Development of high temperature simultaneous saccharification and fermentation by thermosensitive Saccharomyces cerevisiae and Bacillus amyloliquefaciens

Abstract

Scarcity of energy and pollution are two major challenges that have become a threat to all living things worldwide. Bioethanol is a renewable, ecological-friendly clean energy that may be utilized to address these issues. This study aimed to develop simultaneous saccharification and fermentation (SSF) process through high temperature-substrate adaptation and co-cultivation of S. cerevisiae with other potential amylolytic strains. In this study, we adapted our previously screened thermosensitive Saccharomyces cerevisiae Dj-3 strain up-to 42 °C and also screened three potential thermotolerant amylolytic strains based on their starch utilization capability. We performed SSF fermentation at high temperature by adapted Dj-3 and amylolytic strains using 10.0% starch feedstock. Interestingly, we observed significant ethanol concentration [3.86% (v/v)] from high temperature simultaneous saccharification and fermentation (HSSF) of adapted Bacillus amyloliquefaciens (C-7) and Dj-3. We attribute the significant ethanol concentration from starch of this HSSF process to C-7's high levels of glucoamylase activity (4.01 U/ml/min) after adaptation in starch (up-to 42 °C) as well as Dj-3's strong glucose fermentation capacity and also their ethanol stress tolerance capability. This study suggests the significant feasibility of our HSSF process.

Figure 1

Fluorescent microscopic image of thermotolerant adapted strains. The left and right panels represent phase contrast and DAPI images, respectively, to observe the shape, sizes and nucleoids area on to the cell. The procedure for cell culture, preparation and fixation for microscopic study are detailed in the “Methods” section. Scale bar represents 10 μm.

Figure 2

Amylolytic activity of selected starch fermenting microorganisms. Here, in image (a) the left and right panels represent glucose standard solutions (0.0 ml, 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml and 1.0 ml) and experimental samples, respectively. The image in the middle represents the standard glucose curve based on the value estimated from the image on the left. Number 1–4 of every 4-tube from the right image represent samples identified as C-7, M-3, Dfs-1 and P5 respectively. Herein (b), (c) and (d) represents amylase activity of screened strains at 30 °C, 37 °C and 42 °C respectively. One unit (IU) of amylase activity was defined as the amount of enzyme required to release 1 µmole of glucose (from starch hydrolysis) in 1 min.

Figure 3

Growth pattern and their ethanol production capability (%) of selected strains from starch fermentation. Here (a–c) represent growth pattern, where (d–f) represents the ethanol production capability of selected strains from starch fermentation at 30 °C, 37 °C and 42 °C respectively. Samples were collected at 6-h intervals, put on an ice bucket to seize the growth. Subsequently, their growth was measured by a spectrophotometer at 600 nm against the YPS broth as blank. For ethanol concentration measurement, samples were taken at 8 h, 16 h, 48 h, 96 h and 120 h intervals from starch fermentation broth medium.

Figure 4

Growth pattern, amylolytic activity and ethanol production ability (%) of adapted strains from starch fermentation. Here (a–c) and (d–f) represents 37 °C and 42 °C respectively. High temperature and substrate (starch) adaptation process are illustrated in the “Methods” section. Samples were collected at 6-h intervals, put on an ice bucket to seize the growth. Subsequently, their growth was measured by a spectrophotometer at 600 nm against the YPS broth as blank. For ethanol concentration measurement, samples were taken at 0 h, 4 h, 8 h, 12 h, 16 h, 24 h, 48 h, 72 h, 96 h and 120 h intervals from starch fermentation broth medium.

Figure 5

Improvement of ethanol production capability (%) of adapted strains through co-culturing with adapted thermotolerant Dj-3 (Saccharomyces cerevisiae). Here (a) represents growth pattern of Dj-3 at 30 °C, 37 °C and 42 °C, before and after temperature adaptation in YPD medium, where (b–d) represents the ethanol production capability of adapted starch fermenting strains with adapted Dj-3 strains in co-culture conditions at 30 °C, 37 °C and 42 °C respectively. Co-culture process, experimental procedures and conditions have been explained in the “Methods” section.

Figure 6

Ethanol stress tolerance activity of selected bioethanol producing strains before (left panel) and after adaptation process (right panel). Here, top and bottom images of left and right panel represent ethanol tolerance activity of four strains at 37 °C and 42 °C, respectively. In left panel (non-adapted), every 3-spot from left to right represents a sample spot applied for five microliters of 10, 100 and 1000-folds diluted sample, respectively where in right panel (adapted) represents a sample spot applied for five microliters of 10, 100 and 1000, 10,000 and 100,000-folds diluted sample, respectively. Experiments were conducted at least thrice independently and the suitable ethanol tolerant spots are shown.