yields from all five semi-continuous bioreactors (R1 to R5) has been presented in Fig.2 (moving average of 7 days). The reactors R1, R2, R3 and R4 were fed with Lignin A, Lignin B, Lignin C and Lignin E along with 30% clarified manure; R5 was only fed with clarified manure. During the whole experiment the methane production from all the reactors was measured on daily basis. During startup of the experiments, all of the five reactors were only fed with the clarified manure until stable and similar gas production was recorded from all of reactors. Afterwards, the reactors were fed with 70% lignin (A-E) and 30% clarified manure. Methane yield varied during the initial period of experiment (day1-22) from 5 to 94 ml/gVS/day with average values of 35.9±30, 37.8±32, 37.5±31, 38.7±33 and 40.1±32 ml/gVS/day in R1 to R5, respectively. After the gas production stabilized in all the reactors the average methane production from 23-26 days was found to be 93.7±1, 93.9±1, 93.9±0.6, 93.2±0.54 and 92.6±0.92 ml/gVS/day in R1 to R5, respectively. From 27th day Lignin A, Lignin B, Lignin C and Lignin E with 30% clarified manure and 100% clarified manure were used as a feed material in R1 to R5, respectively. The methane yield from the reactors started varying after addition of lignin. The methane yield from all the reactors which were fed by lignin stabilized after 64 days resulting in an average methane yield from R1 to R4 of 128.7±2, 146.4±2, 176.3±2.5,195.4±2.3 respectively, while the R 5 reactor with only clarified manure gave a methane yield of 94.8±2. All the reactors fed with different wet exploded lignin in addition to clarified manure produced higher methane yield compared to the reactor, which were only fed with clarified manure. There was significant differences (T-test) between Reactor 1 (Lignin A), Reactor 2 (lignin B), Reactor 3 (lignin C) and Reactor 4 (lignin E) yielded 36, 54.5, 86 and 106.2% more methane as 9
compared to Reactor 5, which only received clarified manure. The wet exploded lignin showed higher methane yield due to the fact that the small fractions of hemicellulose and parts of the lignin solubilized during the wet explosion pretreatment and could be converted along with some of the solid lignin (Khan and Ahring, 2019). Overall, the secondary wet explosion pretreatment was found to increase the methane yield and the lignin B produced 14% more methane as compared to the primary wet exploded lignin A. The alkali assisted wet exploded lignin showed even higher methane yield as compared to just wet explosion with oxygen at higher temperatures, and Lignin C and Lignin E produced 37 and 52% higher methane yield compared to the lignin A. Moreover the addition of base during the secondary wet explosion pretreatment was found to increase the methane yield and lignin C and E produced 21 and 33% more methane as compared to the lignin B, which was pretreated without any base. The higher concentration of alkali during pretreatment was found beneficial for increasing the methane yield and Lignin E with 2 % alkali added produced 11% more methane compared to the Lignin C, which had only 1% alkali added. The lignin depolymerization is more uniform and the molecular weight of the lignin fragments can be expected to be lower at 2% NaOH as compared to the 1% (Rodriguez et al., 2017), which could explain why the methane yield is higher under these conditions. 3.3 Biogas Composition Methane contents of the biogas in all the reactors has been presented in Fig.3. During the start of experiment the methane contents were found to be very low in all of the reactors. But with the passage of time and as the bacteria started acclimizing the methane contents increased of the biogas produced. After that the methane contents in all of the reactors were stabilized the average methane contents were found to be 59±1, 59±1, 59±1, 60±1, 59±1% in R1 to R5, respectively. After starting the feeding with lignin, the methane percentage started decreasing in all of the reactors, while the methane percentage remained constant in the reactor fed with clarified manure. The average methane contents from 28-95 days was found to be 62.6±1% in the R5 where as in R1,R2,R3 and R4 average methane contents were found to be 54±2,54±2,55±2 and 55±2%, respectively. Overall R5 showed 13.7% higher methane content as compared to R1 and R2 and 12% higher methane contents as compared to R3 and R4.The lower methane contents in the rectors which were fed by lignin can be explained by the higher carbon content as compared to the R5. 3.4 Biodegradation of Substrate The compositional analysis of the solids in the feed and effluent after digestion was determined to understand which part of the raw material carbohydrates or lignin acid-solvable or insoluble fractions, which was converted during anaerobic digestion. As shown in Fig. 4, the volatile solids (VS) destruction for all five feed material i.e. manure, Lignin A, Lignin B, Lignin C and Lignin E was found to be 22.1, 26.9, 37.6, 43.1 and 46.3%, respectively, showing that the wet explosion 10
pretreatment increased the degradation of feed material and that the destruction of VS increased with increased severity of the wet explosion pretreatment. The volatile solids conversion (46.3%) in Lignin E with 2% NaOH in addition to oxygen is 72.1% increased compared to Lignin A, 23.1% increased compared to Lignin B, and 7.4% increased compared to Lignin C. It is also clear from the figure that carbohydrates (23%) are the main portion of the feed material that was converted into biogas in manure during the anaerobic digestion process. In contrast, in all the tested lignin materials i.e. Lignin A, Lignin B, Lignin C and Lignin E, lignin was the major component that was converted to methane with conversion of 32.1, 41.6, 48.8 and 56.3% of the lignin fraction, respectively. It is obvious from these findings that high lignin conversions demand a more severe pretreatment compared to the standard pretreatment used for producing cellulosic sugars from lignocellulosic materials. It was previously found that the wet explosion pretreatment makes significant changes to the lignin structures leading to a higher degree of methoxylation of the lignin molecule (Rana et al.,2015; Ahring et al 2015). Recently, we further discussed the importance of methoxylation of the lignin for biodegradation (Khan and Ahring, 2019). The methoxy-groups was found to be the principal point of attack during microbial degradation of lignin and demethoxylation was the initial process followed by ring cleavage of the aromatic ring of the lignin molecule. It is obvious to assume that the secondary wet explosion further increase the methoxylation of the lignin molecule in excess of the parts, which were methoxylated during the primary wet explosion resulting in even higher methane yield compared to lignin A. The addition of NaOH is known to depolymerize lignin and reduce the molecular weight of the lignin fragments (Rodriguez et al.,2017). Previous studies on anaerobic digestion of smaller fragments of lignin showed that anaerobic bacteria are capable of utilizing the smaller fragments of lignin and convert them into biogas (Jayasinghe et al., 2011). 3.5 Correlation between lignin degradation and methane yield The correlation between methane yield and lignin degradation is shown in Fig.5. As can be seen the methane yield increased linearly with the amount of lignin degradation as previously found by Zhao et al 2014. The results shows that the pretreatment has improved the lignin degradation. The lignin is partly depolymerized during wet explosion pretreatment due to the breakage of the abundantly available beta-aryl ether bonds between the phenyl propanoid units (Biswas et al 2014). The phenolic and non-phenolic lignin fragments can be accessible for further degradation by anaerobic bacteria resulting in increased methane production during the AD process as shown by Yokoyama and Matsumoto, 2010. The primary goal of wet explosion pretreatment is to reduce the recalcitrance of lignocellulosic materials and another important goal is to make changes to the lignin structure, so that cellulose degradation can occur ether by the action of enzymes, or as in this study, due to anaerobic digestion (Khan and Ahring, 2019). During the wet explosion pretreatment of lignocellulosic materials, hemicellulose and parts of the lignin solubilizes while the cellulose fraction remain conserved with minimum structural changes. 11
3.6 Elemental analysis and Methoxyl contents The elemental analysis of the feed and effluent lignin samples is shown in Table 2. As can be seen, carbon is significantly consumed in all of the reactors receiving lignin samples, whereas the manure sample showed far lower carbon consumption. Moreover, the methoxyl group’s percentage was found to be higher in all of the pretreated lignin samples as compared to the clarified manure. This is in accordance with previous findings that the wet explosion pretreatment increases methoxylation of lignin (Rana et al., 2015).The methoxylation of lignin was further found to have increased significantly during the secondary wet explosion pretreatment. The highest degree of methoxylation was found in lignin E. The fact that the largest fraction of lignin has been converted (56.3%) in Lignin E can be explained by the increase of methoxyl groups within this lignin sample. Previously it has been found that the methoxyl groups in lignin can be increased with addition of NaOH (up to 5%)- while higher percentage of NaOH results in decrease of methoxyl groups (Hidayati et al.,2018). The demethoxylation of lignin during anaerobic digestion was also found to be highest for Lignin E (49.54%) as compared to the other lignin samples tested. The highest degree of demethoxylation found in lignin E correlate with the fact that demethoxylation is the primary way for attacking lignin by anaerobic bacteria (Khan and Ahring 2019). The correlation between lignin degradation and demethoxylation has been shown in Fig.5. As shown in the Figure, demethoxylation and lignin degradation are highly correlated in accordance with the general understanding of anaerobic lignin degradation (Wu and He,2013). 3.7 Volatile Fatty Acids Analysis of volatile fatty acids (VFA) was carried out weekly to assess the performance of all the reactors and the data are presented in Table 3. The analysis showed a considerable amount of fatty acids in the feed material but the effluent analysis showed a stable low concentration of VFA in the effluents of all bioreactors throughout the experiment. The VFA concentration for the pretreated lignin with 2% NaOH was found to be considerably higher as compared to the other lignin feed materials. The higher concentration of VFAs in this feed material was due to the higher solubilization during pretreatment with the highest addition of NaOH tested. The results of the VFA concentration in the feed and effluent are comparable with the previous study of Ahring et al.,2015. Conclusion This study demonstrates that biorefinery lignin can be a good substrate for biogas production. The wet explosion pretreatment can increase the biodegradability of the lignin especially when alkali is added in addition to oxygen. Increased severity of wet explosion pretreatment with base addition (2%) increased the biogas yield and lignin conversion (56.3%) during AD. Wet 12
explosion pretreatment resulted in an increase in the methoxylation of the lignin and a clear correlation was found between lignin degradation and degree of methoxylation. The highest degree of methoxylation was found for lignin E, which further was found to have the highest degradation and methane yield. Lignin E produced 106.2% more methane compared to clarified manure alone.
Acknowledgement. This work was supported by a grant from Easterday Farm to support research on Anaerobic Digestion and from The Government of Punjab in collaboration with University of Agriculture, Faisalabad, Pakistan to Muhammad Usman Khan for studies abroad.
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Tables: Table.1.Compositional Analysis of Feed and Effluent Samples Lignin A Feed
Lignin B
Lignin C
Lignin E
Manure
Effluent Feed
Effluent Feed
Effluent Feed
Effluent Feed
Effluent
Carbohydrates (% 42.3 of VS)
46.4
37.9
42
37.7
44.1
35.8
48.61
86.7
82.8
Acid Soluble 1.8 Lignin (% of VS)
1.4
1.7
1.2
1.6
1.1
1.8
1.09
2.7
1.6
Acid Insoluble 55.9 Lignin (% of VS)
52.2
60.4
56.8
60.7
54.8
62.4
50.3
10.6
15.6
Lignin: Carbohydrates
1.15
1.63
1.38
1.65
1.26
1.79
1.05
0.15
0.20
1.36
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Table.2. Elemental analysis and Methoxyl contents of Feed and Effluent samples
Clarified Manure Lignin A Lignin B Lignin C Lignin E
C(%)
H(%)
N(%)
O(%)
S(%)
-OCH3 (%)
Demethoxylation (%)
Feed Effluent
37.1 36.6
3.1 2.8
2.8 2.3
57 58.3
0 0
13.7 12.9
5.6
Feed Effluent Feed Effluent Feed Effluent Feed Effluent
40.5 37.6 41 38 41.5 38 42.4 38.2
5.9 4.7 6.3 4.3 6.8 3.4 6.9 3
1.6 1.1 2.1 1.3 2.1 1.2 1.5 1.3
52 56.5 50.5 56.3 49.5 57.3 49.1 57.5
0 0 0 0 0 0 0 0
23.3 20.0 24.6 18.3 26.3 15.1 26.7 13.4
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14.22 25.58 42.51 49.54
Table.3. Volatile fatty acids analysis for the feed and effluent Samples
Acetic Acid (g/L) Propionic Acid(g/L) Isobutyric Acid (g/L) Butyric Acid (g/L) Isovaleric Acid (g/L) Valeric Acid (g/L) Isocaproic Acid (g/L) Hexanoic Acid(g/L) Heptanoic Acid(g/L) Formic Acid(g/L)
Manure Feed 1.6
Effluent n.a
Lignin A Feed Effluent 1.8 0.3
Lignin B Feed Effluent 3.4 0.193
Lignin C Feed Effluent 2.1 0.6
Lignin E Feed 3.4
Effluent 0.6
n.a
n.a
1.9
0.1
1.8
0.1
3.2
0.1
3.5
0.3
0.3
n.a
0.3
n.a
0.1
n.a
1
n.a
0.7
0.5
0.4
n.a
0.6
n.a
0.2
n.a
3.2
n.a
1.6
n.a
0.3
n.a
0.3
n.a.
0.1
n.a
0.9
n.a
0.6
n.a
n.a
n.a
0.1
n.a
n.a
n.a
0.7
n.a
0.2
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a.
n.a
0.3
n.a
n.a
n.a
0.1
n.a
n.a
n.a
0.4
n.a
0.4
n.a
n.a
n.a
n.a.
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a.
n.a
n.a
n.a
n.a
n.a
n.a
m.a
n.a
n.a
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Figure Captions Fig.1 Wet Explosion pretreatment of lignin Fig.2. Effect of Wet Explosion pretreatment on methane yield Fig.3 Methane content of biogas Fig.4. Volatile solids, carbohydrate and lignin composition for the feed and effluent samples. The results are an average of 2 replicates. Fig.5 Correlation between lignin degradation, demthoxylation and methane yield
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To whom it may concern
The Authors hereby declare that the work presented herein is original and is not published anywhere. They have no conflict of interest. Sincerely, Birgitte K. Ahring, Ph.D. Battelle Distinguished Professor Bioproducts, Sciences and Engineering Laboratory, BSEL
Highlights
Wet Explosion Pretreatment of Biorefinery Lignin Increased methoxylation of lignin increased lignin degradation The addition of NaOH and O2 increased lignin degradation A total of 56.3% lignin was converted in to methane
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