Biohydrogen generation by dark fermentation of starch using bacteria isolated from tapioca wastewater

Abstract

Hydrogen is a desirable alternative energy carrier of the future. Hydrogen can be sustainably produced by microorganisms through biological processes, such as fermentation. Hydrogen produced in this way is termed ‘biohydrogen’. The amount of biohydrogen produced varies between genus and species of microorganisms and also depends on the substrate and experimental physicochemical conditions. Starch is a potentially good substrate that can be used for fermentative biohydrogen-producing bacteria. In this study, tapioca starch wastewater from tapioca processing factory was used as substrate. The aim of this study was to isolate bacteria with the ability to utilise tapioca starch wastewater and produce biohydrogen by dark fermentation. Tapioca wastewater and waste sludge were used as isolation source, giving 45 unique isolates. Fifteen isolates were found to be positive starch degraders. The best starch degrader was identified to be Bacillus sp. strain LFSF20 with GenBank accession number KY399968. However, this isolate is unable to produce biohydrogen. In addition, Acinetobacter sp. AY-SDB4 (accession number KY923069) was found to have both abilities starch degrader and biohydrogen producer. Biohydrogen production was measured using gas chromatography-thermal conductivity detector (GC-TCD). The starter culture medium contained tapioca starch at an initial concentration of 1.0 g/L, initial pH 5.5 with incubation carried out at 30°C. Acinetobacter sp. AY-SDB4 was subjected to further optimisations to investigate parameters affecting biohydrogen production, using onefactor- at-a-time (OFAT) method. The effects of incubation temperature (30°C, 35°C and 40°C), initial pH (5.0, 5.5 and 6.0) and initial starch concentration (0.5, 1.0 and 1.5 g/L) were investigated. Several parameters were analysed during the fermentation process, which are biohydrogen production, starch utilisation, reducing sugar content, cell growth, and a-amylase activity. Kinetic analysis of biohydrogen production by Acinetobacter sp. AY-SDB4 suggested that the optimum conditions for biohydrogen production to be at initial substrate concentration of 1.0 g/L, initial pH of 5.0 and incubation temperature of 30°C. At these conditions, the highest biohydrogen productivity obtained was 3.183 × 10-3 mL/h, highest biohydrogen yield of 34.73 mL/g/L starch, and cumulative biohydrogen production of 19.8 × 10-2 mL. These findings suggest that Acinetobacter sp. AY-SDB4 has the potential to be used to produce biohydrogen using starch wastewater as substrate.