Anaerobic co-digestion of cow manure and microalgae to increase biogas production: A sustainable bioenergy source

2.1 Collection of substrates

Identification of microalgae necessitates a comprehensive procedure involving several approaches. First, samples were collected and examined under a microscope to document morphological features. These samples are then grown to create pure isolates. The identification of microalgae species has been conducted following the recommended procedures by Bouck (1965), Levring (1946) and Coppejans et al. (2009). The microalgae were cultured employing BG 11 media supplemented with vitamin B12 and maintained at a temperature of 25 °C under a light intensity of 45 µmol/m^-2(−|−)S-1 lx for an average of 20 days in order to reach the mid-log phase of growth. By employing FT-IR spectroscopy to examine the microalgae's biochemical composition, complementary data is acquired. The amalgamation of morphological ones such, molecular, and biochemical data ensures accurate and reliable identification, which is necessary for the microalgae species to be utilized successfully in biotechnological processes.

The cow manure was conveyed to the lab after being retrieved from the Ismail cow farm in Dammam City (26.4207° N, 50.0888° E), Saudi Arabia. The tiny plant-based waste materials in the cattle manure have been carefully separated. Then, the collected cattle manure was diluted with de-chlorinated water in equal proportions (1:1), carefully stirred for ten min at 2000 rpm, and subsequently strained through a finer nylon mesh as recommended by Khayum et al. (2018).

The four distinct microalgae species, specifically Anabaena sp., Oscillatoria sp., Chlorella sp., and Tetraselmis sp., were obtained from the Red Sea in Jeddah, Saudi Arabia, at coordinates 21.5292° N and 39.1611° E (Fig. 1). The microalgae species were properly stored in uncontaminated zip-lock plastic bags under controlled cooling conditions and subsequently transported to our laboratory. Next, the microalgae species underwent a thorough washing process using mother seawater to eliminate any extra sand and other components.

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Fig. 1

External morphology of different microalgae. (a) Anabaena sp., (b) Oscillatoria sp., (c) Chlorella sp., and (d) Tetraselmis sp. Scale bar: 10 µm.

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2.2 Pre-treatment process for microalgae species

Prior to the integration of microalgae into efficient biogas generation. Pretreatment methods are necessary to achieve high yields of biogas due to the intricate cell structure of microalgae. In the present study, four different species of microalgae were effectively pretreated using a combination of treatment methods, including ultra-sonication (Brand: VEVOR) with water. Sonication was performed using 10–15 % of the microalgae biomass. Additionally, the microalgae were pretreated employing hot water treatment at 120 °C, in accordance with the method (simple modification) outlined by Saleem et al. (2020).

2.3 Experimental setup

The present research was carried out in our laboratory using pilot-scale anaerobic digesters. Mainly, plastic container with a total volume of approximately 20 L was used to assemble the anaerobic digesters (Fig. 2a). The oxygen molecules have been carefully eliminated from the digester and sealed with butyl rubber caps. Further, it is closed with M−seal to ensure anaerobic conditions. Three distinct concentrations (25, 50, and 75 % v/v) have been employed to produce biogas. The production of biogas was measured daily employing the water displacement method. The entire experimental process was carried out in a mesophilic environment at a temperature of 36.85 °C. The experimental containers were shaken for 1–2 min twice daily before biogas levels were recorded (Fig. 2b) as recommended by Zhai et al. (2015).

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Fig. 2

(a) Anaerobic laboratory digesters; (b) Gas collecting methods (c) Flame experiment.

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2.4 Fourier transform infrared spectroscopy (FT-IR) analysis

In general, FT-IR (Perkin Elmer, USA) is capable of precisely identifying the numerous chemical functional groups in substrate materials. The FT-IR spectra were observed range of 4000 – 450 cm⁻¹.

2.5 Analytical methods

The pH of the substrate materials (1:10 w/v) was measured using a digital pH meter (model − STARA1117). The estimation of total solids (TS) and total dissolved solids (TDS) was conducted following the APHA (2017) guidelines. To determine the TS, samples were collected from experimental glass bottles and subjected to an evaporation procedure utilizing a drying oven. The dried vaporized sample was exposed to a temperature of 105 °C for 1 h, after that it was allowed to cool and subsequently weighed. $$\mathit{Calculation}:mgTS/L=\frac{\left(A-B\right)\times 1000}{\mathit{Samplevolume},mL}$$ Where: A=amount of evaporated residue + dish, mg

B=mass of the dish, mg.

For the analysis of the TDS, samples were meticulously collected from all experimental glass bottles and subsequently cleaned to eliminate any remaining residues. A clean dish (180 ± 2 °C for 1 h in an oven) was utilized. The components that had been filtered were then transferred into the evaporating dish (clean dish), and the evaporation process was then carried out in an oven. To determine the final total dissolved solids, it is necessary to place the sample in an oven at a temperature of 180 ± 2 °C for 1 h. $$\mathit{Calculation}:mgTDS/L=\frac{\left(A-B\right)\times 1000}{\mathit{Samplevolume},mL}$$ Where: A=amount of evaporated residue + dish, mg

B=mass of the dish, mg.

To assess the amount of nitrate, approximately 50 mL of the sample was filtered, and then 1 mL of hydrochloric acid was added, and the mixture was properly mixed. Then, A standard curve was prepared by utilizing the nitrate solution, ranging from 0 to 35.0 mL. The final samples were analyzed at a wavelength of 220 nm employing spectrophotometry to measure the nitrate concentration (Armstrong 1963). The concentration of ammonia was analyzed using the titrimetric method with the use of a boric acid solution as indicated by Meeker and Wagner (1933).

2.6 Flame test

The flame test is an essential component in the analysis of the biogas produced by the experimental digester (Fig. 2c). This evaluation was conducted carefully in a darkened room. The Bunsen burner was linked to one end of the small plastic pipe, and the bio-digester was connected to the other end of the pipe to complete the connection. Further, the quantity of biogas was determined using the flammable nature.

2.7 Seed germination assay using digested slurry

The determination of phytotoxicity activity requires assessing the quality of the final biogas slurry. We obtained four distinct seed varieties, namely Sorgham bicolor, Paspalum scrobiculatum, Oryza sativa, Zea mays, and Vigna unguiculata, from Local Market. Then, the seeds were subjected to a cleaning process using a 2 % solution of sodium hypochlorite (NaClO) for 10 min, followed by rinsing with distilled water. In order to conduct an experiment, the biogas slurry was obtained from biogas treatments and subsequently combined with distilled water in a ratio of 10:1 (v/w) as recommended by Tiquia et al. (1996). About 10 sterilized seeds of each variety were inserted on glass Petri plates containing Whatman number 1 filter paper. Biogas slurry extraction (5 mL) was added to the Petri plates, while distilled water was used as a control. The Petri plates were incubated under a tightly controlled 16-hour dark cycle for a period of 8 to 10 days. The morphometric characteristics of seeds, including germination %, shoot length, root length, fresh weight, dry weight, and number of leaves were quantified.

2.8 Statistical analysis

The statistical analysis was conducted using SPSS software (version 21). The mean ± standard error was used to represent the combined seed germination and chemical characteristics. In addition, One-way analysis of variance (ANOVA) was conducted to assess the differences between the experimental treatments and the control group. HSD multiple comparison tests were performed at a significance level of P<0.05.

3.1 Impact of microalgae on biogas production

There has been a significant increase in the utilization of several types of microalgae in recent years for the production of biogas. In the present study, the production of biogas daily through the use of an anaerobic digester that consists of four species of microalgae (Chlorella sp., Oscillatoria sp., Tetracelmis sp., Anabaena sp.), depicted in Fig. 1, coupled with sewage water and cattle manure. Over the course of six days, experiments were conducted with varying proportions of 25 %, 50 %, and 75 %.

The combination of microalgae and cattle manure can produce a considerable amount of biogas, mainly methane. For example, the Anabaena sp. biomass at a proportion of 50 % yielded a substantial amount of methane, with a recorded value of 345 ± 2.88 mL CH₄/g VS. This was followed by Chlorella at 50 % proportion, which yielded 297.96 ± 0.49 mL CH₄/g VS. Then, Oscillatoria sp. at 75 % proportion, which yielded 185.0 ± 0.288 CH₄/g VS. Tetracelmis sp. at 75 % ratio yielded 100.0 ± 0.577 mL CH₄/g VS and the 75 % proportion of control was produced 138.32 ± 0.50CH4/g VS of methane as presented in Table 1. For biogas generation, there was a statistically (one-way ANOVA) significant difference among the various proportions. The incorporation of microalgae biomass into the anaerobic digester resulted in a substantial increase in the production of biogas. Varol and Ugurlu (2016) demonstrated that the utilization of Spirulina platensis, combined with sewage sludge under two-phase digesting conditions led to a considerable increase in methane yield 640 mL/gVS. The microalgae have a substantial number of polysaccharides, a variety of proteins, lipids, and a minimal amount of lignin, all of which contribute to an increase in the production of methane in anaerobic conditions (Perazzoli et al., 2017; Dębowski et al., 2017). In the present study, the insertion of a co-substrate of Anabaena biomass resulted in a twofold increase in the amount of methane. This may be due to the greater digestibility of Anabaena biomass in comparison to other species of microalgae. The generation of efficient biogas depends on the type of microalgae species employed (Mussgnug et al., 2010a,b). The authors recommend that Anabaena sp. is more efficient than other microalgae species such as Oscillatoria sp., Chlorella sp., and Tetraselmis sp. for biogas production. Further, the C/N ratio of the co-substrate materials plays a crucial role in regulating the production of biogas. When the C/N ratio is at an extreme level, it can potentially affect the biochemical pathways. However, previous reports could not accurately provide the optimal level of C/N ratio (Dębowski et al., 2020). In a study conducted by Deublein and Steinhauser (2008), it was found that maintaining a C/N ratio of 16 to 25 can lead to improved biogas production. This study detected a slight reduction in methane production, which may be due to insufficient water in biogas-producing systems. Anaerobic digestion of microalgae (Scenedesmus sp., Nannochloropsis sp., and Chlorella sp.), poultry manure, and sewage sludge results in a significant reduction in the production of biogas when the water content is reduced as revealed by Torres et al. (2023). The authors assert that the production of biogas is contingent upon the specific kind of substrate materials utilized in the anaerobic digestion system. The employment of inappropriate combinations of substrates can diminish the production of biogas.

According to the experiment, the burning test of biogas showed that a burnable gas was detected on the 10th day of fermentation in Chlorella sp., specifically in the 25 % digesters treatments. The biogas production commenced on the 10th day following the initiation of the digester. On the 16th day, the biogas ignited for the first time, producing a steady blue flame that burned for approximately 10 s (Fig. 4c). This study examined the continuous anaerobic co-digestion of a mixture of microalgae, cow dung, and sewage water. The co-digestion of microalgae with cow dung shown synergistic effects, resulting in a threefold increase in biogas production compared to the mono-digestion of cow dung.

3.2 Analysis of chemical parameters and FT-IR

For determining the quality of the final substrate, it is essential to measure a wide range of chemical properties (total solids, total dissolved solids, nitrate, and ammonia) in sediment produced by biogas treatments. According to the findings of the current study, a significant concentrations of total solids, total dissolved solids, nitrate, and ammonia were found in Anabaena sp., (75 %) and Chlorella sp. (75 %). The high concentration of TS could potentially impact the production of biogas. In a study conducted by Deepanraj et al. (2014), it was found that a TS level of 7.5 % is ideal for maximizing biogas production. A recent study by Torres et al. (2023) has confirmed that a decrease of less than 8.49 % in TS levels can lead to an increase in biogas production.

In this study, the digester sludge was analyzed using FT-IR (Fig. 3). After 25 % digestion with Chlorella sp., the FT-IR spectra revealed peaks at 1645 and 1530 cm⁻¹, indicating vibrations of C=O and N–H bonds of amide, which are related with proteins. The peaks at 3304 cm⁻¹ revealed the presence of C–H bonds associated with polysaccharides and carbohydrates. Khayum et al. (2018) performed a similar FT-IR investigation. Hence, these peaks decrease in the after-digestion suggesting the decomposition of carbohydrates and proteins (Ben Yahmed et al., 2017).

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Fig. 3

(a) Over all experimental set up of water displacement system (a) FT-IR analysis of cow manure sludge (control) from anaerobic digestion (b) Chlorella sp., 50 % (c) Anabaena sp.,50 %.

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Fig. 4

Seed germination percentages for various agriculturally valuable seeds.

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3.3 Germination studies

In order to carry out the seed germination test, it is necessary to investigate the quality of the final biogas slurry. The seed germination assay was conducted using biogas slurry consisting of Anabaena sp. 50 % and a control group. The slurry from microalgae biogas plants shows the highest amount of seed germination when compared to the control (cow manure). The current study found that Sorgham bicolor had the maximum seed germination rate (94.2 %) when subjected to microalgae-associated biogas slurry (Fig. 4). In contrast, the lowest seed germination rate (5.2 %) was observed in the control group of Vigna unguiculata as presented in Fig. Microalgae biogas treatments were significantly (P<0.05) different from control biogas treatments. The seed germination can be significantly improved by using the biogas slurry, which contains several essential enzymes and chemical factors. According to Zhao et al. (2014), 75 percent biogas leachate is capable of promoting the germination of Vicia faba L. seeds. The study conducted by Miyuki et al. (2006) suggested that increasing the germination index by around 50 % is a significant indication of the absence of harmful substances in the substrate materials.

The root length of all the crop seedlings ranged from 0.3 ± 0.78 to 15.5 ± 0.02 cm per seedling. The Sorghum bicolor seeds exhibited the greatest root length, measuring 15.0 ± 0.02 cm per planting. The shoot length of all the crop planting ranged from 0.4 ± 0.0 to 12.5 ± 0.33 cm per seeding. The shoot length of Paspalum scrobiculatum reached a maximum value of 12.5 ± 0.33 cm per seedling, which was higher than the shoot length of the control seedlings (Table 2). The sludge from the Anabaena sp.50 % digester may contain numerous substances that promote plant growth, hence enhancing germination and crop development. Moreover, the authors strongly assert that microalgae-based biogas slurry exhibits a significant concentration of nitrogen and phosphorus, which effectively promotes plant growth. A study conducted by Zheng et al. (2016) found that the application of biogas slurry in the field has the potential to enhance the seedling and production of peanuts. Yu et al. (2010) demonstrated that biogas slurry contains plant vital nutritional elements such as N, P, and K, which actively improve the quality of tomatoes. In light of this, Anabaena sp. (50 %) and Chlorella sp. (50 %) have the potential to be utilized in the rapid production of biogas. Furthermore, the utilization of biogas slurry would enhance crop yield.