Semidry acid hydrolysis of cellulose sustained by autoclaving for production of reducing sugars for bacterial biohydrogen generation from various cellulose feedstock

Introduction

Cellulosic biowastes are of the most abundant renewable biomass available for hydrolysis for a cost-effective fermentation bioindustries. One of these fermentation biotechnologies is the production of hydrogen gas by bacteria. Hydrogen gas is one of the applicable sources of future renewable energy that is potentially promising to replace the present worldwide used fossil fuels (Yokoi et al., 2001; Nath & Das, 2004; Kapdan & Kargi, 2006; Chong et al., 2009; Abd-Alla, Morsy & El-Enany, 2011; Budiman & Wu, 2018; Noblecourt et al., 2018; Morsy et al., 2019). The hydrogen gas production by eubacteria is a cost-effective way where cheap organic wastes can be utilized in such future biological industry  (Yu et al., 2002; Zhang, Brunsb & Logan, 2006; Pattra et al., 2008; Lo et al., 2009; Cheng & Chang, 2011; Noparat, Prasertsan & Sompong, 2011; Hay et al., 2016; Morsy, 2017; Morsy, Elbahloul & Elbadry, 2019). Cellulose feedstock come at the top of interest to be utilized as an organic form source of carbon for producing H₂ by eubacteria, however most hydrogen producing eubacteria cannot utilize cellulosic biowastes. Hydrolysis of cellulose is thus fundamental for an efficient utilization of cellulosic biowastes in the production of hydrogen by many eubacteria. The efficiency of acid hydrolysis of polysaccharides in general depends on its polymeric complexity where cellulose is one of the difficult types of polysaccharides to hydrolyze. Due to abundant cellulose feedstock all over the world (Sukumaran, Singhania & Pandey, 2005), its hydrolysis is of the hot topics for the production of reducing sugars for various biofuels fermentation biotechnological industries. In the past, cellulosic feedstocks were used in heating but now with the modernization of developing countries, it is no longer used and replaced by biogas. Thus, farmers in developing countries sometimes burn most of the crop plants straw in-situ of their farms to get rid of it. This leads to a high-risk pollution and increases the CO₂ in the atmosphere; a trouble that shares to some extent, besides CO₂-liberating industries and extensive use of fossil fuels, in increasing the percent of CO₂ gas in the atmosphere enhancing global warming. Many works of literature seek useful utilization of cellulose feedstocks in producing hydrogen gas biologically. Acid and enzymatic hydrolysis of the polysaccharide cellulose is usually used for producing reducing sugars (Camacho et al., 1996; Iranmahboob, Nadim & Monemi, 2002; Sun & Cheng, 2002; Xiang et al., 2003; Taherdazeh & Karimi, 2007; El-Zawawy et al., 2011; Heinonen et al., 2012; Ni et al., 2013; Pulidindi, Kimchi & Gedanken, 2014; Vo et al., 2014; Yoon, Han & Shin, 2014). The acid hydrolysis is more applicable in high mass of cellulose at industrial scale. Reducing the amount of acid used in the hydrolysis is useful in two aspects; first it reduces the cost by reducing the amount of acid per-se and the amount of base used in subsequent neutralization step; second it will reduce the final ionic strength of the fermentation medium that might adversely affect the fermentation process. In this study a semidry acid hydrolysis of cellulose was conducted and its efficiency in producing reducing sugars was investigated. Applicability of acid hydrolysis of lignocelluloses and other polymeric carbohydrates is extensively reported in many fields of biotechnologies (Cuzens & Miller, 1997; Baruah et al., 2018; Bhatia et al., 2021). Hydrogen gas production by E. coli strain TFYM from hydrolyzed pure cellulose and various cellulose feedstock was investigated as an example of the many biotechnological applications that can be served by semidry acid hydrolysis of cellulose in a cost-effective way.

Materials and Methods

Hydrogen gas production by E. coli TFYM dark fermentation

Prior to fermentation, E. coli TFYM was grown in LB medium [10 g/l tryptone, 10 g/l NaCl, 5 g/l yeast extract] at 35 °C. E. coli TFYM batch dark fermentation experiments were conducted for H₂ formation from reducing sugars prepared as described above by semidry acid hydrolysis of cellulose and cellulosic feedstock biowastes (wheat bran sugarcane bagasse and sawdust). A one-liter glass fermentation bottle was used with a working volume of 970 ml. A volume 870 ml of neutralized hydrolysate was put in the fermentation bottle and supplemented with E. coli TFYM (OD₆₀₀ equal 0.25) 100 ml inoculum. The fermentation bottle was closed with a rubber bung and subsequently sparged with nitrogen gas for 20 min to accelerate installing anaerobic conditions. The fermentation bottle was kept in dark with continuous stirring at 35 °C. The evolved H₂ gas was collected using a CO₂-free H₂ gas collection system (Morsy, 2015) relying on passing the evolved gas on NaOH solution for absorbing CO₂ and subsequently collecting the CO₂-free H₂ gas by water displacement. The collected H₂gas compared to pure H₂ gas was measured using a Clark-type platinum-coated electrode computerized system [purchased from (Hansatech Instruments, Inc.) for Taibah University, Saudi Arabia] according to manufacturer instructions. The rate of H₂production was estimated as previously described (Morsy, Elbahloul & Elbadry, 2019) at each point of measurements of the produced cumulative H₂ gas as follows: ${\displaystyle \text{Rate of}{\mathrm{H}}_2\text{gas production}=\left(V_{\mathrm{x}}-V_{\mathrm{p}}\right)∕\left(T_{\mathrm{x}}-T_{\mathrm{p}}\right)}$ Where (V_x − V_p) is the difference in volume of collected cumulative H₂ gas at measurement time point (x) and the previous measurement time point (p). (T_x − T_p) is difference in time between the two cumulative H₂gas measurement points.

Results

In this study a semidry acid hydrolysis of cellulose and cellulosic feedstock biowastes was conducted for producing reducing sugars that can be used in many fermentation biotechnologies including production of hydrogen gas by bacterial dark fermentation. The semidry acid hydrolysis of cellulose was performed in a ratio of 1:1 [1 g biomass:1 ml of HCl where the cellulose was just wetted by the acid and subjected to autoclaving. The optimum HCl molar concentration for semidry acid hydrolysis of cellulose was 6 M determined first at autoclaving period of 30 min (Fig. 1A). The optimum autoclaving period was subsequently determined as 50 min (Fig. 1B) at optimum 6M HCl. These optimum 6M HCl and 50 min autoclaving conditions was efficient (Fig. 2) in hydrolyzing various cheap cellulosic feedstock biowastes (wheat bran, sawdust and sugarcane bagasse). The percent hydrolysis of wheat bran [65% ± 10.58 (Standard Deviation)] and sugarcane bagasse (46.3% ± 7.18) was more than pure cellulose (41.2% ± 5.31) possibly due to hydrolyzing cellulose and other less complicated more hydrolysable polysaccharides such as hemicellulose and others that is known to be present in natural plant residues. The semidry acid hydrolysis was also applied on non-cellulosic biomass of the potent extracellular polysaccharides producing cyanobacterium Nostoc commune where it produced 0.51 ± 0.12 g reducing sugars/ g dry mass. The ability of E. coli to utilize the reducing sugars produced by semidry acid hydrolysis of cellulose was conducted on Basal Mineral (BM) medium, so that the medium contains only minerals and a soli organic carbon source (the reducing sugars of semidry acid hydrolyzed cellulose). The results showed good ability of E. coli for utilization of the reducing sugars prepared by semidry acid hydrolysis of cellulose as investigated in a basal mineral medium where the bacterium could grow efficiently (Fig. 3) using the hydrolysate reducing sugars as an organic carbon source. The aerobic growth of E. coli on reducing sugars of semidry acid hydrolysis of cellulose in Basal Mineral (BM) medium followed by OD at 600 nm (Fig. 3A) was calibrated (Fig. 3B) versus dry cell weight (DCW) where a calibration value of OD_600 nm of 1 equals 0.368 g/L. E. coli like most other hydrogen producing bacteria cannot utilize cellulose as a carbon source and its utilization of reducing sugars prepared by semidry acid hydrolysis is a representative for efficiency of using such hydrolysate in dark fermentation. The bacterium used in this study was E. coli TFYM as a representative of the hydrogen producers to investigate the suitability of the hydrolysate prepared by semidry acid hydrolysis. This isolated bacterium was identified morphologically and biochemically (Table 1) following standard protocols. The identification was also confirmed by phylogenetic analysis of the 16S rRNA encoding gene sequence (Fig. 4). As the bacterium could utilize the reducing sugars in the hydrolysate prepared by semidry acid hydrolysis of cellulose, it was used for investigating the suitability of the hydrolysate for dark fermentation biohydrogen gas production. For the batch dark fermentation hydrogen production, the semidry acid hydrolysis of 10 g cellulose or various cellulose feedstock of wheat bran, sawdust and sugarcane bagasse was conducted and the hydrolysate retaining the reducing sugars was filtrated and neutralized to be used as an organic source of carbon for hydrogen formation by E. coli TFYM dark fermentation. The rate of hydrogen gas production gradually increased and was maximum (23.3 ml H₂ h⁻¹ L⁻¹) at 10 h after the start of fermentation followed by a decline at the late stationary phase of cumulative hydrogen production (Fig. 5). The reducing sugars produced by semidry acid hydrolysis (4.12 g/ 10 g cellulose) were efficiently used for hydrogen production by E. coli producing 0.4 mol H₂ mol⁻¹ hexose which is comparable to previously reported yield by E. coli from expensive pure sugars and sugar wastes (Table 2). The estimated hydrogen yield by E. coli (Fig. 6) from the reducing sugars prepared by the semidry acid hydrolysis of the cheap cellulosic biowastes of wheat bran, sawdust and sugarcane bagasse was 36, 18 and 32 (L H₂ kg⁻¹ dry biomass) respectively. These results indicate a good feasibility of hydrogen production from reducing sugars prepared by semidry acid hydrolysis of such cheap cellulosic biowastes.

Discussion

Cellulosic biomass wastes are cheap and abundant. Thus, upon hydrolysis, these polymeric renewable organic materials can be used in many fermentation biotechnological industries. This study describes a semidry acid hydrolysis of cellulosic feedstock biowastes where the biomass was just wetted with 6M HCl and subjected to autoclaving for 50 min where a considerable amount of reducing sugars were produced where the percent hydrolysis of pure cellulose was 41.2%. Assessment of semidry acid hydrolysis of cellulose was conducted using pure cellulose in first part of the study where 412 ± 53.1 mg reducing sugars/g of pure cellulose were produced. Subsequently application of semidry acid hydrolysis on crude cellulosic biowastes of wheat bran, sawdust and sugarcane bagasse biomasses was conducted. The cellulose, hemicellulose, lignin and other organic materials composition of these crude biowastes were previously reported (Table 3) where cellulose is basic structural components of these biowastes. In comparison to pure cellulose, semidry acid hydrolysis showed higher percent of hydrolysis in case of wheat bran (65%) and sugarcane bagasse (46.3%) mostly due to presence of complicated more hydrolysable polysaccharides associated with cellulose in these natural plant residues such as hemicellulose which is present along with cellulose in wheat bran (Merali et al., 2015), sugarcane bagasse  (Sanjuán et al., 2001) and almost all terrestrial plant cell walls (Scheller & Ulvskov, 2010). The ground tissue in sugarcane stalks retains abundant parenchyma cells whose cell wall is primary one which is composed mainly of cellulose and hemicellulose. Subsequent to crushing sugarcane stalks for extracting its juice by crusher machine in the initial steps of sugar industry, the sugarcane bagasse obtained is rich in residues of the primary cell walls of parenchyma tissue retaining cellulose and hemicellulose. Thus, these natural waste biomasses, including sugarcane bagasse and also wheat bran retaining both cellulose and the less complicated hemicellulose, are highly susceptible for semidry acid hydrolysis. This indicates that semidry acid hydrolysis is efficient for hydrolyzing cellulose and other less complicated polymeric carbohydrates in the cellulosic feedstock wastes. The semidry acid hydrolysis described in this study was thus highly efficient for hydrolysis of non-cellulosic cyanobacterial biomass. The potent extracellular polysaccharides producing (Hill, Peat & Potts, 1994; Hill et al., 1997; Helm et al., 2000; Tamaru et al., 2005; Morsy et al., 2008) cyanobacterium Nostoc commune biomass was efficiently hydrolyzed (51%) by semidry acid hydrolysis indicating that semidry acid hydrolysis was highly effective for hydrolyzing also this less complicated non-cellulosic renewable biomass. Subjecting cellulosic biomaterials to high concentrated H₂SO₄ in a two-step hydrolysis was described elsewhere (Iranmahboob, Nadim & Monemi, 2002; Chang et al., 2018). A two step acid hydrolysis was described to hydrolyze wood chips where the first step included treatment with high concentrated 80% H₂SO₄ at room temperature with a mass ratio of 500 g H₂SO₄/200 g dry mass of wood chips followed by addition of distilled boiling water to reach 26 wt% H₂SO₄ with boiling for 30 min and stirring followed by filtration where the filtrate containing cellulose was heated in a second step for extra 2 h resulting in 78–82% overall hydrolysis efficiency of cellulose theoretical values in wood (Iranmahboob, Nadim & Monemi, 2002). The Two-Step cellulose hydrolysis was also described elsewhere where in the first step cellulose was treated with high concentrated 72 wt% H₂SO₄ at 30 °C over 2 h in a ratio of H₂SO₄/dry mass of cellulose of 36 followed by a partial second step neutralization through using 20 wt% NaOH at 2.3–2.5 molar ratio for H⁺/OH⁻ with subjecting to more hydrolysis 10 min autoclaving at a temperature of 121 °C (Chang et al., 2018). Cotton cellulose was found to completely dissolve at room temperature in high concentrated sulfuric acid above 55% (by volume) and a reduced sugar yields from the initial cotton cellulose concentrations of 30–70 g/L were varied from 64.3 to 73.9% (g R-sugar/g cotton cellulose) at a temperature of 40 (Chu et al., 2011). The percent hydrolysis of sawdust was lower than cellulose possibly due to presence of highly complicated lignin in wood and lower cellulose content. Around 20% to 30% of sawdust content is lignin (Sınağ et al., 2009) which is highly complicated to be hydrolyzed. However, the use of sawdust for obtaining reducing sugars through semidry hydrolysis would depend in the source of the sawdust where the cellulose content would depend on the type of wood. The resulted reducing sugars were utilizable by E. coli as a representative of the fermenting bacteria indicating that the reducing sugars prepared by semidry acid hydrolysis are fermentable and can be utilized not only for dark fermentation hydrogen production but also for other fermentation-based biotechnologies. In this study, the organic carbon sources (the reducing sugars of semidry acid hydrolyzed cellulose and various cellulosic feedstocks) used for dark fermentation hydrogen production, are already subjected to autoclaving during semidry acid hydrolysis and hence it contains no native microbiota. Thus, the effect of native microbiota described elsewhere (Dauptain et al., 2020) is not applicable in the present study where fermentation was conducted by the supplied E. coli inoculum. Confirmative control experiments with no E. coli inoculated to the fermentor, did not produce hydrogen gas indicating no native microbiota effect is there where E. coli is the soli hydrogen producing bacterium in the fermentor through its utilization of the reducing sugars of semidry acid hydrolyzed cellulose and various cellulosic feedstocks. E. coli produce only hydrogen and CO₂ gas (Penfold, Forster & Macaskie, 2003) where CO₂ is absorbed by NaOH in the collection system and the collected hydrogen gas was fully pure compared to reference samples of pure hydrogen in measurements. The use of the abundant cellulose feedstock for hydrogen production would be cost effective through the semidry acid hydrolysis. The feasibility of such industry would be of importance in future upon exhaustion of fossil fuels (Muradov & Veziroglu, 2008). In fact, more and more research on the valorization of agricultural residual into biofuel has attracted great attention, mainly due to the positive effects from both economic and environmental aspects and long-term energy sustainability with greenhouse gas mitigation (Ho, Ong & Wu, 2019). The attempt to use cellulose feedstock would also be of importance in many agricultural countries. Besides, the re-activation of this biological hydrogen production industry would encourage farmers in developing countries to make use of crop plants straw and avoiding the harsh burning of such straw and cellulosic agricultural wastes. The data shown in this study either for growth of E. coli on the reducing sugars obtained from semidry acid hydrolysis or its utilization for hydrogen production did not show any inhibition or toxicity against the bacterium by the hydrolysate contents. As it requires minimum amount of acid and hence minimum amount of base for subsequent neutralization step, the semidry acid hydrolysis of cellulosic biowastes would be cost effective for bacterial hydrogen production biotechnology. The semidry acid hydrolysis of the cheap and abundant cellulosic wastes feedstock might possibly be applicable not only for bacterial H₂ production but also for other cellulose dependent biotechnologies. The described semidry acid hydrolysis reduces the amount of high molarity HCl required for hydrolysis to minimum. Thus, the amount of NaOH required for the tedious neutralization step required for various fermentation biotechnologies comes to minimum. Further future studies for modification of semidry acid hydrolysis such as combination with other hydrolysis protocols would be of interest for the best making use of the abundant cellulosic biowastes in various fermentation biotechnologies.

Supplemental Information