Assessment of sludge reduction and biogas production in two-step anaerobic co-digestion using sesame oil cake and sewage sludge Open Access

Sesame oil, which is popular in Korea, is oil made by roasting sesame seeds, and sesame oil cake (SOC) is the residue left after squeezing oil from sesame seeds. Korea's SOC production is about 3 million tons annually, the second largest quantity after rapeseed cake (ca. 6 million tons/year) (Lee et al. 2021). A portion of SOC is used as fertilizer or as a feed supplement for poultry, but most of it is disposed of as waste (Nascimento et al. 2012). Accordingly, improperly processed SOC can cause environmental problems such as the generation of harmful insects and odors. SOC consists mainly of protein (about 30%), lignocellulose (44%) and minerals (8%) (Yasothai 2014). The amount of fat in SOC varies depending on the method of squeezing the oil and the equipment used but varies from about 5 to 11% (Nascimento et al. 2012; Yasothai 2014). The yield and composition of biogas are greatly affected by carbohydrates, fats and proteins contents of the co-feedstocks (Chanda et al. 2012). The SOC contains large amounts of components such as protein, carbon, nitrogen, phosphoric acid and potassium, and it is known to have a high carbon content (C/N ratio) (Bigoniy et al. 2012; Yasothai 2014). Therefore, SOC can be an excellent candidate as a co-feedstock for biogas in anaerobic co-digestion.

In recent years, research on the synergistic effects of increasing biogas production and reducing sludge through co-digestion with various co-feedstocks and sewage sludge is underway. Co-digestion is anaerobic digestion in which various co-feedstocks and sewage sludge are combined in a single anaerobic digester (Choi 2021). Co-digestion is a viable option to increase methane yield and promote sludge reduction over anaerobic digestion with a single feedstock (Jeong et al. 2019; Choi 2021). The utilization of a co-feedstock with different chemical compositions, particularly about nutrients, can improve the process due to the positive synergistic effect established in the digestion medium and the supply of scarce nutrients (Jhr et al. 2020). Co-feedstocks used for anaerobic co-digestion should be substances that can increase methanogenesis and reduce sewage sludge, and substances that can interfere with methanogenesis should be avoided (Choi 2020a; Cheng & Brewer 2021). According to recent research, various wastes such as food waste, fish by-products, and manure are mixed as a co-feedstock for anaerobic co-digestion (Hassan et al. 2017; Choi 2020a, 2020b, 2021; Karki et al. 2021). In addition, a synergistic effect on sludge reduction and biogas production efficiency has been reported by adding biomass-based biological auxiliary raw materials such as corn stalk, coffee grounds, wheat straw, sugarcane and corn stover (Wang et al. 2018; Pohl et al. 2019; Cheng & Brewer 2021; Karki et al. 2021).

Co-digestion using co-feedstocks can follow single and/or two-stage digestion systems. According to previous studies, if the single-stage co-digestion system is selected, the rapid decrease in pH due to local acid fermentation and scum conversion due to the difference in acid fermentation rate decrease the digestion efficiency of the digester (Pohl et al. 2019). In particular, ligno-cellulose, protein and fat contained in SOC can inhibit the formation of methane gas due to slow hydrolysis (Choi 2020a, 2021). However, if a two-stage digestion process (acidogenic fermentation and anaerobic co-digestion) is used, the problems appearing in the single-stage co-digestion process can be addressed. In other words, in the first step (acidogenic fermentation), high molecular substances such as fat, lignin, and hemicellulose are hydrolyzed to break down into short-chain fatty acids, and monosaccharides that are easily decomposed (Pohl et al. 2019; Choi 2020b). If it is mixed with sewage sludge for anaerobic co-digestion in the second stage, it can have a synergistic effect rather than inhibiting methane gas formation and sludge reduction (Choi 2020a; Karki et al. 2021). However, to date, no research results have been found to produce biogas using the two-step anaerobic co-digestion process by acidogenic fermentation of sesame oil cake (FSOC). Therefore, this study was conducted to observe the effect of anaerobic co-digestion in the first stage (acidogenic fermentation of SOC) and second stage (co-digestion of sewage sludge and FSOC) on biogas production and sewage sludge reduction efficiency. In addition, the synergistic effect of FSOC on anaerobic co-digestion was also observed to confirm whether it is possible to continuously use FSOC as a co-feedstock for anaerobic co-digestion.

Sewage sludge was collected from the bottom of an anaerobic digester in a sewage treatment plant operated in Gangneung, Korea. The wastewater treatment plant in Gangneung is operated with a capacity of 75,000 (m³/day) to treat domestic wastewater. The Biological Coating Removal (BCR) process is applied for sewage treatment. The daily average sludge inflow into the digester was 25.4–30.2 g/L for chemical oxygen demand (COD), 752.6–798.4 mg/L for total nitrogen (TN), 13.8–19.8 mg/L for total phosphorus (TP), 4800–18,023 mg/L for total solid (TS), 6415–12,657 mg/L for volatile solid (VS), and 63.4–74.8% for VS/TS. SOC was purchased as a co-feedstock from an oil mill in Gangneung city, Korea. After squeezing out the oil, the remaining hot SOC was left at room temperature for 3 hours to cool, and filtered through an 80-mesh sieve to remove contaminants such as small stones, sand, leaves. There were no other contaminants except small stones, sand and leaves. Although they were contained in very small amounts (<1% by weight), they could adversely affect fermentation, so they were removed through sieving before fermentation. Moreover, when the lumps of SOC are uniformly released through the sieve, mass transfer is facilitated, and the metabolism of microorganisms is accelerated and activity is increased, which promotes fermentation. After the pretreatment, SOC was stored in a desiccator for use in the experiment.

The experiment was carried out in a two-step anaerobic co-digestion process in a batch-test. The detailed experimental design of the second stage is summarized in Table 1. The first step was the acidogenic fermentation process of SOC. In the first step, after uniform mixing of SOC and tap water in a ratio of 1:3 with glucose (0.1 g/L), the mixture (40 L) was put into a 50 L cylindrical reactor, and it was stirred at 120 rpm for mesophilic digestion (35±2 °C) until the amount of volatile organic acids (VFAs) became constant. The prepared fermented sesame oil cake (FSOC) was stored at room temperature for use in the experiment. In the second step (anaerobic co-digestion process), sewage sludge and fermented sesame oil cake (FSOC) were mixed in wet-weight ratios of 7:3 (R2), 5:5 (R3), and 3:7 (R4) (Table 1). Then, the mixture (40 L) was put into a 50 L cylindrical reactor and biogas production and sewage sludge were observed for 30 days (Fig. S1). Among the various samples, R1 consisted of only sewage sludge for use as a control. The pH was not artificially adjusted, and the temperature for mesophilization around 35 °C±2.

TS, VS, and total chemical oxygen demand (TCOD) were measured based on standard test methods for water pollution processes (APHA 2012). Each test was conducted in triplicate, and the results were expressed as mean±standard deviation. Carbon, hydrogen and nitrogen contents of Sewage sludge, SOC and FSOC samples were measured using a fully automatic ‘Vario EL’ element analyzer (PerkinElmer, USA). The pH was measured using a pH meter (HM-30R, DDK-TOA). For VAFs, 1 mL of the sample was centrifuged at 2000 rpm for 15 min, and the supernatant was collected and analyzed using a liquid chromatography system (1260 LC system, Agilent, USA). HPX-87H (300X 7.8 mm, Biorad, USA) was used as the analysis column. The mobile phase was 5 mM sulfuric acid (H₂SO₄), the column temperature was 60 °C, and the flow rate was 0.6 mL/min. Biogas was quantified by inserting a syringe into the rubber stopper at the top of the serum bottle at a set time every day. Biogas components (CH₄, CO₂, H₂S, H₂, NH₃, etc.) were analyzed using gas chromatography (GC, Nexis SCD-2030, Shimazu, Japan) according to ASTM D7833-14.

The comparative analysis of the components of SOC and FSOC are summarized in Table 2. In anaerobic co-digestion, the carbon and nitrogen contents (C/N) affecting the growth of microorganisms increased in FSOC more than in SOC. The C/N ratio of SOC increased after the fermentation stage from 6.43 to 16.87. The content of VS also increased and the change of favorable components from SOC to FSOC, which affects biogas production in anaerobic co-digestion. A previous study reported that biogas production increased by 3–5 times when VS was consumed by 2 times (Chanda et al. 2012; Choi 2020b). This is an important reason for performing two-step anaerobic digestion without directly incorporating SOC into the anaerobic tank.

It has been reported that most anaerobic microorganisms, including methanogens, have the best activation within the pH range of 6.8–8.5 (Choi 2020b; Cheng & Brewer 2021). The pH of the anaerobic digester initially decreased due to the production of volatile acids. However, the pH of the digester increased and stabilized as the methanogen consumed volatile acids and alkalinity was created (Chanda et al. 2012; Choi 2020a). With a residence time of about 5 days or more, the methane-forming methanogens begin to consume the volatile acids rapidly. In a properly functioning anaerobic digester, the pH is maintained between 6.8 and 7.2 as the volatile acids are converted to methane and carbon dioxide. The pH of anaerobic systems is strongly influenced by the carbon dioxide content of the biogas. Therefore, a two-step co-digestion process is advantageous to shorten the biogas production lag time and promote the initial activation of methanogens.

Because the growth rate of anaerobic microorganisms for biogasification is slow, the rate is greatly affected by changes in the operating parameters of the anaerobic digester. Therefore, for stable operation of anaerobic co-digestion, it is important to maintain/manage operating factors such as pH, C/N ratio, temperature, and VFAs/alkalinity within an appropriate range (Wang et al. 2018; Cheng & Brewer 2021). In addition, when ammonia and heavy metals exceed a certain concentration, close attention is required because the proliferation and activity of anaerobic microorganisms are inhibited by the dissociation equilibrium and toxicity in the anaerobic digester (Choi 2020a, 2021). The microorganisms in the anaerobic digester show different activities depending on the pH range, and since the dissociation equilibrium of inhibitors such as ammonia and VFAs is controlled by the measured pH, it is necessary to maintain an appropriate pH in consideration of the pH value of the organic waste. The pH of the samples ranged from 6.8 to 7.5, and the pH was the lowest in R4, which had the highest content of FSOC (Table 3). The pH of the anaerobic digester initially increased due to the production of volatile acids, but alkalinity was created as the methanogenic bacteria consumed the volatile acids. Then, the pH of the digester decreased, and the process stabilized. Alkalinity has the ability to neutralize acids. Alkalinity is expressed in mg/L by converting the component contained in the form of OH−, CO₃²⁻, HCO₃₋ in water to the corresponding CaCO₃. In the acid-forming stage, organic acids are produced, but if proper alkalinity is not secured, the pH increases due to the accumulation of the produced organic acids, and the activity of methane bacteria may be inhibited (Cheng et al. 2020; Choi 2021).

The C/N ratio of the samples was in the range of 5.6–15.3, and the C/N ratio increased as the FSOC content increased. The C/N ratio for the growth of microorganisms is an important factor influencing anaerobic digestion. According to previous studies, the appropriate C/N ratio for anaerobic digestion using sewage sludge is in the range of 15–30 (Hassan et al. 2017; Choi 2020a). However, when using a co-feedstock, the appropriate range of C/N ratio may vary depending on the type of co-feedstock. For example, in the anaerobic co-digestion process, cattle manure, poultry manure, pig manure and sheep manure have relatively low C/N ratios of 7–34. In contrast, rice straw, wheat straw, corn waste, and sugarcane have high C/N ratios of 51–151 (Chanda et al. 2012; Siddique & Wahid 2018). Thus, the C/N ratio varies according to the type of co-substrate in anaerobic co-digestion. The VFAs were measured as 0.46–1.89 g/L, and VFAs/alkalinity was calculated in the range of 0.19–1.29, which corresponds to an appropriate ratio for biogas production except for the R4 sample. VFAs are organic acids generated by the decomposition of organic compounds, but the buffering capacity of the fermentation substrate decreases when they excessively accumulate inside the anaerobic digestion tank (Strazzera et al. 2018; Cheng & Brewer 2021). The concentration of VFAs that inhibits methane formation differs depending on the co-feedstocks. According to a previous study, when the concentration of VFAs is above 4000 mg/L and the ratios of VFAs and alkalinity are above 0.8 in anaerobic co-digestion, the pH decreases and methane production is inhibited (Chanda et al. 2012; Choi 2021). However, the ratio of VFAs/alkalinity was determined above 1.2 using food waste in an anaerobic co-digestion process (Jeong et al. 2019; Karki et al. 2021), but the pH and biogas production did not decrease. Therefore, it is necessary to optimize the ratio of VFAs and alkalinity according to the type of auxiliary substrate and the reaction conditions.

The average cumulative methane volumes produced over 30 days of the anaerobic co-digestion process were 0.25–0.56 mL·CH₄/g·VS, and the methane content of the produced biogas was determined to be in a range of 51.91–72.06% for all samples. The maximum average methane content reached 72.06% for R3, and the minimum average methane content was 51.91% for R1. In R2 and R3, both the process stabilization time was fast and the biogas production was high, and the content of methane gas was also higher than that of the R1 and R4 samples. The carbon dioxide content of the produced biogas was measured in the range of 0.08–0.11 mL/g·VS for all samples, which was in the range of 20–32% of the produced biogas. Sample R1 showed the highest ratio of 32%, and R3 was the lowest with 20%. According to the report of previous studies, the methane content was 60.7–68.0% and carbon dioxide content was 31.7–38.3% in biogas from the anaerobic co-digestion of Jaropha oil cake as a co-feedstock (Chanda et al. 2012). After process stabilization, the average proportion of methane produced from biogas measured by Deshpande et al. (2012) ranged from 68 to 72%, and the amount of carbon dioxide was 28–31%. Similar results for the range of methane concentration of biogas in anaerobic co-digestion using various co-feedstocks were also observed (Chanda et al. 2012; Deshpande et al. 2012; Wang et al. 2018; Pohl et al. 2019; Jhr et al. 2020).

According to a previous study, when pig manure, corn stover, and cucumber residues were used as co-feedstocks, the CPIs were determined to be 1.9 and 1.8, respectively, indicating a high synergistic effect (Wang et al. 2018). On the other hand, when food waste, toilet paper, oat straw and cow manure were used as co-feedstocks, CPIs were −1.0 and −1.5, respectively, and no synergistic effect was found in anaerobic co-digestion (Zhao et al. 2018; Kim et al. 2019). As such, the synergistic effect for biogas production is determined depending on which co-feedstock is used for anaerobic co-digestion.

Table 5 summarizes the amount of biogas produced using various co-feedstocks. Sugarcane straw, wheat straw, rice straw and fallen leaves showed low methane production of 0.067–0.174 mL/g·VS, whereas Jatropha and sunflower oil cakes reached high methane production in the range of 0.422–0.460 mL/g·VS. De-oiled seed cakes have a high VS content, including hard lignocellulose, proteins, digestible carbohydrates and lipids (Jhr et al. 2020), with the lipid content contributing the most to CH₄ formation. Higher operating temperatures, lower substrate concentrations and lower lignin content provide higher biogas yields. Although the oil seed cake contains some lignin, most of the lignocellulosic matrix degrades during oil extraction, resulting in a low C/H ratio (9–12) and a high CH₄ yield (Choi 2020a; Paritosh et al. 2021). In this study, 0.56 mL/g·VS of methane production was measured for R3, which produced about twice as much methane gas than sugarcane straw or fallen leaves.

Lignocellulosic biomass feedstock has great potential in anaerobic co-digestion due to its abundance, low cost and high availability throughout the year. However, the slow hydrolysis rate of lignocellulosic-based biomass in general limits the single digestion of these highly recalcitrant feedstocks, which is often addressed by utilizing expensive pretreatment (Cheng & Brewer 2021; Paritosh et al. 2021). Pretreatment to improve the stability of the anaerobic process has been used to loosen the structure of the biomass, release more readily degradable components, increase the accessible surface area, and remove toxic saponins from the oil seed cake (Grübel et al. 2019; Cheng & Brewer 2021). In particular, some methanogenesis-inhibiting compounds, such as furfural and hydroxymethyl-furfural, are inevitably released from the pretreatment of lignocellulosic biomass (Monlau et al. 2013; Jhr et al. 2020; Karki et al. 2021), meaning that not all pretreatments have a positive effect on CH₄ production. It was recently reported that CH₄ production was improved by more than 17% using an efficient two-stage anaerobic co-digestion (Pohl et al. 2019; Cheng & Brewer 2021; Karki et al. 2021). According to a previous study, methanogenesis inhibiting compounds were not significantly produced in the two-step anaerobic digestion (Pohl et al. 2019; Choi 2020a; Paritosh et al. 2021). In addition, two-stage anaerobic co-digestion can reduce the use of chemicals (Siddique & Wahid 2018; Choi 2020a, 2020b).

An analysis of the kinetics during anaerobic co-digestion was conducted to find the optimal conditions for biogas production by analyzing the mechanism of microbial activity and removing factors that interfere with the process (Choi 2020b). Since the production of biogas is closely related to the growth and activation of methane microorganisms, the produced biogas is a result of the growth and activation of microorganisms (Choi 2020a). The modified Gompertz equation (Equation (3)) was used for the kinetic analysis of microorganisms for biogas production in the anaerobic co-digestion process, and the results are summarized in Table 6. The λ (d) representing the reaction delay time was 5.86, 2.89, 2.33 and 3.61 for R1, R2, R3 and R4, respectively, with R1 being the longest and R3 being the shortest. In particular, R2, R3 and R4 samples using FSOC had shorter reaction delay times for biogas production compared with R1 using only sewage sludge. These results confirm that FSOC improves the biodegradability of sewage sludge, and it is also demonstrated that methanogenic archaea are activated in a short time in anaerobic co-digestion. The shorter the reaction delay time of microorganisms in the anaerobic co-digestion process, the faster the adaptation time of microorganisms for biogas production, which is also related to biogas production. This is because the production efficiency of biogas in the anaerobic co-digestion process depends on the activity of microorganisms. The values of M and R_max were inversely proportional to the value of λ. In other words, methanogenic microorganisms were activated earlier for shorter reaction delay times, which led to an increase in methane production. These results are also related to the value of C/N ratio mentioned above. The ratio of the value of M to the M_max value, which can be interpreted as the conversion efficiency of organic matter into methane in anaerobic co-digestion, was in the range of about 97.80–99.34% when using FSOC. This was about 10% higher than the case where only sewage sludge was used (87.88%). These results showed that there is a way to achieve stable digestion conditions by balancing the nutritional status in anaerobic co-digestion of FSOC. The correlation coefficients (R²) showed high significance for all samples in the range of 0.9907–0.9989. In particular, R3 showed the highest correlation of 0.9989. Considering these results, anaerobic co-digestion in which FSOC is acid-fermented and combined with sewage sludge is a new useful method that can simultaneously treat waste and produce biogas.

Energy-economic calculations for anaerobic co-digestion using FSOC may affect the feasibility of anaerobic co-digestion and may influence the assessment of whether FSOC can be used continuously as a co-feedstock (Zhang et al. 2019; Karki et al. 2021). Table 4 summarized an energy-economic analysis that was performed using methane gas produced when biogas was produced by anaerobic co-digestion using FSOC. The amount of energy recoverable was calculated by measuring the amount of methane in the total amount of biogas generated and using the calorific value of methane. The amount of methane produced per input VS showed the highest value in R3, and thus the amount of energy recovery was also highest in R3 at 5.61 kWh, followed by R4 at 4.21 kWh. R4 and R1 showed values of 2.51 and 3.21 kWh, respectively, and R1 showed the lowest energy production. In particular, R3 produced about 2.24 times more energy than that of R1, so a ratio of 5:5 is recommended when biogas is produced by anaerobic co-digestion of FSOC. Combining the experimental results of biogas generation, organic matter removal, and methane content in biogas, it was found that when sewage sludge and FSOC were mixed in anaerobic co-digestion, more energy could be recovered compared to the anaerobic process using only sewage sludge.

Sewage sludge reduction and biogas production were measured in a two-step anaerobic co-digestion process with sesame oil cake. Sewage sludge and FSOC was mixed in various ratios of (100:0 (R1), 70:30 (R2), 50:50 (R3), and 30:70 (R4)) and observed for 30 days at a mesophilization temperature of 35±2 °C. The C/N ratio increased from 6.43 for SOC to 16.87 for FSOC, and the content of carbon and hydrogen that affects the growth of microorganisms in anaerobic co-digestion increased in FSOC rather than in SOC. The average TS removal reached 28.5, 42.6, 53.2 and 34.8%, while VS removal was 37.6, 53.7, 64.9 and 44.9% for R1, R2, R3 and R4, respectively. The cumulative biogas volumes produced in R1, R2, R3 and R4 were 175.21, 305.73, 389.67, and 253.96 mL/g·VS_in, respectively. In addition, the cumulative methane production during 30 days of the anaerobic co-digestion process was confirmed to be 51.91–72.06% of the produced biogas. The CPI values were 1.29 for R2, 1.39 for R3, and 1.10 for R4, indicating that R3 had the highest synergistic effect. In terms of the reaction delay time λ (d), R1, R2, R3 and R4 showed values of 5.86, 2.89, 2.33 and 3.61, respectively, with R1 being the longest and R3 being the shortest. This means that the microorganisms contained in the R3 sample had the shortest adaptation time, and thus the production of methane gas was the highest compared to other samples. Finally, the amount of energy recovery in R3 was the highest at 5.61 kWh. Therefore, when producing biogas by anaerobic co-digestion of FSOC, a mixing ratio of 5:5 is recommended. Overall, the anaerobic co-digestion of FSOC can recover more energy and further reduce sludge compared to an anaerobic process using only sewage sludge based on the combined experimental results of biogas production, organic matter removal and methane content in biogas.

Co-feedstocks used for anaerobic co-digestion should be substances that can increase methanogenesis and reduce sewage sludge, and substances that can interfere with methanogenesis should be avoided. The sesame oil cake (SOC) contains large amounts of components such as protein, carbon, nitrogen, phosphoric acid and potassium, and it is known to have a high carbon content (C/N ratio). Therefore, SOC can be an excellent candidate as a co-feedstock for biogas in anaerobic co-digestion. However, ligno-cellulose, protein and fat contained in SOC can inhibit the formation of methane gas due to slow hydrolysis. However, if a two-stage digestion process (acidogenic fermentation and anaerobic co-digestion) is used, the problems appearing in the single-stage co-digestion process can be addressed.

This research was supported by the Basic Science Research Program and ‘Regional Innovation Strategy (RIS)’ through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (MOE) (2021R111A305924311; 2022RIS-005).

All authors whose names are listed in this manuscript certify that they have participated sufficiently in this work to take public responsibility for content, including participation in the concept, design, collection, analysis, writting, and revision of the manuscript.

This research was supported by the Basic Science Research Program and ‘Regional Innovation Strategy (RIS)’ through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (MOE) (2021R111A305924311; 2022RIS-005).

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The authors declare no competing interests.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.