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Environ Eng Res > Volume 28(2); 2023 > Article
Sharma, Gupta, and Singh: Co-digestion of euryhaline microalgae Scenedesmus sp. MKB. with paddy straw for biogas production


Biogas is a clean and renewable biofuel source produced by anaerobic digestion of organic matter and can replace conventional sources of energy such as fossil fuels. The present study was undertaken to assess biogas production from microalgae Scenedesmus sp. MKB. (MN796425) which was isolated from the saline water collected from the waterlogged area of Southwest Punjab. The biogas production for different concentrations of microalgal biomass, microalgal filtrate, and paddy straw was observed in laboratory conditions and the performance of microalgae was also studied in field conditions. The co-digestion of microalgal biomass and paddy straw under laboratory conditions revealed maximum production of 60.18 L when both were co-digested as compared to 48.56 L during the digestion of paddy straw alone. Kinetics of biogas production studied by modified Gompertz equation revealed that the highest biogas production potential (P) of 68.58 mLg−1 volatile solids at the biogas production rate (Rm) of 1.34 mLg−1d−1 with lag phase (λ) of 0.66 days. Microalgae biomass production under field conditions was carried out in an open raceway pond of 200 L capacity and later biomass was digested in 1 m3 digester that showed cumulative biogas production of 2,407 L and it was 20.44% more than control.

1. Introduction

Advancement in various technologies and demographic explosion caused enhanced energy consumption rate leading to two challenging situations namely energy crisis and environmental pollution [1]. The exhausting supply of fossil fuels extremely hindered this ever-increasing energy demand and caused the research community to search for alternative and environmentally favorable technology [2, 3]. Biofuels are the most viable option as they have many commendatory attributes like low toxicity, renewability, biodegradability and can be produced from substrates like animal fats, starch, vegetable oils, and a newly emerged source of oleaginous microorganisms such as microalgae [4]. Biomass and organic wastes are envisaged as beneficial feedstock for energy production purposes. Besides, biomass to biofuel conversion cycle is carbon dioxide (CO2) neutral process as it emits no net CO2 to the environment [5]. Edible energy crops have many drawbacks like poor energy balances, pose a threat to food security, and cause deforestation [6]. Therefore, micro-algae are being explored globally as an alternative to edible crops for bio-energy production. They are third-generation biofuels. These organisms are photosynthetic and simple nutrients like Nitrogen (N), Phosphorus (P), and Potassium (K) are required for sustaining growth [7]. They have been firstly exploited for biodiesel production but their production at a large scale has not been achieved due to different constraints such as the requirement of extensive cultivation and harvesting technologies for efficient processing. Moreover, in comparison to fossil fuels, biodiesel production from microalgae is still under the developmental phase [8]. It has been reported that significantly more energy has been produced from algal biomass with low lipid content or lipid extracted algal residue through anaerobic digestion (AD) compared to biodiesel production [9]. Also, wet biomass can be used for AD, therefore, no extensive drying methods are needed [10].
Stubble burning, commonly practiced in states like Punjab, Haryana, and Uttar Pradesh (mainly paddy straw) is another major issue [11, 12] that is causing detrimental environmental pollution and results in respiratory as well as other different kinds of health issues. However, lignocellulosic biomass like paddy straw can be effectively managed by anaerobic digestion through the dry fermentation route for producing clean fuel. Paddy straw has a very high carbon:nitrogen (C/N) ratio of 70–90:1 which is not suitable for efficient anaerobic digestion. However, it can be brought down by supplying an exogenous nitrogen source [13] like micro-algae and can make the anaerobic digestion process more efficient. Microalgae are efficient in removing nutrients from waste streams also. Different studies have indicated that algal biomass can be anaerobically digested to obtain biogas. Algae and cow dung (CD) was mixed in three different proportions, viz. 80:20% (S1), 40:60% (S2), and 20:80% (S3) on a mass basis and found that S2 gave the highest methane (CH4) and carbon dioxide (CO2) content in the biogas [14]. Co-digestion of rice husks and microalgae in the ratio of 1:6 carried out in the biodigester at mesophilic temperatures (29–33°C) produced 156.25 L biogas after 75 days [15]. Enzymatically pretreated (1% cellulase and enzyme mix) microalgal biomass when subjected to anaerobic digestion increased the methane content by 8 and 15% for cellulase and the enzyme mix respectively [16]. Microalgae polyculture dominated by Scenedesmus sp. grown on nutrient-deficient medium contained more volatile solids (VS) and thus provided higher biogas yield compared to nutrient-rich medium [17]. Microalgae when added to cassava starch effluent and yeast increased biogas production to 726.43 mL/g total solids (TS) as compared to control (189 mL/g TS) [18]. Microalgae S. subsalsa BGLR6 was used as a substrate for enzymatic, hydrothermal pretreatment, and anaerobic digestion for biogas production. The pretreatments enhanced the ultimate biogas production potential and biogas production rate (Rm) by 1.11–1.65 and 1.07–1.68 folds compared to the untreated raw biomass [10]. Co-digestion of microalgae biomass of Arthrospira platensis and Platymonas Subcordiformis to the agricultural biogas plants using cattle manure and maize silage indicated a systematic increase in the biogas production efficiency when the ratio of microalgae biomass was increased from 0 to 40% VS. A higher microalgae biomass ratio had no significant impact in improving the efficiency of biogas production [19]. Hence, based on the different studies for extracting energy from different microalgae species, the present study was carried out with the objectives to evaluate the biogas production potential of microalgae Scenedesmus sp. MKB. (MN796425) previously isolated from the waterlogged area of Southwest Punjab [20]. Secondly, to conduct the outdoor experiments in raceway pond for microalgal biomass production and its utilization in co-digestion with paddy straw in large capacity biogas plant so that the technology can be disseminated to farmer field. The procedure followed in conducting the present study is shown in Fig. 1.

2. Material and Methods

2.1. Raw Material and Its Source

Microalgae species viz. Scenedesmus sp. procured from Biogas laboratory, Department of Renewable Energy Engineering, Punjab Agricultural University (PAU), Ludhiana, Punjab (India) was used for co-digestion experiments. Microalgae were previously isolated in the lab from saline water (HCO3, Cl and Ca2+ with Mg2+ ions in the range of 7.6–14.0, 5–22, and 2.5–10.1 milliequivalents per L (mEq/L), respectively) collected from South-West districts of Punjab by enrichment in different media such as Algae culture medium [21], Guillard’s F/2 medium [22], Bold’s basal medium [23], BG-11 medium [24], and Conway medium [25]. Pure microalgal colonies were then isolated by various techniques like standard dilution, plating, and streaking methods. Molecular identification of the microalgal species was carried out from Chromous Biotech Pvt. Ltd. Banglore (India). The microalgae identified were Scenedesmus sp. MKB. (MN796425). The identified algae is a robust and dominant type that can tolerate a wider range of environmental fluctuations because when the algae was grown for 3–4 years in an open raceway pond, it did not allow any other microbial species to dominate. The biomass of the microalgae has been used for biogas production both alone and in combination with paddy straw. The paddy straw was procured from research fields of PAU, Ludhiana. It was used in chopped condition having a straw size of 5–10 cm. The straw was chopped to make easy and fast decomposition by bacteria during anaerobic digestion. It is also reported that particle size reduction of paddy straw breaks the cell walls and makes the organic substrate readily available to microbes for decomposition [26]. This in turn helps in the increase of the surface area of rice straw that results in breaking down the polymer structure and hence, there is increase of hydrolysis yield as well as rate during digestion [27]. Cow dung and bio-digested slurry were also used in small quantities to provide the necessary methanogenic bacteria required for anaerobic digestion. They were collected from the dairy farm of Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana.

2.2. Microalgal Biomass Cultivation at Lab Scale

The chemicals used in the experiments were purchased from Hi-media chemicals Pvt. Ltd. ACM media [21] was used for cultivation of microalgae with the following composition (g per L) and specifications: NaNO3 (Sodium nitrate, HI-AR™/ACS, Assay: ≥ 99%): 1.000; K2HPO4 (Di-potassium hydrogen phosphate anhydrous, Hi-AR™): 0.500; MgSO4.7H2O (Magnesium sulphate heptahydrate, Hi-AR™/ACS, Assay: ≥ 99.5%): 0.513; NH4Cl (Ammonium chloride, Hi-AR ™): 0.050; CaCl2.2H2O (Calcium chloride dihydrate, AR™/ACS, Assay: ≥ 99%): 0.058; FeCl3 (Ferric chloride, Hi-AR™, Assay: ≥ 98%): 0.003; NaHCO3 (Sodium hydrogen carbonate, Hi-AR™, Assay: ≥ 99.5%): 16.800. All media constituents were mixed in distilled water and sterilized by autoclaving at the temperature of 121°C and 15 psi pressure for 15 min before inoculation except sodium hydrogen carbonate which was autoclaved separately and mixed later. Microalgae were inoculated at a rate of 10% v/v in fabricated glass ponds filled with 2,000 mL of ACM media. Ponds (made of glass) were maintained at a temperature of 28±2°C with a pH of 11.0. Illumination was provided by a white LED light source having the light intensity of 4,000 Lux as measured by a lux meter and the photoperiod cycle was fixed at 16:8 h (light:dark). Aeration was provided by small air pumps (Venus Aqua AP-308A, 3W) having airflow adjustment mode. Biomass was harvested by filtration using muslin cloth after 30 days.

2.3. Experimental Study at Lab Scale

The experimental setup comprised of glass anaerobic bioreactors of 2 L capacity working at mesophilic conditions under batch mode. The digester was sealed tightly with rubber cork using Araldite as adhesive and had two connections: one with a liquid reservoir and the other with a gas collecting chamber. All the digesters were kept under stationary conditions and shaken manually daily. The biogas production experiment was conducted for 127–130 days from October 2018 to February 2019. A total of six digesters labeled as A to F were maintained in triplicate and the composition of different feedstock fed to each digester is presented in Table 1. The water displacement method was used for measuring the volume of biogas produced after every 24 h. Biogas production was studied as cumulative biogas production in terms of L/kg TS added and L/kg VS added.
Table 1 showed that the first two digesters were taken as control in which digester A was fed with 1,500 g microalgal biomass with the moisture content of 95–97% and digester B had 250 g paddy straw which was pretreated by soaking overnight with water. The other digesters from C to F were fed with different ratios of microalgal biomass, microalgal filtrate (the liquid left after filtration of biomass), and paddy straw. The ratio of microalgal or filtrate used in required digesters as compared to paddy straw was 6:1 and the maximum amount used was 1,500 g. These digesters (C to F) were also provided with 150 mL bio-digested slurry and 300 g of cattle dung that would act as inoculum and inducer respectively to initiate fermentation. The biogas yield of bio-digested slurry and cattle dung was also studied separately and their yield was subtracted from the biogas yield of the substrates in digesters C to F so that the co-digestion potential of microalgae and paddy straw can be measured. The digesters A and B were not fed with any inoculum to observe the fermentation potential of individual materials.

2.4. Kinetic Study of Biogas Production

Biogas production kinetics was studied by using a modified Gompertz equation [28] because the equation can be used to study the kinetics of biogas production as it assumes that under batch conditions, the growth rate of methanogenic bacteria in the bio-digester is directly proportional to the biogas produced. The curve generated through the equation must have the following characteristics: (1) the S type curve model, (2) positive growth rate (3) unique inflection point, and horizontal asymptote otherwise the biogas yield data cannot be successfully fitted. These Gompertz parameters, especially lag phase (λ), are also crucial in determining the efficiency of anaerobic digestion [29]. Experimental biogas production data from different set of digesters was noted after every 24 h. The cumulative biogas (mL biogas g−1 VS) yield was fitted in Eq. (1) for calculating various parameters using MS Solver of Excel 2007.
Where M is the cumulative biogas yield (mL biogas g−1 VS added), P is the enhancement in ultimate biogas yield (mLg−1 VS), Rm is the maximum rate of biogas production (mLg−1d −1) and λ is the lag phase (days) and e = 2.718.

2.5. Analytical Methods

Proximate analysis (Total solids ‘TS’, volatile solids ‘VS’, and ash) of the feedstock fed into the digesters was done by following standard procedures [30]. The chemical composition (cellulose, hemi-cellulose, lignin, and silica) of the feedstock at the field scale was also determined. Daily ambient temperature was noted by the thermometer. The reduction in volatile solids rate (VSR%) was calculated by Eq. (2) as given below:
Where: VSbd and VSad signify volatile solids before and after anaerobic digestion.

2.6. Statistical Analysis

All experiments were conducted in triplicates. IBM SPSS Statistics 22 software was used to find the significant difference between the mean values with the help of Tukey’s HSD Multiple Range test in the case of biogas yield.

3. Results and Discussions

3.1. Cumulative Biogas Production

The cumulative biogas yield from different digesters is shown in Fig. 2. It is clear that the biogas production in digester A loaded with only microalgal biomass was minimum with only 0.80 L gas in 127 days. This may be due to the absence of required methanogens which delayed the fermentation of microalgae. Moreover, the other reason might be the inability of the methanogens that were produced with time to degrade the hemicellulosic wall of microalgae for anaerobic bacteria degradation [31]. However, in the case of paddy straw in digester B, there is a continuous release of biogas up to the last day due to the degradation of paddy straw cellulose and hemicelluloses in anaerobic conditions and it led to the total production of 48.56 L of biogas and 73.16 L/kg VS added. A lesser amount of biogas might be due to the high C/N ratio of 70–90:1 of paddy straw. The biogas production in digesters C to F increases with time and then it becomes constant for the remaining days of observation. The biogas production increased for 45 days in digester C but in digester D, it increased for all the days of observation at a slow pace and the total biogas production was 28.80 and 24.62 L, respectively in digester C and D. There is a decrease in biogas production in co-digestion of microalgae with cow dung and bio-digested slurry because algae contain a higher percentage of proteins that degrade and built high ammonia levels with time which produce a toxic effect to methanogens and stops the production of biogas [31, 32]. The highest biogas was obtained in digester E having paddy straw along with microalgal biomass. This was due to improvement in the C/N ratio of the substrate that enhances biogas yield up to 60.18 L and 88.18 L/kg VS added. Moreover, the paddy straw provides a growth medium for microalgae that in turn helps in the increase of degradability by consuming more nutrients from rice straw. The co-digestion of microalgae with high-carbon biomass such as rice straw causes a more balanced substrate for anaerobic digestion [32, 33]. Scenedesmus sp. has also been reported with good carbon sequestration potential [34, 35] that results in the conversion of microalgal lipids into sustainable biofuels as a substitute for fossil hydrocarbons. The results are also in line with the findings of co-digestion of Chlorella sp. “CD01” substrate with cow rumen fluid and manure as inoculum produced 314.5 mL biogas with the yield of 43.23 mL/g TS and 1,758 mL biogas with the yield of 98.96 mL/g TS, respectively [36]. The positive effect of co-digestion of different organic wastes has been found by other research workers. Lee et al. [37] reported that the co-digestion of Sewage sludge (Ss) improves the performance of anaerobic digestion in terms of high biogas yield and better process stability. Several combinations of organic waste with sludge are operationally possible such as brewery waste and cow dung, Ss and pig manure, agricultural resources, etc. The Biochemical Methane Potential (BMP) of fresh leachate and domestic wastewaters co-digestion over a period of 90 d showed that cumulative biogas production was insignificant in the case of wastewaters mono-digestion while the co-digestion significantly improves the BMP [38]. The co-digestion of rice straw and Hydrilla verticillata (nitrogen-rich co-substrate) at different C/N ratios revealed enhanced process stability and higher volumetric methane yields than mono-digestion (control). Co-digestion showed the highest methane productivity with an increase of 40% than the control at C/N ratio 25. This increase was attributed to the improvement of proper nutritional structure (C/N) [39]. There was minimum biogas production in digester F of 18.43 L and 27.67 L/kg VS added among the co-digested experiments. This was due to the presence of insufficient algal cells infiltrate that could obtain a balanced C/N ratio. Secondly, the filtrate has more concentration of growth media that itself has high pH of up to 11 and it could act as a toxic medium for the methanogens produced during co-digestion. Table 3 also showed that digesters C and D have more biogas production of 800 L and 769.50 L in terms of total solids added, whereas, it was 870 and 855 L in terms of VS added respectively. This showed that the microalgae is a potential source for biofuel production per kg of solids added but it contains total solids of only 2.5 to 3.0% that demand high biomass production which is still a constraint for its use in biofuel applications [40].

3.2. Proximate and Chemical Analysis of Feedstock

Table 2 represents the results of proximate and chemical analysis of feedstock fed into different digesters before and after completion of the anaerobic digestion process. The total solids in microalgae alone were 2.70% and they increased to 3.60% when bio-digested slurry and cattle dung was added to the microalgal biomass. Similarly, the volatile solids increased from 67.45 to 91.87%. There was a significant decrease in total solids and volatile solids of feedstock after the digestion process as compared to initial e.g. in digester C, the TS decreased from 3.60 to 3.30% and VS decreased from 91.87 to 87.45% which indicated the consumption of solids by micro-organisms during digestion for production of biogas. In the process of bio-methanation, after the hydrolysis of the substrate molecules, small molecules are converted to volatile fatty acids and alcohols by fermentative bacteria which by further action of acetogenins are transformed to acetate, carbon dioxide, hydrogen, and finally methanogens convert acetate to methane [41]. After the completion of the anaerobic digestion process, ash content was increased notably. The increase in ash was due to the conversion of volatile matter of biomass to biogas. In addition, there was a significant decrease in cellulose and hemicelluloses of the organic matter used but the lignin content did not change significantly after digestion e.g. in digester D of microalgal filtrate digestion, cellulose, and hemicelluloses decreased from 20.80 to 11.20% and 14.20 to 12.60%, respectively but there was insignificant change in lignin which remained at 0.40%. The decrease is due to the hydrolysis of cellulose and hemicelluloses in the anaerobic digestion process. In bio-methanation, during the initial hydrolysis phase, hydrolase enzymes are released by the facultative and obligate anaerobic bacteria which are responsible for the breakdown of large polymers like cellulose, carbohydrate, proteins, and fats into monomers. However, lignocellulosic material and lignin are broken down occurs slowly and incompletely [42].

3.3. Kinetics of Biogas Production

The results after fitting of data in the modified Gompertz equation for all the digesters are shown in Table 4 and Fig. 3. Table 4 showed that the experimental data was successfully fitted in the Gompertz model with R2 values varying from 0.984 to 0.997 and therefore, the model can be used successfully to explain the anaerobic digestion rate conditions for all the digesters. It is also indicated that digester C has the highest biogas production potential (P) of 30.85 mLg−1 VS with the maximum biogas production rate (Rm) of 0.58 mLg−1d−1 in the mono-digestion study which was higher than the control digester A (P) = 1.23 mLg−1 VS, (Rm) = 0.02 mLg−1d−1). Lower biogas production in control digester A is due to the rigid and recalcitrant nature of the microalgal cell wall. The cell wall lysis for the disintegration of membrane structure is a crucial problem in algal biorefinery and biofuel processes [43]. For the action of anaerobic microorganisms on the internal organic matter of the cell, cell wall hydrolysis is obligatory [44]. The cell wall of microalgae Scenedesmus consists of hexose sugar molecules such as glucose, mannose, and galactose and is rigid in nature [45]. It has three layers: inner cellulosic layer, middle algaenan layer, and outer pectic layer [46]. Many microalgae of chlorophyte division have algaenan which is a recalcitrant, insoluble, and non-hydrolyzable substance and the presence of highly resistant aliphatic polymers makes it extremely difficult to breakdown [43]. Digester E showed the maximum values of all the parameters of the Gompertz equation i.e. biogas production potential (P) of 68.58 mLg−1 VS at biogas production rate (Rm) of 1.34 mLg−1d−1 than control digester B which has P of 54.00 mLg−1 at Rm of 1.00 mLg−1d−1. Before starting biogas production, the initial time required for adaptation of the bacteria to the substrates in media is indicated by the parameter λ i.e. lag phase obtained from modified Gompertz equation [47]. Except in both the control digesters i.e. digester A and digester B, biogas production started shortly after inoculation in all the digesters. For control digesters, A and B lag phase was 15.13 and 8.00 days respectively whereas the lag phase of 1.30, 0.00, 0.66, and 1.31 days was shown by digester C, D, E, and F, respectively. The high correlation coefficient of determination (R2 = 0.984–0.997) showed great agreement between predicted biogas yield (mLg−1VS) (from the model) and experimental biogas yield (mLg−1VS) (Table 4). The difference between predicted biogas yield (mLg−1VS) and experimental biogas yield (mLg−1VS) ranged from 0.00 to 0.08. Modified Gompertz equation is based upon the assumption that bacterial growth under different conditions in the digester is proportional to the rate of methane production and is a sigmoidal function [48]. Similarly, the equation was used by many research workers to study the kinetics of biogas yield in lignocellulosic biomass such as rice straw [49] and wheat straw [50] and the Gompertz equation was found best in revealing the biogas potential of wastes as a function of the digestion time. During anaerobic digestion, volatile solids present in the substrates are degraded by the bacteria for conversion to biogas [51]. The maximum percent reduction in VS was in digester C (7.80%) and digester E (27.01%) in mono-digestion and co-digestion, respectively. Both were higher than respective control digesters (control digester A = 1.91% and control digester A = 20.61%). Osman et al. [52] in their study for evaluation of methane yield from different ratios of alginate-extracted (AEWLJ) and non-extracted (NAEWLJ) waste of Laminaria japonica in mono and co-digestion with rice straw (RS) used the modified Gompertz equation. Syaichurrozi [51] reported the highest biogas yield of 113.92 ± 6.90 mL/g VS in co-digestion of aquatic weed Salvinia molesta (SM) and rice straw (RS) at the mass ratio of 40:60. For predicting biogas yield, the cone model was selected to design the volume of the digester and develop a kinetic model for volatile degradability rate, out of three models (Gompertz model, first-order kinetic model, and cone model).

4. Outdoor Cultivation of Microalgae and Its Performance in Co-digestion

4.1. Raceway Pond

The pilot scale study for the growth of microalgae and its co-digestion was carried out to validate the results obtained in the lab study. The schematic view of the raceway pond used to grow microalgae in the field is shown in Fig. 4. The open algal raceway pond was installed in the field area of the department. The pond was made of a double-walled fiber sheet. The pond had a length of 157 cm with a width of 66 cm. The depth of the pond for microalgae growth was 40 cm while the working depth was kept at 30 cm for allowing sufficient light penetration for microalgal photosynthesis. A partition wall of width 5 mm was also constructed in the middle of the pond and it was provided with 25 cm slits on both ends of the pond to facilitate the circulation of water. The width of chambers for microalgae growth on both sides of the partition wall was 24 cm. Natural light provided the required illumination and shading was done by covering the pond with the green sheet. Thus, imparting the photoperiod cycle of 16:8 hours (light/dark). The shading was made possible by the provision of a frame made from MS pipes along the sides of the pond with a height of 43 cm for constructing the artificial and temporary roofs. One large paddle wheel of diameter 39.6 cm was installed with a center above 7.5 cm from the top of the raceway pond for mixing of feeding material and it was driven by an 850W adjustable speed motor (Model: BLDC NY-850T, 48V DC). The paddle wheel was operated during the daytime. Carbon-dioxide could be injected into the pond through a micro- porous polymer tube (gas diffuser) provided at one side of the pond. A heater with a temperature controller for controlled heating was also provided inside the pond for maintaining micro algae growth in winters. The pH was checked manually by the pH meter. At the start, 150 L of unsterilized ACM media was filled in the pond, and inoculum @ 10% v/v was added. Subsequently, only three chemicals viz. NaNO3 (1.00 g/L), K2HPO4 (0.500 g/L), and MgSO4.7H2O (0.513 g/L) mixed in simple tap water were used for further refilling the pond. The pH was adjusted to 11.0 with carbonate-bicarbonate buffer @ 2.06 g Na2CO3 and 16.8 g NaHCO3 per litre. Finally, harvesting of biomass was done by filtration through the muslin cloth and used further for analysis.

4.2. Co-digestion for Biogas Production

Co-digestion study of microalgae with paddy straw at field scale was conducted for approximately 05 months. The process of anaerobic digestion was carried out at field scale in the double-walled digesters of 1 m3 capacity made up of reinforced plastic with a diameter of 146 cm and height of 104 cm. An electric heater was provided at bottom of the digester to heat the water circulated between two walls was used to maintain the required temperature. A water mist was sprayed inside by an ultrasonic nebulizer through ultrasonic waves for maintaining humidity. The digester lid was made impervious by keeping in a water seal which assists in preventing the leakage of the gas produced. The composition of feedstock added to the experimental digester was 50 kg paddy straw (soaked overnight), 20 kg cow dung, 5 kg bio-digested slurry, and 7.5 kg microalgal biomass. Control digester was run simultaneously containing 50 kg of paddy straw (soaked overnight). The ratio of microalgae biomass and paddy straw was different as used in lab studies due to the limited growth of microalgae biomass obtained. According to Liu et al. [53], there is no established method for estimating the appropriate input ratio. It is also reported that there is no technical basis for selecting the proportions of waste to prepare for co-digestion and improve the performance of anaerobic digestion [54]. Biogas measurement was done by the gas meter (m3/h) attached at the side of the digester. Results revealed the total biogas production was 2,407 L in the experimental digester as compared to 1,915 L in the control. This indicated that there was an enhancement of 20.44% in biogas obtained from co-digestion of microalgae and paddy straw. Cumulative biogas production was 31.67 and 25.87 L/kg in terms of TS and VS added, respectively. Proximate analysis showed TS of 76.00%, VS of 93.06%, and ash content of 6.94%, respectively. Chemical composition revealed cellulose, hemicellulose, lignin, and silica of 40.0, 28.90, 2.56, and 4.30% respectively. After completion of anaerobic digestion, there was a significant reduction in TS and VS to 70.90 and 85.34%, respectively while Ash content increased to 14.66%. Other parameters like cellulose (33.40), hemi-celluloses (22.45%), lignin (2.45%) also decreased while silica content increased (5.10%) compared to control. In control digester TS, VS, ash, cellulose, hemicellulose, lignin and silica content were 74.00, 90.90, 14.00, 35.55, 23.12, 3.15, 14.23, respectively before anaerobic digestion and 68.67, 84.89, 20.34, 30.84, 19.14, 2.90, 15.00, respectively after digestion. The results showed that the addition of microalgae substrate to the paddy straw in co-digestion helped in releasing more volatile solids and thus more biogas release.

5. Conclusions

The proximate analysis showed that the total solids in microalgae alone were 2.70% and there is an increase in total solids as bio-digested slurry and cattle dung was added to the microalgal biomass. The volatile solids also increased from 67.45 to 91.87%. After anaerobic digestion, the total solids and volatile solids of feedstock in co-digested biomass of microalgae and paddy straw decreased from 3.60 to 3.30% and VS decreased from 91.87 to 87.45%. The cumulative biogas production was also highest in co-digested biomass digester as compared to the control of mono-digested biomass of micro-algae and paddy straw. The pilot-scale study for co-digestion of microalgae and paddy straw after algae harvesting from an open algal raceway pond showed an increase of 20.44% in biogas production as compared to anaerobic digestion of paddy straw alone. It can be concluded from the study that co-digestion of algal biomass with paddy straw can be taken as an opportunity for getting more energy through anaerobic digestion to meet the energy needs of rural Punjab and the potential for the growth of sufficient micro-algae biomass can be tapped from the waterlogged area.


The authors are thankful to ICAR for its continuous support to All India Coordinated Research Project on Energy in Agriculture and Agro-based industries that helped in completion of the study.



The authors declare that they have no conflict of interest.

Author Contributions

N.S. (Ph.D. student) conducted all the experiments and wrote the manuscript; U.G. (Principal Scientist) planned and supervised the experiments; I.S. (Scientist) reviewed and revised the whole manuscript.


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Fig. 1
Flow-diagram showing the procedure followed for conducting the study.
Fig. 2
Cumulative biogas production during anaerobic digestion at lab scale in different digesters.
Fig. 3
Fitted Gompterz model for anaerobic digestion at lab scale in different digesters.
Fig. 4
Schematic view of raceway pond used for growth of microalgae at field scale.
Table 1
Amount of Different Raw Materials Used for Anaerobic Digestion at Lab Scale
Digester label Microalgal biomass Microalgal filtrate Paddy straw Biodigested slurry Cow dung
A 1,500 g - - - -
B - - 250 g - -
C 1,500 g - - 150 mL 300 g
D - 1,500 mL - 150 mL 300 g
E 1,500 g - 250 g 150 mL 300 g
F - 1,500 mL 250 g 150 mL 300 g
Table 2
Proximate and Chemical Composition of Feedstock before and after Anaerobic Digestion Process
Digester Proximate composition (%) Chemical composition (%)

Total solids (TS) Volatile solids (VS) Cellulose Hemicellulose Lignin

Before After Before After Before After Before After Before After
A 2.70 2.10 67.5 66.2 2.40 1.8 3.4 2.8 0.70 0.70
B 76.0 65.0 87.3 69.3 33.60 30.4 25.2 22.0 3.40 3.20
C 3.60 3.3 91.9 87.5 17.4 13.6 3.4 3.3 0.20 0.20
D 3.20 3.0 90.0 83.0 20.8 11.2 14.2 12.6 0.40 0.40
E 74.0 70.0 90.0 80.0 37.6 29.6 26.6 20.6 1.60 1.50
F 75.0 68.0 91.0 66.4 37.0 27.4 33.6 30.6 1.80 1.80
Table 3
Biogas Production from Co-digestion of Scenedesmus sp. MKB. Biomass and Paddy Straw at Lab Scale
Digester Composition of feedstock Biogas (in L) Cumulative biogas (L/Kg TS consumed) Cumulative biogas (L/Kg VS consumed)
A Microalgal biomass(Control) 0.80 29.63 43.93
B Paddy straw (Control) 48.56 63.90 73.16
C Microalgal biomass+ Biodigested slurry+ cowdung 28.80 800.00 870.00
D Microalgal filtrate + Biodigested slurry + cowdung 24.62 769.50 855.00
E Microalgal biomass+ Paddy straw+ Biodigested slurry+ cowdung 60.18 80.25 88.18
F Microalgal filtrate + Paddy straw + Biodigested slurry+ cowdung 18.43 24.91 27.67
Table 4
Estimation of Various Parameters of Modified Gompterz Equation for Digesters A–F
Digester P (mLg−1 VS) Rm (mLg−1d −1) λ (d) R2 VSR (%) Predicted biogas yield (mLg−1 VS) Experimental biogas yield (mLg−1 VS) Difference between predicted and experimental biogas yield
A 1.23 0.02 15.13 0.987 1.91 0.71 0.71 0.00
B 54.00 1.00 8.00 0.992 20.61 37.64 37.64 0.00
C 30.85 0.58 1.30 0.991 7.80 23.21 23.18 0.03
D 30.00 0.45 0.00 0.997 4.81 21.02 20.94 0.08
E 68.58 1.34 0.66 0.984 27.01 52.38 52.33 0.05
F 22.00 0.21 1.31 0.997 11.11 11.94 11.90 0.04

[i] P: ultimate biogas yield; Rm: maximum rate of biogas production; λ: lag phase; R2: Coefficient of determination; VSR: volatile solid reduction

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