|Toto Iswanto||Department of Chemical Engineering, Institut Teknologi Sepuluh Nopember, Sukolilo, Surabaya 60111 Indonesia|
|Nuniek Hendrianie||Department of Chemical Engineering, Institut Teknologi Sepuluh Nopember, Sukolilo, Surabaya 60111 Indonesia|
|Maya Shovitri||Department of Biology, Institut Teknologi Sepuluh Nopember, Sukolilo, Surabaya 60111, Indonesia|
|Ali Altway||Department of Chemical Engineering, Institut Teknologi Sepuluh Nopember, Sukolilo, Surabaya 60111 Indonesia|
Biological methods using bacteria and fungi are regarded as more economically viable and environmentally friendly alternatives for improving lignocellulosic degradation. Coffee pulp waste (CPW) as a lignocellulosic biomass is abundant and has potential as a reducing sugar feedstock. However, it contains lignin as a matrix polymer, which associated with pectin and cover the cellulosic microfibrils and make it difficult to be digested during the bioprocess. In this study, the performance of biological pretreatment in reducing lignin and pectin using a co-culture of Bacillus subtilis (BS), Aspergillus niger (AN), or Trichoderma reesei (TR) has been investigated. The pretreatment of the CPW was made using various microbial ratios in an aerobic stirred-bioreactor and incubated at 30o C, pH 5 for 7 days. Removal of lignin and pectin was analyzed during the pretreatment process. PEG 4000 as a surfactant was used and its effect on the yield of reducing sugar production from pretreated CPW using a A. niger and T. viride (TV) co-culture with a surfactant to substrate ratio of 1:1 (w/w) was investigated. A culture without surfactant was used as a control. The results reveal that the best lignin and pectin removal was 99.9%, when using a co-culture of AN and TR with a ratio of 1:1 (v/v) and of BS and TR with a ratio of 2:1 (v/v). The cellulose content of CPW in these co-cultures was 86.99% (w/w) and 81.61% (w/w), respectively, and the reducing sugar concentration obtained was 12.5 g/L and 9.74 g/L respectively. In further hydrolysis of pretreated CPW using a AN:TV (2:1) co-culture with the addition of surfactant, the yield of reducing sugar obtained was higher than that of the control, at 20.69%. Use of PEG 4000 as a surfactant had a positive effect on enhancing the yield of reducing sugar from coffee pulp waste.
Biological pretreatment; Coffee pulp waste; Hydrolysis; Reducing sugar; Surfactant
The utilization of agro-industrial residues as lignocellulosic biomass to produce more valuable material has encouraged the improvement of advanced biotechnological innovations, principally in enzyme and fermentation technology, which have ultimately had an impact on the reduction of environmental pollution problems (Pandey et al., 2000). One of the agro-industrial residues that is available in abundance in Indonesia, but which has particular problems associated with its utilization due to the presence of anti-nutritional factors such as caffeine, tannin, and polyphenols, is coffee pulp waste (CPW). Coffee is an important plantation commodity in Indonesia (Haryuni et al., 2019); the country produces an average amount of 686×106 kg of coffee each year (Widjaja et al., 2017a). Without proper handling, the abundant untreated CPW will release xenobiotics as toxic substances into the soil, which kill the saprophytic microorganisms, causing disruption to important biotransformation stability in the environment (Ibrahim et al., 2014).
In the biotechnological approach, CPW can be converted into reducing sugar as a substrate to produce biofuels such as ethanol and biogas. To obtain the optimum yield of reducing sugar, a pretreatment process is needed to remove the unnecessary matrix polymers such as lignin and pectin surrounding the cellulosic microfibrils. Pectin is a heterogeneous polysaccharide, whose molecular structure, weight and functional properties depend on its agro-waste source (Kusrini et al., 2018). Pectin has the function of cross-linking cellulose and hemicellulose fibers, providing rigidity to the cell wall (Abbott & Boraston, 2008). This is in line with Menon and Rao (2012), who state that hemicelluloses, amorphous polymers of different sugars, and other polymers such as pectin, attach the microfibrils of cellulose, which are stabilized by hydrogen bonds and covered by lignin, making the biomass difficult to be digested in the bioprocess.
Physicochemical pretreatment methods have been widely developed, such as steam explosion (Guo et al., 2011), or use of dilute acid (Dussán et al., 2014), alkali (Menezes et al., 2014) or oxidation, or varied combinations of these, but such processes usually involve high temperature, pressure and cost (Kumar et al., 2009). Since chemical or physical methods are not able to provide selective removal, are expensive, and are not environmentally friendly, microbial and enzymatic techniques for lignin and pectin removal could be alternative advantageous methods.
The co-culture strategy has been assessed as a more efficient way to treat lignocellulosic waste (Menon & Rao, 2012). The combination of cultures can consist of two, three, or a consortium of bacteria or fungi or a mixture of them, which can be applied in many biological production processes. In the case of lignin and pectin removal, previous studies have reported that A. niger and T. reesei are capable of degrading lignin (Adav & Sze, 2014; Asses et al., 2018). Moreover, Roussos et al. (1995) found that some strains of A. niger are capable of degrading caffeine. B. subtilis can degrade lignin and pectin (Gummadi & Kumar, 2005; Torimiro & Okonji, 2013; Cragg et al. 2015). Juliastuti et al. (2018) successfully achieved the combination of three strains, A. niger, Pseudomonas putida, and T. harzianum to treat coffee pulp in degrading lignin and anti-nutritional factor for a biogas feedstock.
Besides applying the pretreatment, the use of surfactants (i.e. PEG 4000) in the hydrolysis of lignocellulosic substrates has produced good effects. Surfactants, as surface-active additives, affect the interaction between enzymes such as cellulase and substrates (Li et al., 2012), leading to an increase in the total amount of reducing sugar after the hydrolysis process. PEG is presented in varying molecular weights (e.g. PEG 4000, 6000 and 8000); at the lowest molecular weight (PEG 4000) the amounts of reducing sugar produced were 7% and 13% higher than those from PEG 6000 and 8000 respectively (Iveti? et al., 2014). However, the effects of the addition of the same surfactants on the obtained hydrolysis product may be different, depending on the operating conditions, initial chemical content in the substrate, and treatment method during the hydrolysis process.
Therefore, this study conducts an investigation to determine the best co-culture ratio involving A. niger, T. reesei and B. subtilis to develop a co-culture for degrading lignin and pectin at a faster degradation rate and which is able to tolerate the presence of the anti-nutritional factors that effect their growth. The obtained CPW from the best pretreatment condition was further hydrolyzed using a co-culture of A. niger and T. viride with the addition of PEG 4000. The effects of the co-culture on the reducing sugar obtained from the hydrolysis process were also investigated and described.
The microbial method in the form of a fungal and bacterial mixture has produced satisfying results in the degradation of lignin and pectin in coffee pulp waste, with more than 99% removal of those compounds. Due to the high cellulose concentration (more than 80%) in pretreated coffee pulp waste, two co-cultures, AN:TR (1:1) and BS:TR (2:1), were chosen for the following hydrolysis process. Subsequently, the hydrolysis of pretreated CPW using A. niger and T. viride with the addition of surfactant resulted a higher yield of reducing sugar than that without surfactant. The highest yield of reducing sugar resulted from the pretreated CPW by AN:TR (1:1) and hydrolyzed by the AN:TV (2:1) co-culture with the addition of PEG 4000, at 20.69%. In this co-culture, PEG 4000 as surfactant made a difference of 3.4% in reducing sugar, compared to without surfactant. Hopefully, the co-culture ratio obtained can indicate a direction for further study, focusing on the kinetics of its cell growth, which is important for developing an economical bioprocess of reducing sugar production.
The authors thank Atikah Badriya Husein and Dwi Ayu Primaningrum who have provided coffee pulp waste and selfless help. We also thank all members of Biochemical Technology Laboratory and Wastewater Treatment Laboratory, Institut Teknologi Sepuluh Nopember for their endless support.
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