Rapid hydrogen generation from cotton wastes by mean of dark fermentation

Dark fermentation of textile wastes is discussed in the paper. In the experiment cotton wastes were fermented. Before fermentation the cotton was hydrolyzed using 0.1 M HCl acidic solution. The inoculum was pretreated by means of heat shock for 0.5 h at 105 °C. The fermentation was carried out under mesophilic conditions at a load of 5 g VSS/L, and pH 5. Oxygen was added in small quantities during fermentation. The oxygen flow rates (OFR) were between 0.3 and 1.0 mL/h. The fermentation was carried out for a few days at temperatures between 40 and 43 °C. Hydrogenesis prevailed at the lower temperature (40 °C) and methanogenesis at the higher (43 °C). Conversion of cotton waste to methane (3.4%) was slightly higher than conversion to hydrogen (2.6%). The highest hydrogen production was obtained for OFR 0.8 mL/h and the percentage of hydrogen in biogas was 43%. At higher temperatures (43 °C) no hydrogen production was observed


Introduction
Cotton waste constitute a high proportion of textile wastes, which account for 50% of world biomass waste [1]. Its utilization, complying with demands of circular economy, can solve a problem especially in the Middle East, including Syria [2] but also in Europe [3]. Poland generates around 236 661 Mg of cotton waste annually, which is clearly a huge amount to process [4]. On the other hand, there exists a high demand for energy [5] and industrial raw materials [6], that increasingly will not be easily met by fossil resources [7]. So, the role of renewable fuels like hydrogen produced from biomass will grow continuously [8]. Cotton plantations can contribute to global world sustainability [9], oil from its seeds may serve as biofuel or lubricant [10,11], and cotton wastes may be transformed into biodiesel [12,13] or as an additive to other biofuels [14,15] with properties similar to FAME [16]. Cotton wastes are used for PET or insulating material production [17] or serve as important substrate for pyrolysis [16,17].
An interesting issue, undertaken in this research, is related to the possibility of using cotton waste, including cotton stalk [18] as a source of hydrogen. Fast depletion of fossil fuels [19] means that there is a growing need to seek renewable ways of hydrogen production, an important resource for chemistry [20] and carbon free fuel [8]. Taherzadeh works [2,21] proved that cotton wastes are a good source of methane generation, but the process includes expensive chemical pretreatment using NMMO, highly concentrated sulfuric acid or sodium hydroxide [2,22]. But the alternative dark fermentation (DF) process may lead to biohydrogen production [23]. DF process, a truncated anaerobic digestion (without final methanisation), converts substrates into gaseous products like hydrogen, carbon dioxide and volatile organic acids (e.g. butyric, acetic or propionic) [24].
According to initial assessments, cotton waste could be a potential source of hydrogen [25]. The aim of this research is to determine the optimal conditions for hydrogen production in the DF process, and its industrial scale viability, with a focus on the effect of temperature variation in the mesophilic range on hydrogen production from cotton waste. Similar studies were performed by Chandra et al. [26] for rice straw anaerobic fermentation. As in earlier research [27], related to sour cabbage fermentation, microaeration (which stimulates hydrogen production) was applied.
Lack of information on successful scaling up of DF processes [28] (except possibly [29] leads Carrillo-Reyes et al. [30] to state that even lower efficiency (15% of practical hydrogen yield) could be considered as feasible. In most cases the optimal dark fermentation process proceeds under acidic conditions (pH ~ 5.0), at mesophilic temperature and using stressed inoculum. However, for some substrates like potato waste [31] or cotton stalk, optimal conditions [18] are closer to pH neutral or basic. Therefore, it is important to clarify these differences and determine conditions (including pH, temperature, microaeration rate, etc.) that are optimal for hydrogen DF production for various available substrates and to scale up process into profitable range. Besides, it is also checked whether the upper temperature limit of mesophilic process (for various aeration rates) stimulates or inhibits hydrogen production [26]. At the same time we check how various considered conditions influence methane production. The studied issues as well as the investigated substrates are rarely discussed, or contradicting results are presented (e.g. pH level). The results obtained here were compared with data from earlier studies [32] for sour cabbage and cotton waste at higher pH value (7.54) and without inoculum stress.

Materials and methods
The fermentation process of cotton wastes was performed in batch reactors of volume 2 dm 3 with working volume 1.2 dm 3 . As inoculum, sludge from an agricultural biogas plant (Pomerania Region) was used, and 5 g VSS/L (VSS = volatile suspended solids) were applied to each batch of cotton waste. A substrate (load 5 g VSS/L), obtained from 100% cotton lab coats, was milled and hydrolysed using 0.1 M HCl acidic solution for 2 h.
Initially, the inoculum was treated by heat shock for 0.5 h at 105 °C. Later, the initial pH 7.84 was lowered by HCl to pH 5.0 and applied to the DF process. The substrate was pretreated analogous to a Nasirian procedure for DF of wheat straw [33] but a 0.1 M solution of H 2 SO 4 , which was the most efficient in the case of hydrogen production from their substrate [34], was replaced here by cheaper HCl (for pH 3).
Then cotton and inoculum samples were added to the reactors. The temperatures 40 and 43 °C (maximum temperature for mesophilic conditions [35] were maintained in the reactors. The oxygen flow rates (OFR) range from 0 to 1.0 mL/h on average was provided for the fermentation process in the reactors. The oxygen was added twice a day (for approximately 2 s) until the fermentation process was stopped.
All the experiments were carried out in triplicate, (see scheme of setup in Fig. 1). The biogas production was determined using the Owen method [36]. The qualitative and quantitative assessment of the gases produced was performed using a gas chromatograph (GC) with a thermal conductivity detector and argon as a carrier (gas flow rate was 0.6 mL/h). A Silco packed column Restek® with characteristics of 2 m/2 mm ID 3 mm OD Silica was used. In order to determine the right amount according to Standard Methods [37], of fresh matter (FM) of inoculum and the substrate total solids (TS) [%FM] and volatile solids (VSS) [%TS] (determined to the [38]. The results are presented in Table 1. Cotton wastes characteristics (TS, VSS) were determined before and after hydrolysis and after fermentations and drying. Cotton wastes were sieved by net with mesh 2 mm after hydrolysis and after DF (taking it from bottom of reactors). The changes of VSS were used to determined degree of utilization of cotton waste in agreement with norms [37,38].

Results
The GC spectra allowed determination of methane, hydrogen, carbon monoxide, carbon dioxide and nitrogen (from process initiation procedure) concentrations. The fermentation process with pretreated inoculum (initial pH ~ 5) was continued for 5 h (later DF process had stopped) at temperature 40 °C, while at 43 °C the fermentation process was continued for 45 h.
In the case of boiled inoculum (pH ~ 8.5) biogas wasn't generated, so process at temperature of 43 °C was checked only for pretreated inoculum (pH ~ 5). In the case of process performed at temperature 40 °C the biogas contained carbon dioxide, nitrogen and hydrogen (no methane generation was observed, in contrast to fermentation at 43 °C). The hydrogen production strongly depends on OFR, see Fig. 2  After 5 h DF process terminated at 40 °C for all OFR rates. Thus the results are not presented in the form of cumulative hydrogen production versus time as is usual [39]. The optimum OFR value lies between 0.3 and 1 mL/h. Fast and effective hydrogen production has already been observed in dark fermentation but with lower rate e.g. in ref. [40], although it continued longer, even for 80 h.
At fermentation temperature 43 °C, biogas comprises: methane, carbon dioxide and nitrogen. No hydrogen was registered. The strictly anaerobic cf an oxygen flow rate ~ 0.3 mL/h conditions were compared-see Fig. 3. Under strict anaerobic conditions 0.92% of methane (0.003 dm 3 ) in 0.34 dm 3 of biogas was found after 45 h fermentation. In the case of OFR ~ 0.3 mL/h, higher concentration  The pH value in the case of fermentation at 40 °C changed from 5.0 to 5.5, under condition of free pH evolution (no pH control measures applied-see e.g. [41]. In the case of fermentation at temperature 43 °C the pH value increased to 5.9 after 45 h for all OFR values, i.e. pH value is higher in comparison to that at 40 °C. The presented results showed that fermentation process (after heat shock pretreatment of inoculum) led to higher hydrogen production than in [42] (and no methane generation) at the lower temperature 40 °C. Actually at 43 °C hydrogen was not generated in significant amount and methane at concentration below 2% of biogas.

Discussion of results
In this study DF proceeds under standard biogas inoculum condition in contrast to isolated bacterial consortium like in Li et al. [18]. It is worth adding that in the presented experiment there were not any nutrients like agar plates [43] or salts applied [44]. Moreover, the discussed process of hydrogen production is rather simple and not demanding; it proceeds without any bacteria isolation or hydrolysate separation. Thus, it is more economical than those already reported [45][46][47][48].
The process of cotton digestion slows down at higher temperature 43 °C (the rate is 9 times lower) when compared with the case at 40 °C. The observed at 40 °C hydrogen yields per day were higher than those reported by Li et al. [18]. The hydrogen production measured here was 6 times higher than in [49]. On the other hand biomass conversion is low (2.6%), 0.9% lower than in production of methane from cotton waste at 43 °C-see e.g. [22].
The experiments pointed to the importance of inoculum preatretment (thermal shock) and process temperature for hydrogen production rate. When DF temperature is higher methane production prevails, bacterial consortium is stimulated to generate methane (although in low quantities if compared to [22] even at low pH [50]. Similar results were reported by Chandra et al. [26] for rice straw anaerobic fermentation. Also Del et al. [51] concludes that stable mesophilic condition with temperature from the range 36-40 °C is necessary to keep process of hydrogen production continuing.
It was also found that in some cases after termination of hydrogen generation, methanogenesis continues e.g. when food and garden waste mixture is fermented at 37 °C [52] or xylose fermented at 30 °C [53]. However, it was not the case for cotton fermented here at 40 °C.
The results obtained here were compared with DF data for other substrates-see Table 2. The hydrogen yields obtained here were larger than from cow dung pretreated using 2% HCl solution [54], microalgal biomass [55] or sunflower pretreated by 4% HCl [56] but less than 50% of hydrogen obtained from cotton stalk after pretreatment in 4% H 2 SO 4 and 121 °C for 30 min. or 80% less than from aspen wood treated using 2% NaOH. Highest yields were obtained for cassava with addition of α-amylase and gluco-amylase after thermal pretreatment at 112 °C for 15 min und using activated sludge by heat shock for 30 min.

Conclusions
Cotton waste at load VSS 5 g/L at pH 5.0 was found to be a good source of hydrogen by mean of dark fermentation, when inoculum is stressed. The process is fast (short) and efficient. The discussed process of hydrogen production is rather simple and not demanding; it proceeds without any bacteria isolation or hydrolysate separation. Thus it is more economical than those already reported.
However, rather low conversion of cotton waste was observed, e.g. conversion of cotton waste in methanogenation 3.4% was higher than in hydrogenation process 2.6% and lower increase of bacterial biomass was registered. So, the process can be used for utilization of cotton wastes as fast preliminary stage of cotton waste utilization followed by methanisation.
The highest production was observed at OFR 0.8 mL/h and DF process temperature 40 °C i.e. 0.168 dm 3 of hydrogen-42% of biogas content. Besides it was found that temperature is a relevant parameter of stimulating or inhibiting hydrogen production in DF. Methanogenesis can revive again after repeated heat shock treatment and temperature of fermentation increase to 43 °C in the case of cotton.
Besides, it should be underlined that that the applied 5 g VSS/L should be probably increased in future studies to check the effects of load increase. The phenomena will be investigated further.

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
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