Effect of Inoculum Concentration and Pretreatment on Biomethane Recovery From Cotton Gin Trash

The potential of cotton gin waste, a considerable challenge to the gin owners, has not been fully investigated as a renewable energy source via anaerobic digestion. The weathered cotton gin trash and inoculum for triplicate biomethane potential assays were obtained from a local cotton gin mill and a municipal wastewater treatment plant, respectively. The moisture, total solids, volatile solids, and C, H, N, S, hemicellulose + cellulose, and lignin contents of gin waste were determined in triplicates. The biomethane potential of untreated and pretreated (hot water and 6% NaOH (wet CGT weight basis) gin waste was determined at different inoculum to substrate ratios. The highest cumulative biomethane yield of 111.8 mL gvs was observed in inoculum to substrate ratio of 2.3, and it was statistically similar to the values; 101.8, 104.7, 100.5, and 108.9 gvs, observed in 0.8, 1.2, 1.5, and 1.9, respectively. The biomethane yield at the inoculum to substrate ratio of 0.4 was significantly lower than all higher ratios. The T80-90 for biomethane production was 26-30 for the ISRs of 1.2, 1.5, and 2.3. The T80-90 for inoculum to substrate ratios of 0.4, 0.8, and 1.9 were 26-31, 27-32, and 27-31 d, respectively. The modified Gompertz equation fitted very well (R = 0.98-0.99) to the anaerobic digestion at all inoculum to substrate ratios and pretreatments as the observed and predicted biomethane values were similar. The model predicted a lag phase of 8-10 days for control and treatments compared to the observed of 10-15 days. The highest biodegradability of 24.8±2.6% was observed at inoculum to substrate ratio of 2.3, which was statistically similar to the values observed in ratios of 0.8, 1.2, 1.5, and 1.9, respectively. Among pretreatments, the highest biodegradability of 33.0±2.4 was observed in 6% NaOH pretreatment, and it was statistically similar to hot water treatment and non-pretreated or control. These research findings advance the knowledge in the anaerobic degradation of cotton gin trash, thus helping to maximize biomethane recovery from this agro-industrial waste.


Introduction
Cotton is one of the world's most important cultivated crops owing to its high-quality natural fiber. According to a recent statistical report, around 26.7 million metric tons of cotton were produced globally during the 2019-20 growing season, and the United States was the major exporter (Cotton Incorporated, 2020). Texas is a major cotton-producing state in the US; other US states producing cotton are Georgia, Arkansas, and Mississippi (Figures 1 and 2). The upland cotton (Gossypium hirsutum) is dominant throughout Texas. Pima cotton (G. barbadense), which is well adapted to the desert, is grown in far western Texas.  separate fiber ginning one b major cotton g upon the cotto nically harveste .1), seed (0-2. nt strategies ha the soil as hu heating value (H e et al., 2020). ney on dispos ude as a sour t of transparen g states, 2018-r, leaving behi ale of cotton y growing US sta on cultivar an ed, and it is m .9%), motes (2 ave been to us umus. The pre Holt, 2006) in . The proper di sing of CGT ( ce of micro a nt plastics (Haq -2020 Vol. 13, No.  However, the urgency to lower fossil fuel consumption had the scientists investigating agro-industrial wastes such as CGT as potential renewable energy sources (Zabaniotou et al., 2010). Consequently, CGT has been investigated as a bioenergy source through ethanol production, gasification, and anaerobic degradation (Hamawand et al., 2016). The high chemical oxygen demand (COD) and volatile solid (VS) contents in CGT can be recovered as biomethane through anaerobic degradation (Hamawand et al., 2016;Wilde et al., 2010).
Anaerobic degradation is widely adopted to convert organic matter into biomethane (USEPA, 2019; Guo et al., 2015). According to the United States Environmental Protection Agency (USEPA), as of October 2019, 287 anaerobic digesters were operating at the animal farms across the nation (USEPA, 2019). They were adopted by the farmers to curb methane emissions from animal farms (manures) as directed by EPA under the regulation of the National Pollutant Discharge Elimination System (USEPA, 2019). Biomethane potential (BMP) assays are used to determine a given substrate's suitability for anaerobic digestion. Anaerobic digestion relies upon the intricate balance of various bacterial groups which carry out the four distinct phases of hydrolysis (substrate break down to simple organic and amino acids), acidogenesis (conversion of simple organic and amino acids to volatile fatty acids, H 2 and CO 2 ), acetogenesis (volatile fatty acids are converted to CH 3 COOH) and finally methanogenesis (Meegoda et al., 2018). The microbial degradation is impacted by inoculum, substrate, experimental and operational conditions (Raposo et al., 2011). Some of the most important factors impacting the process; hence biomethane yield are inoculum to substrate ratio or ISR and substrate composition (Ntiamoah-Ohemeng & Datta, 2019; Raposo et al., 2011).
There are some studies (Cheng & Zhong, 2014;Adl et al., 2012;Isci & Demirer, 2007;Funk et al., 2005) that focused on the BMP and/or ISR of cotton wastes in mono-digestion or co-digestion with manures. Adl et al. (2012) determined the effects of pretreatment, inoculum source, and feed to inoculum ratio (F/I) on the BMP of cotton stalks. Cheng and Zhong (2014) investigated the effects of the F/I, pretreatment, and co-digestion (with swine manure) on the BMP of cotton stalks. Additionally, the authors fitted the modified Gompertz equation to the cumulative experimental data and reported a high correlation between experimental and predicted values. Funk et al. (2005) co-digested CGT with swine manure (the mixing ratios from 1:1-10:1) in a two-stage bioreactor. Isci and Demirer (2007) reported that the addition of basal media yields higher biomethane during the anaerobic digestion of cotton oil cake, seed hull, and stalks.
Once the particle size and crystallinity of the biomass are reduced via mechanical milling, several pretreatment methods are available to remove the lignin fraction of the biomass, as summarized by Hassan et al. (2018). Two of these methods are hot water and alkaline treatments, which remove soluble and whole lignin and hemicellulose fractions, respectively (Hassan et al., 2018). The effect of these pretreatments on BMP of CGT still has not been investigated.
The biomethane potential assays were conducted at different inoculum to substrate ratios utilizing untreated and pretreated (hot water and alkali) cotton gin trash as feedstock. The digester performance at each treatment was evaluated by simulating the process with mainstream mathematical models and comparing them with theoretical values.

Substrate and Inoculum
The aged CGT (from more than 3 months cotton ginning, Figure 3) was collected from Varisco-Court Gin Co.
(5354 Steel Store Rd, Bryan, TX, 77807). The samples were collected from weathered, transition, and core layers and mixed to obtain a uniform gin waste. It was passed through Willy mill, sieved (2 mm) to obtain small particles, and stored at 4 °C in the refrigerator. (1) Where, a = 390.0, b = 450.0, c = 290.8, d = 21.2, e = 0.6.

Biodegradability
The biodegradability (BD) of a substrate is its fraction that is converted to biomethane during anaerobic digestion. The BD of CGT was determined using cumulative biomethane yield (EBY) from the experimental and theoretical biomethane (EMY and TBY) as described by Raposo et al. (2011).

Biomethane Production Kinetics
The anaerobic degradation process or bacterial growth in the bioreactors can be described by fitting the modified Gompertz equation developed by Lay et al. (1996Lay et al. ( , 1997 as described below: Where, P(t) = The cumulated methane (mL g vs -1 , minus the blank) at digestion time t (days); P 0 = Maximum cumulative methane production (mL g vs -1 ); R m = Maximum daily rate of biomethane production (mL g vs -1 d -1 ); λ = lag phase (days), minimum time to produce biomethane; e = Mathematical constant 2.718.

Data Analysis
The experimental data were processed in Microsoft Excel 2010 (Microsoft, USA). The biomethane volume was converted to dry gas volume at STP by multiplying with a dry biomethane factor of 0.838 as described by Richards et al. (1991). The blank value was subtracted from the treatment values. All of the data were analyzed using the general linear model (GLM) and analysis of variance procedure of Statistical Analysis System (SAS® 9.2, SAS Institute Inc., Cary, NC, USA), and statistically significant treatment means were separated using the least significant difference (LSD) test at 5% probability.

Cotton Gin Trash Composition
Carbohydrates (hemicellulose + cellulose) were 56%, while lignin or acid-insoluble fraction was found to be 32.7% (Table 1). Note. All values, except moisture, TS, VS, and ash are mean±SD from triplicate percentages of total sample dry weight basis (w/w). Agblevor et al. (2006) reported 11.2, 37.1, and 21.7 to 27% ash, total carbohydrates, and acid-insoluble fractions in CGT collected from gin mills in the US. The CGT was collected from a pile that had been stored in the open in a local gin mill for 3 months.

Biomet
The princ determined for the gr (Figure 1) yields in b 0.0001) lo between 8 treatment p attributed t Among co NaOH, wh was 5.1±1 equation i Cheng and degradatio org Figure 5. Daily error bars repr ethane recover ne measured fr R, the recovere SR may have c s, 2004). The f the cumulativ Rs of 0.4, 0.8 a ne between 20r the continuo from cotton sta ne yield from mL g vs -1 for the 95.8-111.8 mL g vs -1 for cotton te with the obs ould be avoide

Biodegradability
Without the addition of inoculum, CGT is hardly degradable, and only 1.8±0.1% of the added biomass was recovered as biomethane (Table 2).
Lignin is one of the most recalcitrant components of the plant-based agro-wastes and is not easily degraded during anaerobic digestion . The lignin content of organic manures and energy crops, and animal manures is negatively correlated to their BMP (Kafle & Chen, 2016;Triolo et al., 2011). The CGT in this study had a lignin content of 32.7%, which on solubilization by alkali might have lead to higher BD (although not significant) in 6% NaOH treatment (Hassan et al., 2014). Shi et al. (2009) reported that a combination of fungal and alkali pretreatments in cotton stalks yielded higher biomethane by removing/softening recalcitrant biomass ingredients eg, xylan.

Conclusion
Our study reveals that for proper digestion of CGT the ISR should be more than 0.4. The pretreatments do not enhance biomethane yield and the modified Gompertz equation fits well with the anaerobic digestion of CGT. The gin trash biodegrades better with an increase in volatile solid loading rate from 1 to 7.9%. The T 80-90 for CGT at the ISRs of 1.2, 1.5, and 2.3 was 26-30 days, whereas T 80-90 at ISRs of 0.4, 0.8, and 1.9 were 26-31, 27-32, and 27-31, respectively. The 6% NaOH treatment significantly increased the biodegradability of cotton gin waste. These findings further enhance understanding of the underlying factors in the anaerobic digestion of CGT and will facilitate to maximize biomethane recovery from this agro-industrial waste.