Sugarcane Bagasse as a Co-Substrate with Oil-Refinery Biological Sludge for Biogas Production Using Batch Mesophilic Anaerobic Co-Digestion Technology: Effect of Carbon/Nitrogen Ratio

Man-made organic waste leads to the rapid proliferation of pollution around the globe. Effective bio-waste management can help to reduce the adverse effects of organic waste while contributing to the circular economy at the same time. The toxic oily-biological sludge generated from oil refineries’ wastewater treatment plants is a potential source for biogas energy recovery via anaerobic digestion. However, the oily-biological sludge’s carbon/nitrogen (C/N) ratio is lower than the ideal 20–30 ratio required by anaerobic digestion technology for biogas production. Sugarcane bagasse can be digested as a high C/N co-substrate while the oily-biological sludge acts as a substrate and inoculum to improve biogas production. In this study, the best C/N with co-substrate volatile solids (VS)/inoculum VS ratios for the co-digestion process of mixtures were determined empirically through batch experiments at temperatures of 35–37 ◦C, pH (6–8) and 60 rpm mixing. The raw materials were pre-treated mechanically and thermo-chemically to further enhance the digestibility. The best condition for the sugarcane bagasse delignification process was 1% (w/v) sodium hydroxide, 1:10 solid-liquid ratio, at 100 ◦C, and 150 rpm for 1 h. The results from a 33-day batch anaerobic digestion experiment indicate that the production of biogas and methane yield were concurrent with the increasing C/N and co-substrate VS/inoculum VS ratios. The total biogas yields from C/N 20.0 with co-substrate VS/inoculum VS 0.06 and C/N 30.0 with co-substrate VS/inoculum VS 0.18 ratios were 2777.0 and 9268.0 mL, respectively, including a methane yield of 980.0 and 3009.3 mL, respectively. The biogas and methane yield from C/N 30.0 were higher than the biogas and methane yields from C/N 20.0 by 70.04 and 67.44%, respectively. The highest biogas and methane yields corresponded with the highest C/N with co-substrate VS/inoculum VS ratios (30.0 and 0.18), being 200.6 mL/g VSremoved and 65.1 mL CH4/g VSremoved, respectively.


Introduction
Environmental pollution due to rapid waste generation and energy insecurity has become a global challenge in recent years. The combustion of fossil fuels from five sectors, namely energy generation, food and agriculture, transport, industry and economy, and to remove any trapped impurities and ligneous. The bagasse was then dried in an oven at 105 • C for 24 h to obtain a consistent dry weight. Next, the dried bagasse was ground and milled to smaller than 0.5 mm using a mechanical shredder machine (Granulator WSGP-230) and miller machine (Puluerisette 16). The grinding and milling of the dried bagasse increased its surface area and digestibility by the bacteria during the anaerobic co-digestion process [22,23].
The dried sugarcane bagasse was treated thermo-chemically using sodium hydroxide in different ratios, temperatures, and periods to reduce lignin content as much as possible, which was recalcitrant to the hydrolysis process [24]. Due to the acidic nature of the sugarcane bagasse, mechanical and alkali-thermal pre-treatment methods were selected as effective methods to enhance the delignification and degradation process for better biogas yield and to increase the pH to meet the required pH range for an anaerobic digestion process, between 6 and 8 [19,[25][26][27]. The thermo-chemical pre-treatment ratios were 1% and 2% (w/v) NaOH for 45, 60, and 75 min. The solid/liquid ratio was 1:10, the temperature was 100 • C, and the rpm was 150 rpm.
The oily-biological sludge is a waste-activated sludge that was collected from a petroleum oil refinery wastewater effluent treatment system and stored in a cooling room below 4 • C for less than 24 h before proceeding with the co-digestion process. Pre-treatment for the sludge was needed to enhance the degradation of the organic matter [28]. Due to the acidic nature of the oily-biological sludge, the alkali-thermal pre-treatment method was selected as an effective method to enhance the degradation process for better biogas yield and to increase the pH to meet the required pH range for the anaerobic digestion process, between 6 and 8 [19,29,30]. The sludge was thermo-chemically pre-treated using sodium hydroxide 1 g/L, at 100 • C, 150 rpm for 1 h to increase its pH as it has an acidic nature to comply with the required range of pH during anaerobic digestion process, between 6-8.

Analytical Methods, Design and Operational Setup
Proximate analysis for the treated oily-biological sludge (TOBS) was conducted according to the American Public Health Association (APHA), 1998 methods to determine the total solids (TS) and volatile solids (VS). The ultimate analysis was conducted to determine the carbon, hydrogen, nitrogen, and sulfur (CHNS) elements using the CHNS elementar machine (Vario MICRO) to design the mixing ratios according to the C/N ratio for the batch experiments.
The proximate analysis for the treated sugarcane bagasse was performed in accordance with the American Society for Testing and Materials (ASTM) D3172-89 standard. The pH of the sugarcane bagasse was determined according to Hach method. The ultimate analysis was performed according to ASTM D5373 standard using the same elementar machine to determine the C/N ratio for the mixtures design. The chemical composition to determine hemicellulose, cellulose and lignin was measured according to Chesson's method to compare and analyse the effect of thermo-chemical pre-treatment method on lignin content.
The functional groups present in the raw and treated sugarcane bagasse were determined by Fourier transform infrared (FTIR) analysis. FTIR analysis was conducted to investigate the effects of thermo-chemical pre-treatment on the functional groups, namely hemicellulose, cellulose, and lignin, using a PerkinElmer analyzer. Supra 55 VP (Carl-Zeiss AG, Oberkochen, Germany) was used to analyse the surface morphology of raw sugarcane bagasse (RSCB), treated sugarcane bagasse (TSCB), raw oily-biological sludge (ROBS), and treated oily-biological sludge (TOBS) using a variable pressure scanning electron microscope. The images were taken for both materials before and after the thermo-chemical pre-treatment at different magnifications to observe and analyse the effects of the pre-treatment method. The design of mixtures was conducted according to the following equation [31], in order to achieve mixtures for both materials between 20 and 30.
where: R = C/N ratio; Q 1 , Q 2 = mass of materials "as is" or wet weight; C 1 , C 2 = carbon content of materials (%); N 1 , N 2 = nitrogen content of materials (%); M 1 , M 2 = moisture content of materials. The SOLTEQ TR37 sludge anaerobic digester was used to carry out this study [32]. The size of the digester used for this study was 2.5 L, with a working volume of 2 L. The digester's temperature was set manually using the digester control panel at 35.0-37.0 ± 0.5 • C, with a mixing speed of 60 rpm for 33 days. The produced biogas was measured every day using a water displacement method, and the content was measured every three days using the German Optima 7 biogas analyzer. The one-way ANOVA test using Microsoft Excel 2010 was used to compare the biogas and methane yield from different reactor groups based on the C/N ratio and co-substrate VS/inoculum VS increments. The central point C/N ratio mixture was repeated four times to verify the experiment's design. In addition, validation for the maximum biogas and methane yield was carried out using 3 replications for the best ratios. After mechanical pre-treatment of RSCB was carried out to below a 0.5 mm particle size, thermo-chemical pre-treatment was conducted using sodium hydroxide to minimize the lignin content and enhance biogas production. Sodium hydroxide was used in different ratios and periods. The hemicellulose, cellulose, and lignin contents before and after the pre-treatment are tabulated in Table 1. The results show that 1% sodium hydroxide at 100 • C and 150 rpm for 1 h was the best combination pre-treatment to minimize the lignin content from sugarcane bagasse. The lignin content decreased to 13.50%, while the cellulose content increased to 62.05%. This will enhance the hydrolysis process and provide a good source of a digestible, accessible fraction for the microorganisms, which is the cellulose portion [24,25]. Three replicates for the best pre-treatment ratio were conducted and the average hemicellulose, cellulose, and lignin contents were 10.55, 62.45 and 13.60%, respectively, with the significance level being more than 95%. Therefore, this ratio was selected for the ultimate and proximate analysis of TSCB, and consequently for the batch design mixtures. Scanning electron microscopy (SEM) and FTIR were used to show the effect of the pre-treatment through surface morphology and functional group changes. According to Table 2, the pH for TOBS and TSCB has a base nature induced by chemical pre-treatment using NaOH. This will provide a pH of 6.2 to 8, as recommended for an effective anaerobic co-digestion process; during anaerobic co-digestion, the acetogenesis phase has an acidic nature, and that will balance the base nature of the mixtures to be within the recommend pH range [19,33]. The VS for both materials comprise a high portion of the total solids, and this is a good indicator for high biogas yield, as most of the volatile matter will be converted to gas. From Table 2, the C/N ratio for TOBS and TSCB was found to be 14.42 and 132.692, respectively, at base. The high C/N ratio for TSCB will balance the low C/N ratio of TOBS to provide a proper environment for microorganisms, with a C/N ratio between 20 and 30. These analyses were used in the study of methane modelling through surface response methodology for the anaerobic co-digestion of both materials [32,34] This analysis was conducted to determine the functional groups of RSCB and TSCB to analyse the changes induced by thermo-chemical pre-treatment. Figure 1a,b show the FTIR spectra for RSCB and TSCB, respectively. The variation bonds given by the FTIR spectra for the RSCB are shown in Table 3. The variation bonds mainly corresponded to cellulose, hemicellulose, and lignin. Figure 1a shows a strong peak at 3405.76 cm −1 ; this indicates the presence of the O-H stretching and NH group. The OH is related to polyphenol and alcohol, which was present in cellulose and lignin [35]. The peak at 2918.97 cm −1 reflects the C-H stretching, indicating the presence of methylene in the cellulose and hemicellulose [36,37]. The peak at 1733.42 cm −1 reflects the C=O stretching of acetal groups available in hemicellulose [38]. The peak at 1633.70 cm −1 reflects the C=O stretching group carbonyl of ketonic groups found in lignin [37]. The peaks at 1605.30 and 1513.89 cm −1 represent the aromatic skeleton C=C available in lignin [39][40][41]. The peaks at 1427.82 and 1375.33 cm −1 signify the CH2 asymmetric stretching and C-H bend available in lignin and cellulose, respectively [42,43]. The peaks at 1249.50 and 1162.91 cm −1 describe the C-O stretching and C-O-C asymmetrical stretching available in hemicellulose and cellulose, respectively [38,40]. Finally, the peak at 1052 cm −1 may be due to the C-O stretching group available in cellulose [44].

Results and Discussion
The variation bonds given by the FTIR spectra for the TSCB are tabulated in Table 4. The strong peak at 3432.35 cm −1 , as shown in Figure 1b, indicates that the O-H stretching and NH group, and the OH is related to the polyphenol and alcohol present in the cellulose and lignin [35]. The peak at 2912.80 cm −1 reflects the C-H stretching found in methylene, which is present in the cellulose and hemicellulose [36,37]. The peak at 1600.03 cm −1 reflects the C-H skeletal variation available in lignin [34]. The peak at 1421.81 cm −1 presents the C-H bending present in cellulose [45]. Finally, the peaks at 1163.45 and 1057.39 cm −1 indicate the C-O-C asymmetrical stretching and C-O stretching available in cellulose and hemicellulose [38,44,46]. The FTIR spectra analysis for RSCB and TSCB proved that the thermo-chemical pre-treatment for the RSCB induced a positive effect in the chemical structure, where TSCB had a lower lignin content compared to RSCB. Therefore, the thermo-chemical pre-treatment for the RSCB was successful in reducing the lignin content, which inhibited the anaerobic co-digestion process. The TSCB also enhanced the biogas production as the major fraction contents were cellulose and hemicellulose, which were the primary sources for the microorganism activities during the anaerobic co-digestion process with oily-biological sludge.
Water 2021, 13, x FOR PEER REVIEW 6 of 24 cellulose, respectively [42,43]. The peaks at 1249.50 and 1162.91 cm −1 describe the C-O stretching and C-O-C asymmetrical stretching available in hemicellulose and cellulose, respectively [38,40]. Finally, the peak at 1052 cm −1 may be due to the C-O stretching group available in cellulose [44].     The SEM observations for the RSCB and TSCB depicted in Figures 2 and 3 show an apparent physical change in the inner and outer surface induced by the thermo-chemical pre-treatment method. Figure 2a shows the general view for RSCB. There are two structures indicated by arrows S and P.
The arrow S indicates the fiber structure for the outer surface of the RSCB, which is usually the lignin portion. In contrast, the arrow P indicates the pith structure of the RSCB, which is the cellulose and hemicellulose fraction. Figure 2b shows that the TSCB also had two structures, indicated by the arrow S for fiber and P for pith. Based on Figure 2a,b, it was evident that RSCB had a higher fiber structure portion compared to TSCB, which indicated that the thermo-chemical pre-treatment method had a significant effect in reducing the lignin content.
The fiber structure of RSCB was more structured and attached compared to TSCB, which was independent and partially unattached. This indicates that the thermo-chemical pre-treatments broken down the bonds of lignin content at the outer surface, and reduced the inhabitation effect of the low unstructured lignin content during the anaerobic codigestion process. Apart from that, it also increased the accessibility to the cellulose fraction during the hydrolysis process by bacteria [47]. Figure 3c,d show the magnification of pith structures for RSCB and TSCB. It is apparent that the pith structure of TSCB was more fragile and fragmented compared to the pith structure of RSCB. This indicates that the thermal pre-treatment also affected the pith structure, and a portion of the hemicellulose fraction was removed during the pre-treatment process which led to small pores, as shown in Figure 3d. The arrow S indicates the fiber structure for the outer surface of the RSCB, which is usually the lignin portion. In contrast, the arrow P indicates the pith structure of the RSCB, which is the cellulose and hemicellulose fraction. Figure 2b shows that the TSCB also had two structures, indicated by the arrow S for fiber and P for pith. Based on Figure 2a,b, it was evident that RSCB had a higher fiber structure portion compared to TSCB, which indicated that the thermo-chemical pre-treatment method had a significant effect in reducing the lignin content.
The fiber structure of RSCB was more structured and attached compared to TSCB, which was independent and partially unattached. This indicates that the thermo-chemical pre-treatments broken down the bonds of lignin content at the outer surface, and reduced the inhabitation effect of the low unstructured lignin content during the anaerobic co-digestion process. Apart from that, it also increased the accessibility to the cellulose fraction during the hydrolysis process by bacteria [47]. Figure 3c,d show the magnification of pith structures for RSCB and TSCB. It is apparent that the pith structure of TSCB was more fragile and fragmented compared to the pith structure of RSCB. This indicates that the thermal pre-treatment also affected the pith structure, and a portion of the hemicellulose fraction was removed during the pre-treatment process which led to small pores, as shown in Figure 3d.

Scanning Electron Microscopy of ROBS and TOBS
The SEM observations for the ROBS and TOBS, as illustrated in Figures 4 and 5, show a clear physical change in the flocs surface induced by thermo-chemical pre-treatment method. Figure 4 shows the general view for ROBS and TOBS, whereas Figure 5 shows the magnification of flocs surfaces for ROBS and TOBS. The alkaline-thermo pre-treatment induced a physical effect in the surface flocs      Twelve batch experiments, including four replicates for the central point (medium C/N ratio 24.2) to increase the significance and confidence levels, were designed using Equation (1), based on the ultimate and proximate results shown in Table 2, to obtain C/N ratios between 20 and 30. Each batch contained 2 L of TOBS and a matching portion of sugarcane bagasse to meet the C/N ratio, as shown in Table 5. The density of the TOBS was similar to water. There were three main responses in this study, namely, biogas yield, methane content, and VS removed. Prediction of the total methane yield has been published as a part of this project [32]. Table 5 shows the ratios of TOBS and TSCB added to the batch to obtain the desired C/N ratios. Examples to obtain C/N ratios of 20 and 30 using Equation (1)  The biogas then started to slightly decrease from day 26 to the end of the reaction period, day 32. The rapid increase initially until day 12 may be due to the high availability of accessible organic matter from TOBS and TSCB as well. From day 13 to day 25, the biogas yields were almost constant; this may due to the acclimatization of microorganisms with the substrates and operation environment. Meanwhile, there is still a high amount of organic matter to be consumed by the bacteria. However, the biogas yield slightly decreased from day 26 to the end of the reaction period; this indicated that most of the organic matter that can be consumed by the microorganisms under the current operation conditions was already consumed and the biogas yield started to decrease to the end of the reaction period.   The results indicate that the cumulative biogas yield from the reactor with 21.1 C/N is higher than cumulative biogas yield from the reactor with 20 C/N by 8.32%. In addition, the results indicate that the cumulative biogas yield from the reactor with 22.8 C/N is higher than cumulative biogas yield from the reactor with 20 C/N by 1.59%. The increasing biogas yield may be due to volatile solid increment together with the C/N ratio. Overall, there is increasing of biogas yield together with C/N ratio increment. However, the behavior of biogas yield is still not clear and needs more investigation in terms of methane yield and increasing of C/N ratio. Figure 6c shows the daily biogas yields from the central point 24.2 C/N ratio with four replicants. The behavior of daily biogas yield from the four reactors was quite similar. The biogas yield started from day 1 and increased gradually until day 14-this may be due to the high availability of degradable organic matter consumed by the microorganisms. After that, the biogas yield started to decrease from day 15 to day 18 and then remained constant with some fluctuations until day 29; this may due to a decrease in the organic matter content and the microorganisms acclimatized to the reaction environment. Then, a sharp decrease started from day 30 to day 32; this may be due to a sharp decrease in the available organic matter to be consumed by the microorganisms under the current operation conditions. When this C/N ratio is compared to previous C/N ratios, it is obvious that the duration of biogas' sharp increase as well as the constant period of biogas yield also increased. This may be due to the increasing of the C/N ratio by increasing the dosage of TSCB, as therefore the content of organic matter will increase, and the microorganisms will work more effectively. Figure 6d shows the cumulative biogas yield from four reactors with the same C/N ratio, which is the central point 24.2. The cumulative biogas yields were 3638, 3459, 3863, and 3700 mL respectively. The average biogas yield is 3665 mL; this is higher than cumulative biogas yield from reactors with 20.0 and 22.8 C/N ratios by 24.23% and 23.0%, respectively. It is clear that the increasing biogas yield in this C/N ratio was enhanced greatly compared to the enhancement among the previous ratios. This indicates that the behavior of biogas is much clearer when increasing the C/N ratio by increasing the dosage of TSCB. Figure 6e presents the daily biogas yield from the 25.1, 26.5, 27.1, and 30.0 C/N ratios. The behavior of biogas yield from the four reactors was quite similar. The biogas yield started increasing gradually from day 1 to day 7; this may be due to the high availability of degradable organic matter consumed by the microorganisms. After that, the 25.1 and 26.5 C/N ratio reactors almost remain constant, with a slight decrease and some fluctuations from day 8 to day 30, which may be due to a decrease in the organic matter content and the microorganisms acclimatized to the reaction environment. The 27.1 and 30.0 C/N ratio reactors remain constant, with a slight increase and some fluctuations from day 8 to day 30. This may be due to the acclimatization of microorganisms with the reaction environment and more availability of organic matter compared to 25.1 and 26.5 C/N reactors due to the increasing TSCB dosage. Then, a sharp decrease started from day 30 to day 32; this may due to the sharp decrease in the available organic matter to be consumed by the microorganisms under the current operation conditions. When these C/N ratios are compared to previous C/N ratios, it is obvious that the constant period of biogas yield increased. This may be due to increasing the C/N ratios to high levels by increasing the dosage of TSCB, as therefore the content of organic matter will increase and the microorganisms will work more effectively compared to lower volatile solids reactors. Figure 6f shows the cumulative biogas yield from 25.1, 26.5, 27.1 and 30.0 C/N ratios. The cumulative biogas yields were 4867, 3683, 6808, and 9268 mL, respectively. The results indicate that the cumulative biogas yields from reactor with 30.0 C/N is higher than the cumulative biogas yields from reactors with 25.1, 24.2 and 20 C/N by 47.39%, 60.46%, and 70.04%, respectively. It is clear that increasing the biogas yield by gradually increasing the C/N ratio to higher levels is highly enhanced compared to the biogas yield from the lower C/N ratios. Overall, the yield of biogas increased gradually while increasing the C/N ratio by increasing the dosage of TSCB. This may be due to the fact that increasing the dosage of TSCB will result in increasing the volatile solids content. Therefore, the microorganisms' activity is enhanced and they are better acclimatized to the reaction environment, and the biogas yield will enhance as well. In addition, at high C/N ratios between 25.1 and 30.0, the pH after 5 days of the reaction was stable between 6.9 and 7.3, which is within the ideal range of pH for better biogas production during anaerobic digestion, namely between 6.8-7.4, which provides a more healthy environment for bacteria [50,51]. Meanwhile, the lower C/N ratios cause ammonia inhabitation and the pH values increased and become toxic and inhibit the growth of methanogenic bacteria [52][53][54][55]. C/N ratios between 25 and 30 were also commonly found during previous studies to be better ratios during the anaerobic digestion process, with the C/N 30 ratio being the most common ratio [53,[56][57][58][59], and this is consistent with the results of this study. Therefore, C/N 30 was selected from this study for the maximum biogas and methane yield and was validated using three replicates. The average after three-replicate validation of the maximum biogas yield form C/N 30 was found to be 9035.0 mL (195.6 mL /g VS removed ), with the significance level being more than 95%.

ANOVA Analysis for Biogas Yield from C/N 20-30
Appendix A, Table A1 summarizes the ANOVA analysis for 20.0-22.8 C/N ratios; it shows that there is no significant difference in the daily biogas yield, with a p value > 0.05. This indicates that the dosage of TSCB added is still not enough to achieve a significant difference in the biogas yield from reactors with a low C/N ratio. Appendix A, Table A2 summarizes the ANOVA analysis for 20.0-24.2 C/N ratios; it shows that there is a significant difference in daily biogas yield, a with p value < 0.05. This indicates that the dosage of TSCB added can cause a significant difference in the biogas yield from reactors when the C/N ratio increases to 24.2. Appendix A, Table A3 summarizes the ANOVA analysis for 25.1-30.0 C/N ratios; it shows that there is a significant difference in daily biogas yield, with a p value < 0.05. This indicates that a larger dosage of TSCB added can achieve a highly significant difference in the biogas yield from reactors when the C/N ratio increases to 30.0. Appendix A, Table A4 summarizes the ANOVA analysis for 20.0-30.0 C/N ratios, and it shows that there is a significant difference in daily biogas yield, with a p value < 0.05. This indicates that the overall increment for the dosage of TSCB added can achieve a significant difference in the biogas yield from reactors. Therefore, the C/N ratio is a key factor affecting the production of biogas; increasing the C/N ratio by adding more TSCB results in an increase in the biogas yield, and vice versa. Figure 7a presents the results of the 3-day interval methane yield for four reactors with a low C/N ratio. The C/N ratios were 20.0, 21.1, 22.6, and 22.8, respectively. The measurement of the methane yield started from day 3 and the methane yield increased gradually until day 24, with some fluctuations. The methane content mainly related to the biogas yield quantity, as well the duration of reaction. When there was an increase in biogas yield, the methane content also increased and continued to gradually increase until day 24. Then, from day 25 to the end of the reaction, the methane yield sharply decreased; this may due to the fact that the biogas yield also sharply decreased as a result of the fact that most of the organic matter that can be consumed by the microorganisms under the current operation conditions was already consumed and the biogas yield started to decrease to the end of the reaction period. Figure 7b presents the cumulative methane yield from the four reactors. The cumulative methane yield was 980.0, 1023.80, 1051.28, and 1067.28 mL, concurrent with 20, 21.1, 22.6, and 22.8 C/N ratios, respectively. The results indicate that the cumulative methane yield from the reactor with 21.1 C/N is higher than cumulative biogas yield from the reactor with 20 C/N by 6.78%. In addition, the results indicate that the cumulative biogas yield from reactor with 22.8 C/N is higher than cumulative biogas yield from reactor with 20 C/N by 8.18%. The increase in the methane yield is not high. However, the overall view of methane yield from these reactors shows that there is gradual increase in the methane yield together with the C/N ratio increment. The stability of the increase in the methane yield is greater compared to the biogas yield. This may be due to the fact that the C/N ratio increment highly affects the methane yield compared to the biogas yield.  The results indicate that the cumulative methane yield from the reactor with 25.1 C/N is higher than cumulative biogas yield from the reactor with 24.2 C/N by 18.29%. In addition, the results indicate that the cumulative biogas yield from the reactor with 30.0 C/N is higher than cumulative biogas yield from  The methane content is mainly related to the biogas yield quantity as well the duration of the reaction. The biogas yield from day 16 until day 30 experienced a constant production rate with some fluctuations. Therefore, the methane yield was constant as well during this period. Then, the methane content from day 30 to the end of the reaction shows a sharp decrease in methane content, and this is mainly related to the sharp decrease noted in the biogas yield during this time. Figure 7d presents the cumulative methane yield from the four replicate reactors. The cumulative methane yield was 1257.29, 1317.53, 1393.38, and 1317.57 mL, concurrent with the same C/N ratio of 24.2. The average methane yield is 1321.42 mL. The results indicate that the average cumulative methane yield from the reactor with 24.2 C/N is higher than the cumulative biogas yield from the reactor with 20 C/N by 25.84%. In addition, the results indicate that the average cumulative biogas yield from reactors with 24.2 C/N is higher than cumulative biogas yield from the reactor with 22.8 C/N by 19.24%. The increase in the methane yield from this ratio is high and promising compared to previous ratios. Figure 7e presents the results of a 3-day interval methane yield for four reactors with a low C/N ratio. The C/N ratios were 25.1, 26.5, 27.1, and 30.0, respectively. The measurement of the methane yield started from day 3 and the methane yield increased gradually until day 15 with some fluctuations. The methane content was mainly related to the biogas yield as well the duration of reaction. The methane yield then experienced stability with some fluctuations until day 30. This may be due to the constant production of biogas during this period. Then, from day 30 to the end of the reaction, the methane yield sharply decreased due to the sharp decrease in the biogas yield. Figure 7f presents the cumulative methane yield from the four reactors. The cumulative methane yield was 1617. 18, 1723.40, 2007.84, and 3009.46 mL concurrent with 25.1, 26.5, 27.1, and 30.0 C/N ratios, respectively. The results indicate that the cumulative methane yield from the reactor with 25.1 C/N is higher than cumulative biogas yield from the reactor with 24.2 C/N by 18.29%. In addition, the results indicate that the cumulative biogas yield from the reactor with 30.0 C/N is higher than cumulative biogas yield from the reactor with 25.1 C/N by 46.26%. The results also indicate that the cumulative biogas yield from the reactor with 30.0 C/N is higher than the cumulative biogas yield from the reactor with 20.0 C/N by 67.44%. The increase in the methane yield is very high compared to previous C/N ratios. Overall, the methane yield was more stable and gradually increased in accordance with the C/N ratio increment compared to the biogas yield. The average of three-replicates validation for the maximum methane yield form C/N 30 was found to be 2996.07 mL (64.85 mL CH 4 /g VS removed ), with a significance level of more than 95%.

Effect of Carbon/Nitrogen (C/N) and Co-Substrate VS/Inoculum VS Ratios on Methane Yield
The thermo-chemical pre-treatment of the sludge also contributes to the increased convertibility of the organic matter to biogas, which is similar to the observation by David C [60] during his study on the effect of thermal pre-treatment on waste activated sludge. He found that the increment of methane content increased concurrently with a temperature increment up to a maximum of 175 • C. Apart from that, different studies showed that the thermal and thermo-chemical pre-treatment increased the digestibility of the sludge during the anaerobic digestion process [61,62]. The thermo-chemical pre-treatment for sugarcane bagasse had a significant effect on enhancing biodegradability rate, which resulted in higher biogas production as well as methane content. This was also noticed by Costa [63] during his study on the effect of thermo-chemical pre-treatment to improve the anaerobic digestion of sugarcane bagasse.

ANOVA Analysis for Methane Yield
Although ANOVA analysis showed a non-significant increase in the methane yield during the lower increment of C/N ratios, as shown in Appendix B, Table A5, Table A6, and  Table A7, there was a significant difference in the methane yield during the high increment of C/N ratios, as shown in Table A8, with a p-value of less than 0.05. This indicates that the increment of C/N and co-substrate VS/inoculum VS ratios has a higher effect on biogas yield than methane content. However, the methane yield production is more stable and gradually increased together with C/N ratios increment without fluctuations compared to the biogas yield.

Effect of Carbon/Nitrogen (C/N) and Co-Substrate VS/Inoculum VS Ratios on Volatile Solids Removed
The VS removed increased progressively with C/N and co-substrate VS/inoculum VS ratios. According to Table 6, the minimum VS removed was met with minimum C/N and co-substrate VS/inoculum VS ratios. In contrast, the maximum VS removed was met with maximum C/N and co-substrate VS/inoculum VS ratios (30 and 0.18). The removal of volatile solids between minimum and maximum biogas and methane yields increased by 30.09% at C/N 30. The stable range of pH for all reactors was found to be between 6.9 and 8.0, and this was required for a proper environment for better microorganism activities. Therefore, the stability of a pH of 6.9-7.3, which is healthier for microorganisms at C/N 25.1-30, contributed to the higher removal of volatile solids and resulted in higher biogas and methane production. Alagöz and Maragkaki [64,65] indicated that VS removal improved during the co-digestion process for sludge with agricultural wastes, while the production of methane yields improved.  Table 7 shows the comparison of the current study with different studies used anaerobic co-digestion technology. This study shows better results than other studies in terms of biogas yield. However, the methane yield was lower than other studies. This may be due to the differences in materials' characteristics and co-digestion operational conditions. The present study indicated that anaerobic co-digestion using a co-substrate with a high C/N ratio and VS content to balance the lower C/N ratio of the oily-biological sludge would enhance the digestibility, resulting in higher biogas and methane yields in case we used mono-digestion. However, to increase the methane yield from oily-biological sludge with sugarcane bagasse, the pre-treatment method or parameters for both materials can be changed and tested. For instance, the delignification process for sugarcane bagasse using thermo-chemical method can be optimized using the response surface methodology to minimize the lignin content. In addition, another pre-treatment method can be applied. In addition, for the oily-biological sludge, the pre-treatment parameters and method can also be changed. For example, we can increase the temperature and reduce the time when using thermo-chemical method, or use a different pre-treatment method such as ultrasonic pre-treatment. In addition, we can test the methane yield by changing the operational condition during the anaerobic co-digestion process or by changing the type of digestion method. For instance, we can change the temperature from a mesophilic thermophilic condition or decrease the mixing speed during the co-digestion process. In addition, changing the type of digestion method could enhance the methane yield, such as using the two-stage anaerobic co-digestion method.

Conclusions
In this study, sugarcane bagasse was used as a high C/N ratio co-substrate to treat the low C/N ratio oily-biological sludge through anaerobic co-digestion technology. Thermochemical pre-treatment using NaOH was carried out for sugarcane bagasse to reduce the lignin content as well as to increase the pH. The best ratio for the sugarcane delignification process as a part of pre-treatment was 1% (w/v) sodium hydroxide, 1:10 solid-liquid ratio, at 100 • C, and 150 rpm for 1 h. In addition, thermo-chemical pre-treatment were carried out for the oily-biological sludge using NaOH to increase the sludge digestibility and to increase the pH as well. The gradual increase in the C/N ratio resulted in a gradual rise in sugarcane bagasse content along with co-substrate volatile solids/inoculum volatile solids. Therefore, the biogas yield and methane content increased when higher C/N and co-substrate VS/inoculum VS were used. The biogas yield from C/N 30 was higher than the biogas yield from C/N 20 by 70.04%. Meanwhile, the methane yield from C/N 30 was higher than the methane yield from C/N 20 by 67.44%. The maximum biogas and methane yield coincided with the highest C/N and co-substrate VS/inoculum VS ratios (30.0 and 0.18) with 200.6 mL/g VS removed and 65.1 mL CH 4 /g VS removed , respectively. This increase was attributed to the increment of volatile solids content while increasing the C/N ratios. In addition, it was due to the stability of pH for high C/N ratios reactors between 6.9 and 7.3, which is more healthy for microorganisms activities. The lower C/N ratios cause an increased ammonia concentration, which is toxic to the bacteria, resulting in a lower biogas and methane yield.  Data Availability Statement: This project still on going and the data cannot be published at the current stage because there might be more analysis and comparison of the data after anaerobic co-digestion process under thermophilic condition. However, in case any researcher needs the data can either contact us via email or use Digitizer Plot for the graphs to extract the data.
Acknowledgments: Authors would like to thank Department of Civil and Environmental Engineering, Universiti Teknologi PETRONAS and Yayasan UTP for their support, funding and contribution to this work under grant cost center 015LC0050. Also, the authors would like to acknowledge Universiti Tenaga Nasional (UNITEN).

Conflicts of Interest:
The authors declare no conflict of interest.
Novelty Statement: To the authors' best knowledge, there is no published analysis of the anaerobic co-digestion of oily-biological sludge generated from petroleum refineries' wastewater treatment system with sugarcane bagasse under mesophilic or thermophilic conditions. This study used mesophilic anaerobic co-digestion technology. Both mechanical and thermo-chemical pre-treatment methods were applied to the raw materials prior to the digestion process to enhance the digestibility and improve microorganism activities by reducing the lignin content of sugarcane bagasse and increasing the sludge solubilization and stabilization. Due to the acidic nature of the sludge and sugarcane bagasse, NaOH was used during the pre-treatment process to adjust the pH during the anaerobic co-digestion process within 6-8. The design for the batch mixtures was determined based on C/N ratios and co-substrate VS/inoculum VS ratios to analyse their effects on biogas yield, methane content, and VS removed. A part of this project was recently published in the Sustainability journal, which contains the prediction of methane yield from this design [32]. However, this study focused and extended on the analysis, the characterization of thermo-chemically pre-treated materials, as well as the kinetics of daily biogas yields, along with interval methane yields using one-way ANOVA analysis, and the effects of C/N ratios with co-substrate VS/inoculum VS ratios on total biogas and methane yield.