# Cellulolytic Potential of Newly Isolated Alcohol-Tolerant Bacillus methylotrophicus

## TL;DR

A new ethanol-resistant bacteria produces cellulase that can break down agricultural waste for biofuel production.

## Contribution

A novel cellulase-producing strain of Bacillus methylotrophicus with ethanol tolerance is isolated and characterized.

## Key findings

- The optimal cellulase activity was 0.469 U/mL under specific culture conditions.
- The enzyme showed broad pH and temperature tolerance and was effective in hydrolyzing pretreated substrates.
- The strain's ethanol resistance makes it suitable for biofuel production from lignocellulosic waste.

## Abstract

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.