Processing of Agricultural Residues with a High Concentration of Structural Carbohydrates into Biogas Using Selective Biological Products
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1. Introduction
Additional input into the anaerobic process of biochemical and biological complex bioproducts accelerates and intensifies the decomposition of cellulose and hemicellulose into glucose, galactose, and arabinose. At the same time, the yield of biomethane and the intensity of its release would increase, and it would be possible to form the desired quality of digestate. Thus, using biochemical methods of anaerobic process activation in biogas production could increase the sustainability of the technology, as the amount of product (in this case, energy) would increase with the same input of raw materials and energy.
There is a lack of scientific literature on changes in organic waste with a high concentration of structural carbohydrates in biogas production using biochemical methods, as well as on changes in structural carbohydrates using bioproducts in various organic wastes, not limited to plant biomass. So far, few studies have been conducted on the influence of biochemical methods on the degradation of structural carbohydrates, biogas production rates, and digestate composition using chemical compounds, biological additives, and micro/macro-elements.
The researched technological process of processing hard-to-degrade raw materials into biogas is innovative in that the intensity of biogas production and the stability of the process will be increased by using selective bioproducts for the treatment of hard-to-degrade (structural carbohydrates) organic materials. The knowledge gained will enable the development of innovative technologies for processing organic waste into biogas, which would increase the potential of renewable energy resources and reduce pollution of the atmosphere, soil, and water. The aim of the present study is to determine the effect of employing selective biological products in the processing of raw materials with a high concentration of structural carbohydrates on the biogas production process and biomethane potential.
2. Materials and Methods
2.1. Feedstock Characteristics
The concentration of TS in the studied straw was 92.215%. Total nitrogen was 0.564% in TS, phosphorus was 0.041% in TS, and potassium was 1.341% in TS. Total nitrogen was 0.52% in natural matter, phosphorus was 0.038% in natural matter, and potassium was 1.237% in natural matter.
The proper composition of trace elements effectively eliminates all contributors to process instability, leading to a notable improvement in process reliability.
2.2. Characteristics of Biological Product (BP)
In order to improve the abundance of microorganisms and operational efficiency, samples of hard-to-degrade raw material were inoculated with a biological product. The biological product consisted of microorganisms of the Trichoderma spp. genus belonging to the collection of microorganisms of the company “Bioenergy LT”. The concentration of microorganism spores in the sample was at least 1 × 109 mL−1. These microorganisms are characterized by the synthesis of proteases, cellulases, amylases, lipases, and other enzymes that ensure efficient decomposition of biomass. The BP was prepared from the company’s collection of microorganisms stored at −80 °C. Microorganism cultures included in the product were revived on rigid LB- (for bacteria) and PDA-fed (for fungi) media. For bacteria, under sterile conditions, we added 200 mL of sterile culture medium to a 1 L Erlenmeyer flask and introduced one colony of bacteria using a sterile loop. The flask was sealed, and the prepared inoculum was incubated in a shaker for 24 h at 30 °C and 130 rpm. After incubation, the inoculum was microscopically examined and inoculated onto rigid LB media using the serial dilution agar plating method. The purity of the prepared inoculum was checked, and the number of viable cells was evaluated. For fungi, 200 mL of sterile nutrient medium was added to a 1 L Erlenmeyer flask under sterile conditions. Mycelial discs 5 mm in diameter were cut from the grown mycelial colonies and one of them was introduced into an Erlenmeyer flask with nutrient medium using a sterile loop. The flask was sealed, and the prepared inoculum was incubated in a shaker for 7 days at 30 °C and 130 rpm. After incubation, the inoculum was inoculated on solid PDA nutrient media, and the purity of the prepared inoculum was checked. For inoculation, the tested inoculum was dosed into a reactor containing a sterile nutrient-enriched nutrient medium.
2.3. Methodology for the Preparation of the Studied Biological Product without pH Regulation
2.4. Methodology for the Preparation of the Studied Biological Product with pH Regulation
2.4.1. Influence of BP on the Development of Microorganism Cultures
Straw and monocrystalline cellulose were chosen as reference materials to evaluate the effect of the BP. Organic acids were used to prepare acidic solutions: lactic acid, formic acid, citric acid, and acetic acid. The organic acids were diluted with deionized water until a pH of 5.00 was reached. Eight different variants were tested: WWS control, monocrystalline cellulose control, WWS+BP, monocrystalline cellulose+BP, WWS+propionic acid+BP, WWS+formic acid+BP, WWS+citric acid+BP, and WWS+acetic acid+BP. Each variant was tested with 3 replications. Plastic 100 mL screw-on containers were used for testing. An 8 mm-diameter hole was drilled in the caps of the containers for gas diffusion. In order to prevent intensive loss of moisture, the stoppers were covered from above with cotton swabs. For each tested variant, 2 g of WWS or monocrystalline cellulose was used. Tests were performed in a Memmert Model 100–800 drying cabinet (Memmert GmbH + Co. KG, Schwabach, Germany) at a constant temperature of 25.0 ± 0.5 °C.
Before starting the study, containers of WWS and monocrystalline cellulose (not wetted) were disinfected by keeping them for 1 h at 105 °C. Subsequently, irrigation and inoculation of the BP were performed on WWS and monocrystalline cellulose. The test mass was moistened to 70% moisture using 4 mL of deionized water or acidic solution, of which 1 mL was used as the inoculum. A concentrated solution of the BP was used for the inoculation of the BP, which was diluted with deionized water at a ratio of 1:250, and only then was 1 mL of the prepared solution used for inoculation. The biological product was diluted 1:10 with deionized water using 1 mL for inoculation.
2.4.2. BBP Tests
At the beginning of the tests, the straw was treated with the prepared BP solution for 7 days. To prepare the BP solution, a concentrated BP preparation was used, which was diluted at a ratio of 1:250 (2 mL of BP preparation, 500 g of water) with a stabilized citric acid solution (5 kg of deionized water + 14.66 g of citric acid (C6H8O7∙H2O) + 28.498 g of sodium citrate dihydrate (C6H5Na3O7∙H2O)), with a pH of 5.10. In each of the replicates, 5 g of air-dried WWS was used, which was treated with 50 g of the prepared BP solution. Nine repetitions were performed. After processing, studies were continued in the incubator. For 48 h, the contents of uncovered containers with treated straw were kept at 25.0 ± 0.5 °C to evaporate excess moisture. After 48 h, the containers were covered with appropriate stoppers and further (5 days) kept in an incubator maintained at a temperature of 25.0 ± 0.5 °C.
Plastic 100 mL screw-on containers were used for testing. An 8 mm-diameter hole was drilled in the caps of the containers for gas diffusion. In order to prevent intense moisture loss, the corks were covered from above with cotton swabs. Tests were performed in a Memmert Model 100–800 drying cabinet (Memmert GmbH + Co. KG, Schwabach, Germany) at a constant temperature of 25.0 ± 0.5 °C. Before starting the study, the containers were disinfected by keeping them for 1 h at 105 °C.
2.4.3. Periodic Loading Studies
At the beginning of the tests, the straw was treated with the prepared BP solution for 7 days. To prepare the BP solution, a concentrated BP was used, which was diluted at a ratio of 1:250 (2 mL of BP, 500 g of water) with a stabilized citric acid solution (5 kg of deionized water + 14.66 g of citric acid (C6H8O7∙H2O) + 28.498 g of sodium citrate dihydrate (C6H5Na3O7∙H2O)), with a pH of 5.10. The treatment was carried out using 174 g of WWS and 1740 g of stabilized acid solution with preparations (9 mL) in a 10 L container. The prepared contents of the container were stored for 7 days at a temperature of 25 °C. After treatment, the content of the treated straw was dosed into 15 plastic resealable 1 L containers (127 g each) and stored until used. This pretreatment was repeated three times, but the method of storage of the prepared samples was different. The storage of pretreated WWS was performed at storage temperatures of +6 °C (sample variant WWS+BPT1), −18 °C (WWS+BPT2), and +25 °C (WWS+BPT3).
An 8 mm-diameter hole was drilled in the cap of the 10 L container for gas diffusion. In order to prevent intense moisture loss, the cap was covered from above with a cotton swab. Tests were performed in a Memmert Model 100–800 drying cabinet (Memmert GmbH + Co. KG, Schwabach, Germany) at a constant temperature of 25.0 ± 0.5 °C. Before starting the study, the container was disinfected by keeping it for 1 h at 105 °C.
2.5. Research Equipment
Experimental research was carried out in the Biogas Laboratory of the Vytautas Magnus University Agriculture Academy.
Samples of the raw material under investigation were weighed with an electronic scale, KERN EG4200-2NM (Kern & Shon GmbH, Balingen, Germany), with an accuracy of ±0.02% and resolution of 0.01 g. The pH of the raw material and processed substrate was determined during each run with a Hanna PH213 (Hanna Instruments Ltd., Woonsocket, RI, USA), with a measurement accuracy of ±0.01 and a resolution of 0.01.
The biogas production rate was continuously recorded and automatically adjusted according to standard conditions (1 bar and 0 °C).
Biogas potential determination study experiments were performed in triplicate to ensure experimental data reliability.
Sludge from an anaerobic reactor operating at a sewage treatment plant (Kaunas, Lithuania) was used for the inoculation of biogas production studies. The inoculum was stored in a 19 L anaerobic reactor in the laboratory at mesophilic temperature (37.0 ± 0.5 °C) and used 5 days after the sludge was collected from the sewage treatment anaerobic reactor to ensure sludge deactivation and degassing.
Statistica 10 software (StatSoft®, Hamburg, Germany) was used for final data collection and processing. The mean of the study results was compared using the t-test criterion. Differences were considered significant when p-values were less than 0.05.
2.6. Determination of Biogas Yield and Energy Value of Feedstock
here, bM is the biogas yield from fresh matter, L/kg, and bVS is the biogas yield from VS, L/kg.
3. Results and Discussion
3.1. Influence of the BP on the Development of Microorganism Cultures
Samples of the affected areas were sent to Bioenergy LT for microscopic examination, during which the activity of the BP preparation was characterized and confirmed.
3.2. The BBP Tests
The concentration of TS in the studied straw was 92.215%. Total nitrogen was 0.564% in TS, phosphorus was 0.041% in TS, and potassium was 1.341% in TS. Total nitrogen was 0.52% in natural matter, phosphorus was 0.038% in natural matter, and potassium was 1.237% in natural matter. The proper composition of trace elements effectively eliminates all contributors to process instability, leading to a notable improvement in process reliability.
The coefficients of determination (R2) for the modified Gompertz model were 0.984 and 0.996 for untreated WWS and treated with the BP, demonstrating significant reliability of the attained factors.
It can be said that the use of BP during pretreatment ensured a 20.8% higher biogas yield compared to untreated wheat straw.
The pH measurements were conducted for each reactor before and after the test. It was found that the pH value of rectors with WWS treated with the BP was higher (+0.42) at the beginning (7.88 ± 0.03) of the BBP study than at the end of the study (7.46 ± 0.05). The difference between the means of the tested variants was statistically significant at the 95% confidence level (p < 0.05). At the start of the study, the pH of raw WWS was measured at 7.82 ± 0.02. After the BBP tests were conducted, the pH of the raw WWS was slightly lower, with a value of 7.47 ± 0.04.
Results suggest that the biological product affects the biogas yield and biogas composition of WWS.
3.3. Results of the Processing of Straw Treated with the BP into Biogas in the Continuous Load Mode
The continuous load test was carried out in 5 stages and lasted for 115 days.
From the 73rd day (WWS+BPT1), WWS treated with the BP was added, and the prepared samples were kept in a refrigerator at a temperature of +6 °C before being loaded into the bioreactor. At this stage, 553.6 ± 11.8 L/kg VS of biogas was produced. The increase in biogas yield was 11.4% compared to untreated straw, and this difference was significant at the 95% confidence level (p > 0.05). The methane concentration was 51.8 ± 0.5%.
From day 83 to day 93 (WWS+BPT2), WWS treated with the BP was added to the reactor. After 7 days of treatment, the samples were stored in a freezer at −18 °C. During this period, 582.0 ± 24.1 L/kg VS of biogas was obtained. The increase in biogas yield was 14.7% compared to untreated straw, and these differences were significant at the 95% confidence level (p < 0.05). The methane concentration was 52.0 ± 0.4%.
From day 93 to day 103 (WWS+BPT3), wheat straw treated with the BP was added to the reactor. After 7 days of processing, the samples were stored in a thermal chamber at a temperature of +25 °C. During this stage, 577.4 ± 15.5 L/kg VS of biogas was obtained. The increase in biogas yield was 14.0% compared to untreated straw, and these differences were significant at the 95% confidence level (p < 0.05). The methane concentration was 52.3 ± 0.8%.
4. Conclusions
The scientific conclusions drawn from this comprehensive study provide valuable insights into the effectiveness of the biological product in multiple aspects of sustainable biogas production. Firstly, this study underscored the influence of BP on the development of microorganism cultures on winter wheat straw, as evidenced by the emergence of green mold-like zones on the straw’s surface. Microscopic examination confirmed the activity of BP, establishing its role in promoting microbial growth. This demonstrates the potential of BP in enhancing the microbial ecosystem, a key component in biogas production.
Moreover, this study highlighted the significant impact of BP treatment on biogas production from winter wheat straw. The addition of BP during pretreatment resulted in a substantial 20.8% increase in biogas yield compared to untreated straw. The biogas yield from untreated WWS was 364.1 ± 5.6 L/kg natural matter, while the WWS treated with the BP showed a notable increase, reaching 439.9 ± 9.0 L/kg natural matter. Remarkably, the biogas produced from BP-treated straw exhibited a higher methane concentration, indicating its potential as a valuable source of biomethane. This finding holds promise for enhancing the energy output of biogas facilities using agricultural residues.
Furthermore, the application of BP treatment led to a remarkable 27.4% improvement in biomethane yield compared to untreated wheat straw. There has been an improvement in enzymatic digestion of straw as a result of disruption of straw structural components, which has made it easier for cellulose and hemicellulose to be accessible for enzymatic digestion. These results suggest that BP treatment has the potential to contribute to improved energy recovery from wheat straw and support the transition to more sustainable and efficient biogas production processes.
In addition to examining the impact of BP-treated straw on biogas yield, this study also explored the continuous loading mode as a means of demonstrating the compatibility of BP-treated straw with the biogas production process. The introduction of BP-treated straw led to an 11.4% increase in biogas yield without significant changes in the methane concentration. The robustness of the system was further confirmed by the consistency of the biomethane yield and methane concentration under varying storage conditions, underscoring the reliability of using BP-treated straw in biogas production processes. BP treatment provides valuable guidance for optimizing biogas production efficiency and contributes to the broader goal of sustainable energy production from biomass resources.
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