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.

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.

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.

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), R_m is the maximum rate of biogas production (mLg⁻¹d ⁻¹) and λ is the lag phase (days) and e = 2.718.

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: VS_bd and VS_ad signify volatile solids before and after anaerobic digestion.

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.

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].

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].

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 (R_m) 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, (R_m) = 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 (R_m) of 1.34 mLg⁻¹d⁻¹ than control digester B which has P of 54.00 mLg⁻¹ at R_m 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).

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.

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.