Prospecting Agro-waste Cocktail: Supplementation for Cellulase Production by a Newly Isolated Thermophilic B. licheniformis 2D55

Abstract

Bacteria isolated from thermophilic environment that can produce cellulase as well as utilise agro-waste biomass have a high potential for developing thermostable cellulase required in the biofuel industry. The cost for cellulase represents a significant challenge in converting lignocellulose to fermentable sugars for biofuel production. Among three potential bacteria examined, Bacillus licheniformis 2D55 (accession no. KT799651) was found to produce the highest cellulolytic activity (CMCase 0.33 U/mL and FPase 0.09 U/mL) at 18-24 h fermentation when grown on microcrystalline cellulose (MCC) as a carbon source in shake flask at 50 °C. Cellulase production process was further conducted on the untreated and NaOH pretreated rice straw (RS), rice husk (RH), sugarcane bagasse (BAG) and empty fruit bunch (EFB). Untreated BAG produced the highest FPase (0.160 U/mL), while the highest CMCase (0.150 U/mL) was supported on the pretreated RH. The mixture of untreated BAG and pretreated RH as agro-waste cocktail has remarkably improved CMCase (3.7- and 1.4-fold) and FPase (2.5- and 11.5-fold) compared to the untreated BAG and pretreated RH, respectively. The mechanism of cellulase production explored through SEM analysis and the location of cellulase enzymes of the isolate was also presented. Agro-waste cocktail supplementation provides an alternative method for an efficient production of cellulase.

Keywords: Agro-waste cocktail; Cellulase production; Composting; Enzyme location; NaOH pretreatment; Scanning electron micrograph (SEM); Thermophilic bacteria.

Fig. 1

Profiles of temperature, moisture content and bacterial count during composting of OPEFB and chicken manure. Diamond indicates temperature, circle moisture content and triangle bacterial count

Fig. 2

Quantitative screening for cellulase production by bacterial strains grown on microcrystalline cellulose. Values are means of (n = 3), ±SD (vertical bars)

Fig. 3

Phylogenetic dendogram showing relatedness between 16s rRNA of the isolated bacterium and related bacteria strains from gene data base using the neighbour joining method

Fig. 4

a CMCase, FPase, xylanase, β-glucosidase production by Bacillus licheniformis 2D55 grown on agro-waste cocktail (AWC) containing a combination of untreated bagasse and pretreated rice husk ratio 1:1. Values are means of (n = 3), ±SD (vertical bars). Circle indicates xylanase activity, square CMCase activity, triangle FPase activity and diamond beta-glucosidase activity. b Growth profile of B. licheniformis 2D55 grown on agro-waste cocktail. Values are means of (n = 3), ±SD (vertical bars)

Fig. 5

SEM image of cell suspension sample from culture grown on UBAG (a, b), UBAG before inoculation (c) and residue from culture grown on UBAG (d)

Fig. 6

SEM image of cell suspension sample from culture grown on TBAG (a, b), TBAG before inoculation (c) and residue from culture grown on TBAG (d, e)

Fig. 7

SEM image of AWC residue of culture grown on AWC (a) and AWC residue degradation (b)

Fig. 8

Localisation of enzyme produced by B. licheniformis 2D55 grown on mixed agro-waste material