3.1. Isolation and identification of lactic acid bacteria
Isolation and Characterization of potential lactic acid bacteria:
Identification of the isolate AG1 on the basis of 16s rRNA analysis
After identifying the genera of isolated bacteria AG1 by basic characterization described in Bergey's Manual of Systemic Bacteriology, the isolates was further identified to species level by 16s rRNA sequencing. For this, the bacteria were sent to “Genexplore Diagnostics & Research Centre Pvt. Ltd.” Ahmedabad, Gujarat, India.
Alignment report - 16S rRNA sequencing of isolate L. delbrueckii ssp. Indius AG1
TTCCTTCGGGATGATTTGTTGGACGCTCGCGGCGGATGGGTGAGTAACACGTGGGCAATCTGCCCTAAAGACTGGGATACCACTT
GGAAACAGGTGCTAATACCGGATAACAACATGAATCGCATGATTCAAGTTTGAAAGGCGGCGCAAGCTGTCACTTTAGGATGAG
CCCGCGGCGCATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCAATGATGCGTAGCCGAGTTGAGAGACTGATCGGCCACAT
TGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACG
CCGCGTGAGTGAAAAAGGTTTTCGGATCGTAAAGCTCTGTTGTTGGTGAAAAAGGATAGAGGCAGTAACTGGTCTTTATTTGAC
GGTAATCAACCAGAAAGTCACGGCTAACTACGTGCCAGCACCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGG
GCGTAAAGCGAGCGCAGGCGGAATGATAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGAAACTGCATCGGAAACTGTCATT
CTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCG
GCTCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAAC
GATGAGCGCTAGGTGTTGGGGACTTTCCGGTCCTCAGTGCCGCAGCAAACGCATTAAGCGCTCCGCCTGGGGAGTACGACCGCA
AGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTA
CCAGGTCTTGACATCCTGCGCTACACCTAGAGATAGGTGGTTCCCTTCGGGGACGCAGAGACAGGTGGTGCATGGCTGTCGTCA
GCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCTTTAGTTGCCATCATTAAGTTGGGCACTCTAGA
GAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTAC
AATGGGCAGTAC
ACGAGAAGCGAACCCGCGAGGGTAAGCGGATCTCTTAAAGCTGTTCTC
3.2 VOLUME OF BIOGAS PRODUCED FOR EACH CO-DIGESTION WASTE
Figure 5 shows the volume biogas produced from sterile agriculture waste & L. delbrueckii ssp. indius AG1, control within the retention period 20 days. Production of gas from sterile agriculture waste &L. delbrueckii ssp. indius AG1, started on day 9 of the retention period by producing average biogas of 45 ml, thereafter increases to 97.5 ml on day 10 and reduces to 185 ml on day 12. At day 13, the biogas produced was 255 ml in which decreases back to 185 ml on the next day and increases thereafter until it reached the peak on day 16 with 575 ml biogas production after which it begins to reduce till the completion of the retention period which is similar to the work of Aremu and Agarry, 2012).
Temperature of Slurry for Co-digestion waste
Figure 6 indicates the temperature of the co-digestion of sterile agriculture waste &L. delbrueckii ssp. indius AG1, control. The temperature varies from 25.1 - 27.4 for agriculture waste &L. delbrueckii ssp. Indius AG1, 25.2 – 27.8°C and remained stable on day 15 to day 17. These temperature ranges also signifies a mesophilic thermal stage of biogas production (25 - 45°C. The maximum biogas produced for each co-digestion was attained at day 20, day 14 and day 19 respectively in which the temperature for these days was 27.1°C, 26.4°Cand 26.9°C respectively. Temperature has been observed by most biogas researchers to be quite critical for anaerobic digestion, since lactic acid bacteria –L. delbrueckii ssp. Indius AG1 operate most efficiently at temperatures 30.0 – 40.0°C. For this study, the four digesters operated under mesophilic condition which is similar to the temperature 30.0 – 40.0°C .The temperature of below 30 in which this experiment was operated, could have contributed to the slow development of methanogens and consequently low methane production. This is similar to the report of (Iloriet al., 2007) that the recovery time for biogas production as well as the quality and quantity of biogas produced from agricultural materials are a function of the nature, and composition of the digester feedstock.