Using Microbial Inoculants for Enhancing Decomposition of Citywaste and Biohythane Production

doi:10.21203/rs.3.rs-5290259/v1

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Using Microbial Inoculants for Enhancing Decomposition of Citywaste and Biohythane Production

https://doi.org/10.21203/rs.3.rs-5290259/v1

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Biohythane is a renewable energy source that carries great importance owing to its capacity to tackle energy security, environmental issues, and sustainable development goals. The current research focus on the the advantages of biohythane use as compared to the use of hydrogen and methane, as separate biofuels.The attention has been focused on the biohythane production from organic wastes, the most abundant organic substrates treated by anaerobic digestion, reporting the main milestones and the future trends. This research has primarily centered on the utilization of city waste to produce biohythane gases with the help of different consortia of microbes. Four treatments were used: T₁: Control, T₂: Organic waste + Acidithiobacillus thioxidans/Acidithiobacillus ferroxidans,T₃: Organic waste + Methanosarcina thermophila / Methanobacterium beijingense and T₄: Organicwaste + Acidithiobacillusthioxidans)/Acidithiobacillus ferroxidans) + Methanosarcina thermophila / Methanobacterium beijingense.The results indicated that the mean maximum value of CH₄ was 74.2% in T₄ where organic waste was used along with bacterial consortia followed by 64% in T₂.The highest value of H₂ was 14.96% in T₄ followed by 10.53% in T₃. The maximum value of H₂S was 63.3ppm in T₄ followed by 54.66ppm in T₂. The highest value of CO was 850ppm in T₂ followed by 680ppm in T₃. Sulfur-oxidizing bacteria, iron-oxidizing bacteria, and methane-producing bacteria were also used to enhance decomposition. Their cultures were obtained from local sources, and they were applied before the start of composting. It was concluded that the highest percentage of gases (CH₄ ,H₂S, H₂ and CO) were obtained from the anaerobic digestion where hydrogen-producing bacteria, methane-producing bacteria, and organic waste were applied together.

Biohythane

Anaerobic digestion

Bacteria consortia

Citywaste

Waste management

Solid waste management is a major global concern. The proper use of waste resources is critical to fulfilling the United Nations' seventh Sustainable Development Goal (SDG), which is to ensure affordable and clean energy [1, 2]. The progressive depletion of fossil fuels, which is expected to occur between 2069 and 2088, makes it critical to shift away from traditional fossil fuels and towards alternate energy sources [3, 4]. This transformation has resulted in the generation of biogas and compressed natural gas (CNG) from agricultural waste, the organic fraction of municipal solid waste (OFMSW), effluents from wastewater treatment facilities, agricultural waste, and animal manure using anaerobic digestion (AD) [5, 6].The present food waste generation in eight Pakistan’s mega cities is approximately 9.9 metric tonnes per day, with the rate anticipated to rise to 15 tonnes by 2030 [7]. In optimum conditions, this amount of food waste might create 1.96 million m³ and 2.96 million m³ of biogas per day by 2017 and 2030, respectively [8, 9]. Another alternative fuel, hydrogen, can be created from both organic and inorganic wastes by gasification and electrolysis [10].To take advantage of the benefits of both fuels, Hydrogen Component Inc. (HCI) developed the name Hythane [11]. It is a combination of CH₄ and H₂ that exhibited a higher reduction in the release of pollutants such as oxides of nitrogen (NOX) into the atmosphere when tested on internal combustion engines (ICE)[12]. The term Bio-Hythane refers to the production of both H₂ and CH₄ using bioresources[6]. Hythane manufacturing using chemical and physical methods is not a sustainable strategy because it takes more input energy [13, 14]. Researchers in renewable resources are interested in the prospect of producing H₂-enriched CH₄ through a separate fermentative phase [13, 15]. Bio-Hythane is a gaseous fuel composed of H₂, CH₄, and CO₂ gases at varying concentrations (5–10%, 50–60%, and 35–45%).Anaerobic digestion has been used to produce methane gas from organic residues for a long time [9, 11]. When hydrogen gas is mixed with methane gas, a new gas fuel called biohythane is generated. Because of its enhanced fuel and heat efficiency, biohythane, which is made up of hydrogen (10–25%) and methane (75–90%), is a promising alternative to conventional fossil fuels. To assist methane combustion in automotive engines, the addition of hydrogen gas can decrease carbon emissions, boost burning speed, extend the combustibility range, and enhance combustion efficiency [15].Anaerobic digestion is a biochemical process that involves a wide range of microbial groups, microorganisms are crucial to the digesters [14].The microorganisms play a significant role in CH4 production, therefore, it is important to determine their functions and the optimal operating parameters to ensure efficiency and stability of the anaerobic digestion process [16].One of the most significant advantages of this research is that it can aid in the transforming of bioreactors to optimize the maintenance of useful microorganisms in order to increase the anaerobic digestion of organic wastes for renewable energy [17].

It would be more beneficial to use a microbial consortia that is enriched from nature and typically exposed to particular environmental modifications in order to produce biohythane from natural biowastes [18].Many attempts have been made in the field of microorganisms to enhance the efficiency and stability of anaerobic digestion. [19, 20].Clostricdium, Bacteroides, Bifidobacterium, Butyrivibrio, Proteobacteria, Pseudomonas, Bacillus, Streptococcus, Eubacterium and other bacteria have an essential part in the hydrolysis and acidogenesis mechanisms. [21].Methanogens are archaea, and 66 species have been discovered so far, belonging to three orders, seven families, and 19 genera. Methanobacterium, Methanococcus, Methanobrevibacter, Methanomicrobium, Methanosarcina, and Methanosaeta are the main types of microbes that produce methane. [22, 23].One major requirement is to determine which component of the microbial community is critical in the high-efficiency or failure process of FW digesters [24].Moreover, the qualitative and quantitative relationship between microbial community structure and anaerobic digestion process function needs to be investigated [25]. In this research, four kinds of microbes were used for the decomposition and production of biohythane and other biogases including hydrogen sulphide and carbon monooxide. The Sulphur Oxidizing Bacteria Acidithiobacillus thiooxidans and Iron Oxidizing Bacteria Acidithiobacillus ferrooxidans for hydrogen production, and Methanosarcina thermophila and Methanobacterium beijingense for methane production from city waste (organic) to produce biogas and compost.Anaerobic digestion makes the management of the organic waste cycle completely sustainable [26].When the biogas produced is used properly and in many cases, the costs are significantly lower than aerobic treatment, anaerobic digestion does not represent a potentially damaging process when contrasted to incineration or landfilling. The treatment of organic waste through anaerobic digestion is a feasible and long-lasting method [24]. When processing the biogas adequately, there is a net reduction in the methane emissions into the atmosphere, thus lowering greenhouse emissions, and odors and the hygienic problems of landfills are avoided. Hydrolysis breaks down complicated macromolecules into smaller, simpler ones. Acetogenesis uses the residues of the preceding step as substrates to synthesize acetic acid (CH₃COOH)[11]. Methanogenesis is the process through which methane and carbon dioxide are synthesized [27].A green and clean substitute from organic biomass biohythane is produced. In addition to the utilisation of waste biomass, this also indirectly provides the benefit of lowering greenhouse gas emissions [28].Therefore, the present study was conducted with the following objectives: (i) To determine the efficiency of anaerobic solid-state digestion using metropolitan garbage to produce biohythane and compost. (ii) To study the potential of sulfur/iron-oxidizing bacteria and methanogenic bacteria in the production of biohythane. (iii) To examine the quality of anaerobically produced composts and their impact on soil quality and maize growth.

Preparation of city waste for biogas production

Preparation of city waste was done by manual mixing of kitchen waste (40%), lawn clippings (20%), wastepaper mix (20%), wood chips (10%), and sawdust (10%). The addition of microbial inoculants included SOB (Acidithiobacillus thiooxidans) and FeOB (Acidithiobacillus ferrooxidans) for hydrogen production, and Methanosarcina thermophila and Methanobacterium beijingense for methane production from city waste (organic) to produce biogas and compost. Compost was used for maize production in the greenhouse.

Description of Study Area

The location of this experiment was the biogas unit at PMAS Arid Agriculture University, Rawalpindi. Four plastic drums (digesters) were utilized in this biogas unit for biogas production (biohythane). Various types of city waste were used to produce biogas and compost, including kitchen waste, waste paper, wood chips, and sawdust. City waste was collected from municipal corporation dustbins and included stale vegetables and fruits. Leaf litter and paper waste were gathered from the university premises, while sawdust was sourced from the local market. These different types of waste were handled separately. The digestion mixture was prepared by manually mixing the various waste materials with a combination of sulfur/iron-oxidizing bacteria and methane-producing bacteria, along with sugar syrup (broth). The materials were mixed thoroughly and added to the respective plastic drums at a quantity of 20 kg per drum. Hot water was added to the drums to enhance decomposition activity and maintain temperature. Digital hygrometers were installed to monitor moisture and temperature readings in each drum. Each drum was fitted with two drilled holes: one small hole in the lid for inserting a digital hygrometer to measure moisture and temperature, and a second hole near the top on the side for an outlet valve. These holes were sealed properly to prevent air entry. A black perforated nylon cover was also used to help maintain the internal temperature of the container before it was covered with a lid. Moisture in the drum was maintained at 75%. Mixing of materials was conducted weekly by turning the drums horizontally on the floor. The experiment lasted for 63 days.

Biogas Measurement

Measurement of biogas was carried out by using a Biogas Analyzer (BH-4S portable Multi-Gas Detector). Goabroa Mini Hygrometer Thermometer Digital Indoor Humidity Gauge Monitor with Temperature Meter Sensor Fahrenheit (℉) was used for temperature and moisture.

Experimental Design and Treatments

In this study a Completely Randomize Design (CRD) was used to assess the effect of various microbial treatments on organic waste decomposition. CRD was chosen because it assures that each experimental unit has an equal chance of getting any of the treatments, reducing any possible biases and assisting for an accurate evaluation of the impact of different microbe combinations.The Institute of Soil and Environmental Sciences provided sulfur-oxidizing bacteria (SOB) and iron-oxidizing bacteria (FeOB), ensuring access to well-studied microbial strains for the experiment. The National Agriculture Research Centre (NARC) provided the necessary methanogenic bacteria for the research, specifically Methanosarcina thermophila and Methanobacterium beijingense.For each treatment, broth media containing the particular microbial strains were prepared and consistently applied to the organic waste. The use of broth media assured that the bacteria were growing rapidly and capable of producing the intended impact on the organic material.According to the treatments, broth media containing these microbes was applied and their discussion below in Table 1.

Table 1

Treatments of Biohythane Production
S.No	Treatments
T₁	Organic waste (Control)
T₂	Organic waste + Acidithiobacillus thioxidans/Acidithiobacillus ferroxidans
T₃	Organic waste + Methanosarcina thermophila / Methanobacterium beijingense
T₄	Organic waste + Acidithiobacillus thioxidans)/Acidithiobacillus ferroxidans) + Methanosarcina thermophila / Methanobacterium beijingense

Statistical Analysis

Analysis of Variance (ANOVA) was calculated for all set of data using software Statistics 8.1.

The major objective of this research was to produce biogas, primarily biohythane, from organic waste. The organic waste used in this study was food waste, which included fruits, leaves, paper, and sawdust. To facilitate the experiment, four plastic drums were specially constructed and used. City-generated organic waste was utilized to estimate the generation of various bio gases such as methane (CH₄), hydrogen (H₂), hydrogen sulphide (H₂S), and carbon monoxide (CO).

Measurement of biogas

The biogases were measured after two days. This experiment lasted about nine weeks. The biogases from each plastic drum were analyzed with a portable gas analyzer.

Methane production (%) from organic waste

During the process of decomposition, the generation of CH₄ is very important as it is the main ingredient for bio energy. The methane emission was measured through a digital portable gas analyzer. Figure 1 display that the highest value of CH₄ was 74.2% in T₄ Organicwaste + Acidithiobacillus thioxidans)/Acidithiobacillus ferroxidans) + Methanosarcina thermophila / Methanobacterium beijingense, 69% in T₃ Organic waste + Methanosarcina thermophila / Methanobacterium beijingense, 64% in T₂ Organic waste + Acidithiobacillus thioxidans/Acidithiobacillus ferroxidans, and 57.6% in T₁ (Control) on week 8th. While the lowest value of CH₄ was 32% in T₁ Control, 38.6% in T₂ Organic waste + Acidithiobacillus thioxidans/Acidithiobacillus ferroxidans, 35% in T₃ Organic waste + Methanosarcina thermophila / Methanobacterium beijingense, and 37.6% in T₄ Organic waste + Acidithiobacillusthioxidans)/Acidithiobacillus ferroxidans) + Methanosarcina thermophila / Methanobacterium beijingense at week 2nd. The application of iron and sulfur-oxidizing bacteria in biohythane generation, especially in the context of methane production, absorb their distinctive metabolic pathways to enhance the anaerobic digestion process. The mechanism of iron and sulphur oxidizing bacteria includes Iron oxidizing bacteria oxidizing ferrous iron (Fe²⁺) to ferric iron (Fe³⁺), which has multiple benefits, including the oxidation process can help break down complex organic materials, making it more accessible to methanogens. IOB precipitates sulfides as iron sulfides, which reduces sulfide inhibition of methanogenic bacteria. Iron Oxidizing Bacteria oxidative activity can boost hydrogen-producing bacteria, resulting in increased hydrogen yields .Sulfur oxidizing bacteria oxidizes sulphur molecules like hydrogen sulfide (H₂S) to sulfate (SO₄²⁻). Sulfide control involves converting harmful H₂S to sulphate, which decreases sulphide toxicity and inhibits methanogenesis. By eliminating hydrogen sulfide, Sulphur Oxidizing Bacteria creates a more approving environment for methanogens, increasing methane production. Sulphur Oxidizing Bacteria and methanogens can develop syntrophic partnerships, which boosts overall process efficiency. Iron Oxidizing Bacteria and Sulphur Oxidizing Bacteria activities reduce sulfide toxicity and increase substrate availability, resulting in more stable anaerobic digestion. The presence of these bacteria may result in greater hydrogen and methane yields. Reducing hydrogen sulfide content enhances biogas quality, making it more acceptable for energy uses.By converting hydrogen and carbon monoxide into methane, Methanobacterium beijingense contribute to the ultimate methane yield in the biohythane mixture [29, 30]. Methanosarcina thermophila can convert many metabolites produced during the fermentation stage into methane, making them incredibly beneficial for use in mixed-substrate anaerobic digesters. The presence of both Methanobacterium beijingense and Methanosarcina thermophila can improve anaerobic digestion stability by assuring efficient hydrogen and substrate consumption.These methanogens complementary metabolic pathways help maximize methane synthesis from a wide range of substrates.The combined activity of these methanogens increases biogas quality, resulting in increased methane content and lower hydrogen sulfide levels, especially when paired with iron and sulfur-oxidizing bacteria [31, 32].

Hydrogen production (%) from organic waste

Figure 2 showed that the highest value of H₂ was 14.96% in Organic waste + Acidithiobacillus thioxidans)/Acidithiobacillus ferroxidans) + Methanosarcina thermophila / Methanobacterium beijingense in the 5th week, 10.53% in T₃ Organic waste + Methanosarcina thermophila / Methanobacterium beijingense in the 5th week, 8.8% in T₂ Organic waste + Acidithiobacillus thioxidans/Acidithiobacillus ferroxidans in 6th week, and 7.4% in T₁ (Control) on 5th week. While the lowest value of H₂ was 3.2% in T₁(Control) on 1st week, 2.8% in T₂ Organic waste + Acidithiobacillus thioxidans/Acidithiobacillus ferroxidans on 9th week, 3.6% in T₃ Organic waste + Methanosarcina thermophila / Methanobacterium beijingense on 9th week, and 3.5% in T₄ Organic waste + Acidithiobacillusthioxidans /Acidithiobacillus ferroxidans) + Methanosarcina thermophila / Methanobacterium beijingense on 9th week.In terms of its potential to reduce global carbon dioxide (CO₂) emissions, hydrogen gas is a clean energy source and a promising substitute for fossil fuels. It is less energy-intensive and more environmental friendly.Research indicated that pure cultures were preferable to mixed cultures, particularly in terms of substrate selectivity and relatively simple growth manipulation. Iron-oxidizing bacteria oxidize ferrous iron, generating electrons that hydrogen-producing bacteria can use to convert protons (H+) to hydrogen gas (H₂).Sulfur oxidizing bacteria can oxidize organic molecules and create hydrogen sulphide. Hydrogen-producing bacteria can then use hydrogen sulphide to make hydrogen gas [12, 33] .

Hydrogen Sulfide (ppm) from organic waste

The amount of hydrogen sulfide generated during the breakdown of city waste is entirely dependent on the waste's composition. Anaerobic digestion was found to generate more hydrogen sulfide than aerobic digestion. Figure 3 revealed that the maximum value of H₂S was 63.3ppm in T₄ Organic waste + Acidithiobacillus thioxidans)/Acidithiobacillus ferroxidans) + Methanosarcina thermophila / Methanobacterium beijingense, 54.66ppm in T₂ Organic waste + Acidithiobacillus thioxidans/Acidithiobacillus ferroxidans, and 45.33ppm in T₃ Organic waste + Methanosarcina thermophila / Methanobacterium beijingense, and 28.66ppm in T₁ Control at 3rd week. While the minimum value of H₂S was 13.6ppm in T₁ Control, 24ppm in T₃ Organic waste + Methanosarcina thermophila / Methanobacterium beijingense, 27.33ppm in T₂ Organic waste + Acidithiobacillus thioxidans/Acidithiobacillus ferroxidans, and 31.66ppm in T₄ Organic waste + Acidithiobacillusthioxidans)/Acidithiobacillus ferroxidans) + Methanosarcina thermophila / Methanobacterium beijingense in the 1st week.The combination of IOB and SOB can effectively regulate hydrogen sulphide levels through precipitation and oxidation mechanisms.Iron Oxidizing Bacteria can provide ferrous iron for sulphide precipitation, whereas SOB can oxidize the remaining hydrogen sulphide, resulting in complete sulphide control. Lower hydrogen sulphide levels decrease corrosion and toxicity, leading to a more stable and effective anaerobic digestion process [29, 34].

Carbon monoxide (ppm) from organic waste

Figure 4 indicated that the mean maximum value of carbon monooxide was 855 ppm in T₃ Organic waste + Methanosarcina thermophila / Methanobacterium beijingense, 680 ppm in T₂ Organic waste + Acidithiobacillus thioxidans/Acidithiobacillus ferroxidans 561.6 ppm in T₄ Organic waste + Acidithiobacillusthioxidans)/Acidithiobacillus ferroxidans) + Methanosarcina thermophila / Methanobacterium beijingense, and 460 ppm in T₁ Control at 4th week. While the mean minimum value of CO was 461.6ppm in T₃ Organic waste + Methanosarcina thermophila / Methanobacterium beijingense, 562.3ppm in T₁ Control, 681.6ppm in T₂ Organic waste + Acidithiobacillus thioxidans/Acidithiobacillus ferroxidans, and 565ppm in T₄ Organic waste + Acidithiobacillusthioxidans)/Acidithiobacillus ferroxidans) + Methanosarcina thermophila / Methanobacterium beijingense on the 7th week.Iron and sulfur-oxidizing bacteria can help produce biohythane, a combination of hydrogen and methane from organic materials. These bacteria can help manage and reduce carbon monoxide (CO) levels, which can be toxic to many microorganisms involved in biogas production. These bacteria play an important role in iron oxidation and can influence the cycling of metals and other element cycles in various situations. Their metabolic activities can be leveraged in biohydrogen and biogas generation processes due to their propensity to oxidize iron compounds, which can ultimately impact microbial populations participating in biohythane production. Acidithiobacillus ferrooxidans are sulfur-oxidizing bacteria that contribute to the generation of sulfate and protons. These metabolic processes can be integrated into biohythane synthesis, where the oxidation of sulphur compounds may contribute to maintaining the redox balance and providing substrates for other microorganisms involved in biogas production .Carbon monoxide dehydrogenase (CODH) enzymes enable certain bacteria and archaea to use CO as both a carbon and an energy source. These enzymes aid in the oxidation of CO to CO₂, which can subsequently be used in a variety of biosynthetic pathways, including methane and hydrogen synthesis.These microbes' metabolic plasticity makes them beneficial in biohythane production, as carbon monoxide can be a by-product of organic matter decomposition [35, 36].

Measurements of Temperature (⁰C)

Figure 5 illustrated the outcomes of the temperature were recorded in T₄ Organic waste + Acidithiobacillus thioxidans)/Acidithiobacillus ferroxidans) + Methanosarcina thermophila / Methanobacterium beijingense, which revealed that the maximum peak of the temperature was 50.2 ⁰C on day 36th, while treatment (control) showed the lowest temperature of 27 ⁰C on the 62nd day. Temperature effects on biogas generation were discovered at 20°C, 35°C, and 45°C. According to studies, thermophilic bacteria Methanosarcina thermophila are more efficient in terms of loading rate, gas yield, and hydraulic retention period, but require a larger temperature input. Furthermore, thermophilic bacteria are more sensitive to temperature variations and environmental factors than mesophilic bacteria.Several microbial communities increased and became more prevalent as the temperature increased, whereas others were carried away by anaerobic digestion [20, 24].Methanogens are categorized as mesophilic (37°C) and thermophilic (55°C) due to their capacity to function well over a variety of temperatures [37, 38] .

Challenges and Future consideration

Biohythane production facilitates the recovery of valuable resources from organic waste, reducing landfill use and associated environmental impacts. By converting organic waste into biohythane, methane emissions from landfills are reduced, contributing to lower overall greenhouse gas emissions. Advances in microbial strains, enzymes, and reactor designs can enhance the efficiency and yield of biohythane production. Integrating biohythane production with other renewable energy technologies can create more resilient and efficient waste management systems. By addressing these challenges and focusing on future considerations, biohythane can offer significant benefits for waste management, contributing to a more sustainable and efficient system for handling organic waste.The use of methanogens microorganisms, specifically Methanosarcina thermophila and Methanobacterium beijingense, in the combined application for city waste decomposition and biohythane synthesis, provides substantial advantages. Iron- and sulfur-oxidizing bacteria facilitate the breakdown of complex organic compounds, thus promoting methanogenesis. Methanosarcina thermophila and Methanobacterium beijingense, both essential methanogenic bacteria, efficiently convert the resulting simpler substances into methane, which is the main component of biohythane.This integrated microbial strategy accelerates the waste breakdown and increases biohythane yield, helping to promote sustainable waste management and renewable energy production. The improved decomposition process minimizes odorous emissions while limiting the production of persistent and toxic organic compounds, addressing environmental and public health issues. Furthermore, this technology promotes the circular economy by converting city waste into valuable bio-energy. Future research should focus on optimizing the microbial consortia ratios, researching the capacity for growth of this technique, and assessing the financial potential of large-scale deployment. By improving these processes, combining iron and sulphur oxidizing microorganisms with Methanosarcina thermophila and Methanobacterium beijingense can completely transform urban waste management and biohythane production, leading the way for more sustainable and efficient waste-to-energy solutions.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose. This work was specifically performed to reduce the city waste and produce the alternative sources of energy as in last few years the consumption of energy is going to increase day by day.

Funding

This work is not supported by any national, international or private organization and the authors declare that no funds, grants, or other support were received during the preparation of this manuscript

Author Contribution

All authors contributed to the study conception and design. The concept of experiment was designed by Muhammad Fayyaz Farid. Material preparation, data collection and analysis were performed by Amna Nisar and Memoona Ijaz. The first draft of the manuscript was written by Muhammad Fayyaz Farid and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Using Microbial Inoculants for Enhancing Decomposition of Citywaste and Biohythane Production

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Abstract

Figures

INTRODUCTION

MATERIAL AND METHODS

Preparation of city waste for biogas production

Description of Study Area

Biogas Measurement

Experimental Design and Treatments

Statistical Analysis

RESULTS

Measurement of biogas

Methane production (%) from organic waste

Hydrogen production (%) from organic waste

Hydrogen Sulfide (ppm) from organic waste

Carbon monoxide (ppm) from organic waste

Measurements of Temperature (⁰C)

Challenges and Future consideration

Declarations

Competing Interests

Funding

Author Contribution

Data Availability

References

Additional Declarations

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