Anaerobic Digestion of Pineapple Waste for Biogas Production and Application of Slurry as Liquid Fertilizer Carrier for Phosphate Solubilizers

Waste management in fruits and vegetable refining factories was one among the challengeable jobs around the world. Scientists had centred their attention on the utilization of pineapple waste preferably for biogas and fibre production (Upadhyay et al., 2013). Some of these wastes had been used in industrial applications like gas generations (Mbuligwe and Kassenga, 2004). Bio-methanation of fruit wastes was a simple waste treatment as it both adds energy in the form of methane and also results in a highly stabilized effluent fertilizer with almost neutral pH and odourless property (Bardiya et al., 1996). Rani and Nand (2004) reported that different treatments of pineapple peels gave biogas yields ranging from 0.41-0.67 mg/kg volatile solids with methane content of 41-65%. This proved pineapple pulp could be used successfully for biogas production. The use of pulp and peel together gave higher biogas production (Pimjai et al., 2012). The performance of biogas production depends on biomass chemical composition as well as standardized conditions of feed concentration, hydraulic retention time, pH and temperature (Boe, 2006; Turovskiy and Mathai, 2006; Rehm et al., 2000).
           
Bio-methanation of fruit wastes was the best suited waste treatment as it both adds energy in the form of methane and also results in a highly stabilized effluent with almost neutral pH and odorless property (Bardiya et al., 1996). They utilized pineapple waste for the production of methane using semi-continuous anaerobic digestion which could produce up to 1682 ml/day of biogas with methane content of 51% in maximum.
       
 The lowest possible HRT for banana peel was 25 days, resulting in 36% substrate utilization and with maximum gas production of 0.76 vol/day, while pineapple processing digestors could be operated at 10 days HRT, with 58% substrate utilization and maximum gas production rate of 0.93 vol/day (Bardiya et al., 1996). Gas production over 3 days incubation period was found to be the highest with cow manure followed by orange rind, with the lowest value for papaya peel and banana skins (Inthapanya et al., 2013). In this experiment, we had screened the efficient treatment with cow dung and biogas slurry for maximum methane gas production. The slurry was developed as liquid fertilizer carrier for phosphate solubilizer- Providencia rettgeri, a bacterial solubilizer and Meyerozyma gullerimondi, a yeast solubilizer.

Anaerobic digestor
 
The biogas plant was installed at the household premises at Mannuthy, Thrissur. The work was carried out in Pineapple Research station Vazhakulam, Ernakulam and Nehru Arts and Science College, Coimbatore in year 2019 to 2020. The floating drum biogas plant of 0.5 m³ (Plate 1) capacity was used for anaerobic digestion of pineapple substrate. The substrates were added through the inlet pipe. The gas produced inside the digestor was collected in gas holder and the bottom of the gas holder was dipped into the substrates to create an anaerobic condition. The gas collected in the gas holder was used daily through gas outlet. When substrates got completely digested, slurry flowed through slurry outlet. Regular feeding had done with the slurry at the rate of 1 litre per day to the biogas plant.
 
Inoculation of phosphate solubilizers
 
The phosphate solubilizers selected were Providencia rettgeri, a bacterial solubilizer and Meyerozyma gullerimondi, a yeast solubilizer isolated from rhizosphere soil of pineapple plants from Vazhakulam, Ernakulam district, Kerala. The ratio of 6×10⁴ CFU/ml of slurry was the inoculum used for both organisms. The fermentation was carried out for overnight.
 
Regular monitoring of biogas plant
 
During the anaerobic digestion period, hydraulic retention time (HRT) and daily temperature inside the digester, volume of gas produced and quantity of slurry generated were determined regularly. Hydraulic Retention Time (HRT) was defined as the maximum time taken by the substrates for maximum gas production. The daily temperature of the biogas unit was noted by using digital thermometer for the entire period of study.
       
The gas volume was recorded every day. The gas produced was measured and used for burning the stove. The increase in height of the gas holder was recorded daily and volume of gas was calculated using the formula,
 
Where
V denotes volume, r denotes radius of gas holder and h denotes height increased after gas production. The slurry output from the digester was also measured daily for all the treatments using measuring cylinder (Enaboifo and Adadu, 2020).
 
Analysis of biogas and biogas slurry
 
The gas produced during the first three days was discarded for a stabilized biogas production. Biogas samples were collected after nine days at three days intervals in gas collection bladders (Hans Seamless latex valve bladders) and analysed. The Biogas composition in laboratory test (CH₄, CO₂, H₂, H₂S and O₂) was measured using an automated gas analyser according to Brettschneider et al., (2004). The slurry generated in each treatment were tested for profiling the quantity of macronutrient and micronutrients present in it using FESEM-EDX and Elemental mapping. 
 
Biometric observations of seedlings
 
Biometric observations were recorded biweekly for three months. Plant height, number of leaves per plant, plant girth was recorded. Vigour index was also calculated from the biometric observations (Kaur and Phutela, 2014).
 
Statistical analysis
 
Data were subjected to analysis of variance (ANOVA) (Panse and Sukhatme, 1985) using statistical package ‘MSTAT-C’ package (Freed, 2006). Wherever the F test was significant (at 5% level) multiple comparison among the treatments were done with Duncan’s multiple range test (DMRT).

Composition of biogas
 
The optimum combination of cow dung and pineapple waste for maximum gas production was standardized using completely randomized design with five treatments and three replications for a period of 18 months. All the six treatments were done separately on all five weeks of 6 months from October 2018 to June 2020 at Mannuthy, Thrissur, Kerala. The readings were taken every week.
       
The highest methane content of 60.49% was recorded in T₄ which was on par with T₅ (63.81%) and T₃ (60.29%) and was significantly higher than T₁ (50.00) and T₂ (46.65). From the results it is clear that co-digestion of cow dung with fruit waste increased the methane content in 1:1.5 ratio. With the increase of pineapple fruit waste proportion with cow dung as 1:2 methane generations decreased to 50.86% (Fig 1a). It was evident that CO₂ concentration varied significantly between the treatments. It was found to be highest in T₂ (50.86%) and the lowest in T₄ (32.00%). The recorded CO₂ concentration in T₆, T₁, T₃, T₅ were 45.37, 37.48, 37.31 and 34.47 respectively (Fig 1a). The major nutrients and heavy metal composition of biogas slurry and  substrate was shown in Table 1.
 

 

 
Hydraulic retention time (HRT) and volume of gas generated
 
The hydraulic retention time (HRT) was minimum (15 days) in the treatment T₃ (cow dung + fruit waste, 1:0.5) followed by T₄ (cow dung + fruit waste, 1:1) with 17 days. The highest HRT of 28 days was observed in treatment T₂ (fruit waste alone) whereas the treatments T₁ (cow dung alone) and T₅ (cow dung + fruit waste, 1:1.5) was recorded 23 days and 19 days. The HRT of T₆ was 25 days (Fig 1b). The volume of gas was maximum (0.43 m³/day) in the treatment T₄ (cow dung + fruit waste, 1:1) which was followed by T₃ (cow dung + fruit waste, 1:0.5) with 0.41 m³/day. The lowest volume of 0.29 m³/day was observed in T₂ (fruit waste alone) whereas the treatments T₁ (cow dung alone) and T₅ (cow dung + fruit waste, 1:1.5) was recording 0.35 m³/day and 0.39 respectively and the total volume in the treatment T₆ was 0.36 m³/day (Fig 1 b).
 
Total quantity of slurry
 
The quantity of biogas slurry generated in each treatment was analysed. The T₆ treatment recorded the highest quantity of slurry (96 L) followed by T₅ (78 L), T₄ (62 L), T₃ (57 L) and the treatments T₁ and T₂ were on par recording 40 L (Fig 1c). The nutrients present in the slurry was recorded using FESEM-EDX. The FESEM analysis and EDAX for elemental analysis of the crude extracts and its biofertilizers i.e., biogas slurry and biogas substrate were shown in Fig 2 and Fig 3.
 

 

 
Ananas comosus (Pineapple) and Passiflora edulis (Passion fruit)
 
The germination studies of biogas slurry were done alone in passion fruit seedlings since tissue culture pineapple variety, MD₂ was selected to determine the biometric observations like length and leaf number of the plant and weight of the fruit. This MD₂ pineapple plants treated with biogas slurry at 15,30,45,60 DAP was shown in fig 4a, 4b, 120 DAP in 4c, 365 DAP in 4d. The germination percent was recorded higher for treatment, T₃ (49.42%) which had pre-soaking with biogas slurry having maximum manurial value (12 h) (Table 3, Fig 5a). The minimum per cent (40.20%) was observed for storage in shade. The coating treatments also followed the pre-soaking treatments but the effects were comparatively less. The passion fruit seeds pre-soaked with pineapple fruit waste slurry recorded highest vigour index (197.68). Coating with biogas slurry also showed marginal improvement (177.44) compared to gober gas slurry (Table 3, Fig 5a).
 

 

 

       
The length of pineapple plants (MD₂) was calculated for 365 days with different batches of different plants. The highest plant height (85.33 cm) was observed for pre-soaking treatment with biogas slurry (Table 4, Fig 5b). The lowest height was recorded for pineapple plants coated with gober gas slurry (69.33 cm). The plant stored in shade as control without any treatments recorded 82.33 cm length after 365 days.
 

       
The effect of treatments on number of leaves of pineapple plant were significant for 180 days after planting and found to be non-significant there after. The highest number of leaves was recorded in T₅ at 180 and 365 days after planting (Table 5, Fig 5c). Among the treatments, the plants treated with pineapple waste biogas slurry were significant throughout and found to be the best, recording the maximum number of leaves. The lowest number of leaves was observed for control plant in shade, T₁.
 

       
The average weight of the fruits obtained were also calculated, the maximum fruits were obtained in the case of pre-soaking treatment with biogas slurry (Table 6). The average weight obtained was 1.28 kg for this treatment. The lowest value obtained for control plant, 1.02 kg.
 

 
Anaerobic production of methane from pineapple waste
 
Pineapple peels had been found to be promising feed for biogas generation, since they were rich in carbohydrates and proteins. Their disposal possessed a serious environmental pollution problem. Since pineapple peel was rich in cellulose, hemi-cellulose and other carbohydrates, it was found to be a potential substrate for methane generation by anaerobic digestion (Rani and Nand, 2004). Among the fruit waste, pineapple generate enormous amount of fermenting waste particularly its core and peel, henceforth this fruit waste was selected for producing methane in pilot scale digester (Malik et al., 2001). The experiment was conducted for a month and the maximum amount of methane was generated in treatment with 1:1.5 composition of cow dung and pineapple fruit waste. The nutritional value (NPK) was high in slurry obtained from treatment having 1:2 ratio of cow dung and fruit waste. This slurry (1:2 treatment) generated was applied as foliar and drenching to pineapple (MD₂)- Ananas comosus var L. plants itself to study their height, leaf number and average weight of the fruit. In recent studies, pineapple waste and cow dung were used in raw form for methane gas generation (Hamzah et al., 2020), here we had utilized cow dung slurry instead and maximum production of methane gas was achieved in 1:1 ratio of slurry application. Similar studies were done by Mugerwa., (2018) where 2:1 ratio of pineapple waste and cow dung slurry generated highest methane content of 64% whereas here highest of 63.41% of methane gas was obtained from 1:1 ratio of pineapple waste and cow dung slurry.
 
Effect of sustainable liquid fertilizer made from environmental waste to crops
 
The plant Ananas comosus (pineapple) was usually treated with chemical fertilizers like Urea, potash etc. Fungicides ike Phytrax, SAAF, Bavistin and hormonal treatments like Ethephon for uniform inflorescence. Comparatively, we had designed a recyclable organic fertilizer from pineapple waste for pineapple plants. The core and peel waste biogas slurry were applied to pineapple plants. Pre-soaking with the slurry regulates the transport of water to seed which acts as water reservoir (Aswathy and Sushama, 2015). In this way slurry soaking might had improved germinating ability of seeds. In a current study it showed that pre-treatment of pineapple peel using the alkali enhanced enormous lignin reduction and enhanced biogas production (Dahunsi, 2019), similarly palm oil mill effluent containing various microbial consortium were used for anaerobic fermentation of pineapple pulp and peel waste for biogas production at a range of (40.5 to 70.1%) (Aziz, 2017; Azouma et al., 2018). Likewise, in this research we had fermented pineapple waste using cow dung slurry and phosphate solubilizers for methane gas production and had a recovery percentage ranging from (46.65 to 63.49%).

Production of biogas from Ananas comosus waste (peel and core) were done and the treatment, T₅ with 1:1.5 ratio of cow dung and pineapple fruit waste resulted in 63.81% methane and 34.47% carbon dioxide which was closely followed by T₄ (60.49%, 32.0%) having cow dung + pineapple fruit waste in ratio 1:1. Throughout the study period the volume of gas was generated in the order T₅>T₄>T₃> T₆>T₁>T₂. The temperature inside the digester was always found to be higher compared to the atmospheric temperature. The HRT reduced from 23 days to 19 day by co digestion of pineapple fruit waste with cow dung in 1:15 ratio as compared with either of the substrate. Similarly, with biogas slurry (T₆=1:2 ratio of cow dung and fruit waste), the germination % (49.42) and vigour index (197.68) was studied with Passiflora edulis (Passion fruit seedlings) and were recorded highest in pre-soaking treatment, T₃ with biogas slurry having maximum manurial value (12 h); So, the growth rate order for passion fruit seedlings in terms of germination is T₃>T₂>T₄>T₅>T₁. In addition to this, Seedling height (85.33 cm), Leaf number (69.33) of Ananas comosus (Pineapple) was more in presoaking treatments of both fruit waste and gober gas slurry. For leaf the growth rate order was T₅>T₃>T₂>T₄>T₁ and for height the growth was reported as T₃>T₁>T₂ >T₅>T₄. This data infers the treatments of pre-soaking with fruit waste slurry or gober gas slurry with phosphate solubilizers- Meyerozyma gullerimondi, a yeast solubilizer and Providencia rettgiri, a bacterial solubilizer induces maximum growth rate to the crop.

The authors acknowledge that this research was technically supported by Nanoscience department of Bharathiar University, Coimbatore and Soil Science Department of Kerala Agricultural University, Thrissur. This study was financially supported by Fiber Tech Manufacturing and Trading, Ajman-United Arab Emirates.