Cellulolytic Potential of Newly Isolated Alcohol-Tolerant Bacillus methylotrophicus

Reprocessing lignocellulosic waste to obtain new products for industrial purposes is a vital part of circular economy. This paper reports the cellulase production by newly isolated Bacillus methylotrophicus cultured on lignocellulosic agro-industrial by-products, out of which brewer's spent grain (BSG) was selected as most beneficial. Plackett-Burman design was used for screening medium components, while Box-Behnken design was further applied to model the impact of the three most influential variables. The maximum approximated cellulase activity was 0.469 U/mL (1 U = 1 µmol of reducing sugars/1 min), at 48.6 g/L substrate, 5.3 g/L ammonium sulfate, pH 6.1. The partially purified cellulase was characterized, which demonstrated broad range of optimal pH (6.5-9.4), temperature (50-60 °C), and sensitivity to metals. Changes in lignin and pentosans content was demonstrated as a result of BSG hydrolysis with a cell-free cellulase preparation. The produced enzyme was used for hydrolysis of various chemically pretreated (NaOH and H₂SO₄) cellulosic substrates, where for reused alkali-pretreated BSG (after microbial enzyme production) the saccharification efficiency was at a level of 25%. The cellulolytic potential of the bacterial strain, along with its resistance to ethanol, present a beneficial combination, potentially applicable to aid saccharification of lignocellulosic by-products for biofuel production.