Lignocellulosic bioethanol production employing newly isolated inhibitor and thermotolerant Saccharomyces cerevisiae DBTIOC S24 strain in SSF and SHF

Abstract

Bioethanol is a renewable alternative to fossil fuels which facilitate energy security and reduce greenhouse-gas emissions. High gravity fermentation employing a thermo and inhibitor tolerant strain is a promising technology to reduce fermentation time as well as cost. The present study investigates lignocellulosic ethanol production using inhibitor and thermotolerant S. cerevisiae DBTIOC S24 from non-detoxified and unsterilized rice straw hydrolysate. Efficient ethanol production was observed at a wide range of pH (3–7) and temperature (25–42 °C) using S. cerevisiae isolate. In the presence of lignocellulosic derived inhibitors, a maximum of 75.33 g L⁻¹ (85.56%) and 73.30 g L⁻¹ (79.93%) ethanol was produced at 30 °C and 42 °C, respectively. During fermentation, pH plays an important role in overcoming the synergistic effect of inhibitors. More than 80.65% and 73.5% ethanol yield was achieved employing this isolate with high solid loading (20%) and 20 FPU g⁻¹ of solid loading via simultaneous saccharification and fermentation (SSF) and separate hydrolysis and fermentation (SHF), respectively. While, 91% ethanol yield was obtained during fermentation using rice enzymatic hydrolysate. These values are comparable to the best results reported. Therefore, this isolate has great potential due to its inhibitor and thermotolerant characteristics for lignocellulosic ethanol production at the industrial scale with a lower process time and cost.

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1. Introduction

The global energy demand has inexorably increased over the last couple of years and is projected to further increase of more than 37% by 2040.¹ Presently, the global energy demand is fulfilled mainly by fossil fuel, which is a major concern at present due to its limited availability and high negative impact on the environment.² Biofuel, especially bioethanol, looks a promising alternative to fossil fuel. At present, most of the supply comes from first generation, which is limited by production cost, competing with land, water, food and fiber.³ An alternative, second generation bioethanol from lignocellulosic biomass is fascinating due to its surplus availability in worldwide. Among the lignocellulosic biomass, rice straw is the most abundant in world.⁴ In addition, rice straw has a significant content of silica and is therefore not suitable for the pulp & paper industry or animal fodder.⁵ Hence, rice straw could be considered as a potential agriculture residue for lignocellulosic ethanol production.

Lignocellulosic biomass is a recalcitrant complex structure, which requires pretreatment before enzymatic hydrolysis to make the cellulose accessible for enzymatic hydrolysis. Among the available pretreatment technologies, dilute acid pretreatment is considered one of the most efficient, cost effective and closest to commercialization.⁶ However, the inherent disadvantage of this technology is the production of inhibitors which reduces enzyme activity as well as fermentation efficiency.^7,8 The various detoxification strategies for hydrolysate or slurries includes alkali or sulfite treatment, liquid–liquid extraction, ion exchange and treatment with enzymes but these detoxification methods are costly and causes in loss of sugars, hence they are not considered suitable as an economically viable technology.^8,9

The fermentation process is exothermic in nature and causes a rise in temperature during industrial scale fermentation because these are operated adiabatically due to a much lower surface to volume ratio compared to laboratory fermentors.¹⁰ Therefore, application of a thermotolerant strain for ethanol production is highly warranted with its potential to reduce cooling cost along with its cessation during ethanol fermentation due to the overheating problem.¹¹ Among ethanologenic organisms, S. cerevisiae is considered as an industrial strain for bioethanol production however, it is susceptible to lignocellulosic inhibitors and other stress conditions viz. pH, temperature, substrate concentration, etc.⁸ Various studies have shown that ethanol yield and productivity were significantly reduced below 35 °C using thermotolerant strains and vice versa. Significant effort has been made to develop thermotolerant and inhibitor tolerant traits in ethanologenic microorganisms using protoplast fusion, genetic engineering, genome shuffling, mutation and evolutionary engineering.¹² However, most of these studies addressed for tolerance of one or two specific stresses, thus are not useful for combined tolerance for multiple stresses including temperature and inhibitors. Therefore, employing natural thermo and inhibitor tolerant ethanologenic yeast with tolerance for multiple stresses is a realistic approach for the viable economics of a second generation biofuel production.¹³

Considering the above facts, the present study focuses on isolation of thermo and inhibitor tolerant yeast strain from distillery spent wash as this habitat is acidic in nature, high organic material loading, exposed directly to sunlight leading to increase in temperature, low dissolved oxygen and high solid contents.^14,15 The isolated strain was thoroughly examined for the synergistic effect of inhibitors on ethanol fermentation at high temperature. Further, isolate was evaluated for ethanol fermentation via separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) using unsterilized and non-detoxified rice straw hydrolysate at elevated temperature. To the best of our knowledge, this is the first report using non-detoxified and unsterilized rice straw hydrolysate for ethanol production employing inhibitor and thermotolerant S. cerevisiae at 42 °C in a bioreactor.

2. Materials and methods

2.1 Sample collection, media and chemicals

To isolate ethanol fermenting yeasts, eight soil and spent wash samples were collected in sterile bottles from various sites of three different distilleries and sugar mills from the National Capital Region, India. YPD broth (yeast extract 10 g L⁻¹, peptone 20 g L⁻¹, glucose 20 g L⁻¹) was used for isolation of yeast. For solid medium, 2% (w/v) agar was added into the broth. Two other media were used for screening processes. One was a pre-culture medium (PCM) consisting of yeast extract 10 g L⁻¹, peptone 20 g L⁻¹, glucose 50 g L⁻¹ and ethanol 80 g L⁻¹. The second yeast fermentation medium (YFM) was used to evaluate the fermentation efficiency of yeast isolates. YFM media consisted of yeast extract 2.5 g L⁻¹, yeast nitrogen base 1.7 g L⁻¹, ammonium sulfate 5.0 g L⁻¹, magnesium sulfate 6.0 g L⁻¹ and glucose 60–180 g L⁻¹. All the media were adjusted to pH 5.0 ± 0.2 with 1 M HCl and 1 M KOH, autoclaved at 121 °C and 15 lb. pressure for 20 min.

2.2 Isolation, screening and selection of yeast isolates

Soil/spent wash sample (10 g) was dispersed in 100 mL saline (0.85%) and mixed thoroughly. After appropriate dilution, 100 μL samples were spread on YPD agar plates containing 60 g L⁻¹ ethanol to enrich only ethanol tolerating strains and 0.05 g L⁻¹ streptomycin to prevent bacterial growth, and incubated at 30 °C for 72 h. Twenty six yeast colonies appeared through the soil serial dilution plate method. All 26 yeast colonies were subjected to further screening and selection using PCM broth and agar plates. Selected yeast colonies were evaluated for their fermentation ability using YFM broth amended with 180 g L⁻¹ glucose at 42 °C for 48 h and 180 rpm. The seed culture for fermentation was prepared by growing yeast in 2 liters of YPD media for 16 hours (mid-log phase culture) and centrifuged at 5000 rpm for 10 min. The cell pellet was dissolved in 0.85% saline. The inoculum was transferred to screening media, to give an initial DCW of 1.0 g L⁻¹.

2.3 Identification and characterization

Based on the fermentation efficiency, a yeast strain (DBTIOC S24) was selected for further studies and characterized by sequencing. Yeast DNA was isolated using DNeasy blood & tissue kit (Qiagen). The internal transcribed spacer region (ITS) 5.8S rDNA and the adjacent ITS1 and ITS2 regions were amplified by polymerase chain reaction (PCR) using the primer pairs ITS1 5′-TCCGTAGGTGAACCTGCG-3′ and ITS4 5′-TCCTCCGCTTATTGATATGC-3′.¹⁶ PCR amplification was performed in 50 μL reaction mixtures containing approximately 20 ng of genomic DNA template, 1× PCR buffer with 2.0 mM MgCl₂, 0.2 mM of each dNTP, 0.1 μM of each primer and 1 U Taq polymerase. PCR cycling conditions were 35 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 45 s, followed by an extension step of 72 °C for 10 min. The amplified DNA was purified using Qiaquick PCR Purification Kit (Qiagen) and sequenced. The sequences were BLASTn against NCBI data base. The 99% and higher sequence match were considered for species identification. The phylogenetic dendrogram was prepared using Molecular Evolutionary Genetics Analysis (MEGA) software version 5.0.¹⁷ Substrate utilization profile of the yeast isolate was studied using a HiCarbo™ Kit (HiMedia) following the manufacturer’s instructions.

2.4 Ethanol fermentation at different pH, temperatures and substrate concentrations

To study the influence of pH on fermentation efficiency, the initial pH of the YFM supplemented with 180 g L⁻¹ glucose was adjusted to the desired value (3–7) using sterile HCl and KOH solutions. The flasks were inoculated and incubated at 30 °C and 180 rpm for 72 h. Similarly, a temperature variation experiment was set up at pH 5.0 and incubated the flasks at 25 °C, 30 °C, 37 °C and 42 °C for 48 h. At different time intervals, samples were withdrawn and analyzed for cell growth, residual glucose and ethanol content. Similar to above, the effect of initial glucose concentration (60, 120 and 180 g L⁻¹) on the fermentation efficiency of the selected strain at 30 °C and 42 °C was also evaluated.

2.5 Synergistic effect of ligno-cellulosic inhibitory compounds on ethanol fermentation

In order to study the synergetic effect of inhibitors on the fermentation efficiency of isolated yeast, four inhibitors were used viz. acetic acid (0–4.5 g L⁻¹), formic acid (0–1.0 g L⁻¹), 5-HMF (0–3.0 g L⁻¹) and furfural (0–2.0 g L⁻¹) in 15 different combinations with and without pH adjustment (5.0 ± 0.2) by using HCl or KOH (Table 1). For fermentation experiments, a 250 mL Erlenmeyer flask with 100 mL YFM was inoculated with overnight grown culture (initial cell concentration 1.0 g L⁻¹). After 48 h, samples were withdrawn and analyzed for cell growth, residual glucose and ethanol content.

Table 1 Effect of synthetic inhibitor cocktails at different concentrations on the fermentation efficiency of S. cerevisiae DBTIOC S24 isolate at 30 °C after 46 h using 180 g L⁻¹ initial glucose

Conditions	S. no.	Inhibitor cocktails (g L^−1)				Ethanol concentration (g L^−1)	Biomass (g L^−1)	Yield (g g^−1)	Productivity (g L^−1 h^−1)	Specific productivity (g g^−1 h^−1)
		Acetic acid	Formic acid	5-HMF	Furfural
Without pH adjustment (3.0–4.0)	1	0	0	0	0	76.81	2.17	0.430	1.670	0.77
	2	0	0.33	1	1.32	74.30	1.82	0.436	1.615	0.89
	3	0	0.66	2	0.66	36.69	1.40	0.367	0.798	0.57
	4	0	1	3	2	2.24	0.58	0.086	0.049	0.08
	5	1.5	0	2	2	4.85	0.65	0.147	0.105	0.16
	6	1.5	0.33	3	0.66	1.42	0.49	0.053	0.031	0.06
	7	1.5	0.66	0	1.32	1.83	0.57	0.071	0.040	0.07
	8	1.5	1	1	0	0.98	0.53	0.038	0.021	0.04
	9	3	0	1	0.66	2.30	0.63	0.083	0.050	0.08
	10	3	0.33	0	2	1.31	0.57	0.044	0.029	0.05
	11	3	0.66	3	0	9.56	0.55	0.349	0.208	0.38
	12	3	1	2	1.32	3.25	0.51	0.113	0.071	0.14
	13	4.5	0	3	1.32	1.93	0.51	0.076	0.042	0.08
	14	4.5	0.66	1	2	1.23	0.55	0.041	0.027	0.05
	15	4.5	1	0	0.66	0.64	0.54	0.025	0.014	0.03
With pH adjustment at 5.0 ± 0.2	1	0	0	0	0	77.04	2.05	0.453	1.675	0.82
	2	0	0.33	1	1.32	76.72	1.83	0.440	1.668	0.91
	3	0	0.66	2	0.66	72.69	1.66	0.436	1.580	0.95
	4	0	1	3	2	47.85	1.47	0.374	1.040	0.71
	5	1.5	0	2	2	72.45	1.79	0.427	1.575	0.88
	6	1.5	0.33	3	0.66	67.00	1.79	0.396	1.456	0.81
	7	1.5	0.66	0	1.32	76.71	2.04	0.430	1.668	0.82
	8	1.5	1	1	0	78.89	2.06	0.440	1.715	0.83
	9	3	0	1	0.66	77.47	2.09	0.431	1.684	0.81
	10	3	0.33	0	2	73.10	2.08	0.409	1.589	0.76
	11	3	0.66	3	0	41.24	0.83	0.423	0.896	1.08
	12	3	1	2	1.32	50.77	1.33	0.419	1.104	0.83
	13	4.5	0	3	1.32	8.56	0.71	0.205	0.186	0.26
	14	4.5	0.66	1	2	68.28	1.82	0.410	1.484	0.81
	15	4.5	1	0	0.66	73.89	2.29	0.418	1.606	0.70

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2.6 Ethanol fermentation in the absence and presence of inhibitor cocktails

To mimic actual inhibitor concentration present in hydrolysates, batch fermentation was performed at 30 °C, 37 °C and 42 °C in 7.5 liter NBS Bioflow 115 bioreactor using 3 L YFM (180 g L⁻¹ glucose) amended with inhibitors (acetic acid 2.5 g L⁻¹; formic acid 0.25 g L⁻¹; 5-HMF 0.75 g L⁻¹ and furfural 0.6 g L⁻¹) at pH 5 ± 0.2 and 200 rpm. A parallel bioreactor was also run without any inhibitor as a control. Samples were withdrawn at different time intervals and analyzed for cell growth, residual glucose and ethanol content. The fermentation was started by inoculating yeast cell at an initial DCW of 1.0 g L⁻¹.

2.7 Ethanol fermentation using hydrolysate via SHF and SSF

Dilute acid pretreatment of rice straw was performed in a screw type pilot scale continuous pretreatment reactor using 0.3% sulphuric acid at 162 °C for 15 min as described by Saini et al.¹⁸ and the resulting pretreated slurry contained 28.4% of total solid. The water insoluble solid (WIS) of slurry consisted of 51.4% glucan, 3.9% xylan and 28.9% lignin. Dilute acid pretreated rice straw slurry was enzymatically hydrolyzed without any detoxification, washing, sterilization and filtration step at 20% (w/w) solid loading [water insoluble solid (WIS) 15.2%, glucose 8 g L⁻¹, xylose 32 g L⁻¹] using 20 FPU SacchariSEB C6L (Advanced Enzymes, India) per g of solid loading in NBS Bioflow 115 bioreactor (3 L working volume, pitch blade impeller) at 50 °C. The 20 FPU of enzyme SacchariSEB C6L contains 48.09 U of β-glucosidase, 160.00 U of endoglucanase and 7.34 mg protein.¹⁹ The resulting enzymatic hydrolysate was used in the fermentation experiments employing isolated yeast in a bioreactor containing 3 L hydrolysate amended with yeast extract (2.5 g L⁻¹), yeast nitrogen base (1.7 g L⁻¹), ammonium sulphate (5.0 g L⁻¹) and magnesium sulphate (6.0 g L⁻¹). Yeast cells were inoculated with an initial DCW of 2.0 g L⁻¹ and incubated at 30 °C and 42 °C for 48 h at 200 rpm. Two parallel bioreactors were also run using YFM amended with 80 g L⁻¹ glucose employing yeast isolate at 30 °C and 42 °C as a control. Samples were withdrawn at different time intervals and analyzed for glucose and ethanol concentration.

Similarly, an SSF experiment was also executed in a NBS Bioflow 115 bioreactor (2 L working volume, pitch blade impeller) at 42 °C, 20% (w/w) solid loading and 20 FPU SacchariSEB C6L per g of solid loading. Pre-saccharification was performed at 50 °C for an initial 3 h followed by SSF for 45 h. Another set of SSF was carried out with 25% solid loading without a pre-saccharification step. The rest of the experimental conditions were the same as previously. Samples were withdrawn at different time intervals and analyzed for glucose and ethanol.

2.8 Analytical techniques

To determine the sugar, inhibitors and ethanol concentration in media, samples were collected and centrifuged at 5000 rpm for 10 min. Supernatant was kept at −20 °C until analysis. Quantitative analysis of sugars and inhibitors (5-HMF, furfural, acetic acid) were conducted by HPLC equipped with an Aminex HPX-87H column 300 mm × 7.8 mm ID (BioRad Labs). Sugars and acetic acid were quantified by a refractive index detector while, 5-HMF and furfural were quantified through a PDA detector (UV/Vis detector at 254 nm). The mobile phase was 50 mM H₂SO₄ with an elution flow rate of 0.6 mL min⁻¹. Column and detector temperatures were 50 °C and 30 °C, respectively.

Ethanol estimation was performed on a gas chromatograph (Clarus 680 Perkin-Elmer) fitted with an Elite-5 MS column (30 m × 0.32 mm × 0.25 μm) using the following temperature program: initial 60 °C (held for 3 min) to 150 °C at the rate of 10 °C min⁻¹. Injector and detector temperatures were 150 °C and 250 °C, respectively. Helium was taken as a carrier gas at 2 mL min⁻¹ flow rate. The dry cell weight (DCW) was measured by converting cell absorbance (λ₆₀₀) using a 5 point calibration standard.

2.9 Statistical analysis and equations

All the studies were conducted in triplicate and the results are presented as means of replicates along with their standard deviation (represented as error bars). Data were analyzed by using a one way ANOVA.

Eqn (1)–(4) were applied for synthetic media and SHF (considering glucose in enzymatic hydrolysate) process whereas, eqn (5) was applied to calculate ethanol yield in the both SSF and SHF (considering glucose and cellulose in pretreated slurry) processes.

where, E indicates the total ethanol produced during fermentation (g L⁻¹), X_t indicates biomass (g L⁻¹) after time t, G_t indicates consumed glucose (g L⁻¹) after time t, TS is the total solid biomass (g L⁻¹), WSS indicates water soluble solid, ‘f’ is cellulose fraction of dry biomass, S is glucose content in biomass before pre-saccharification, 0.51 is the conversion factor for glucose to ethanol based on stoichiometric biochemistry of yeast and 1.111 is the conversion factor for cellulose to equivalent glucose.

3. Results and discussion

3.1 Screening, isolation and identification of inhibitors and thermotolerant yeast

In the present study, 26 morphologically yeast colonies were isolated from eight samples on YPD agar medium supplemented with streptomycin and ethanol. In order to obtain ethanol tolerant yeast strains, enrichment isolation were carried out in PCM agar and broth medium at 30 °C. Five yeast strains among all grown on PCM agar and broth medium. These strains were compared on the basis of their fermentation efficiency along with the reference strain S. cerevisiae NRRL2034 at 42 °C for 48 h. Comparative ethanol production results are shown in Fig. 1. Remarkably, the DBT-IOC S24 strain performed much better in ethanol production (69.47 g L⁻¹) and yield (81.16%) than other isolates and reference strain S. cerevisiae NRRL2034. No strains were capable of growing and fermenting sugar at 45 °C (data not shown). Distillery/sugar mill spent wash is a good habitat for efficient inhibitor and thermotolerant natural strains.

Fig. 1 Fermentation profile of yeast isolates in YFM media (180 g L⁻¹ initial glucose concentration, 42 °C, 180 rpm, pH 5.0 ± 0.2, and 48 h) reference strain SCY2034: S. cerevisiae NRRL2034.

To identify DBTIOC S24 isolate, a highly variable region of partial 18S rRNA, ITS1, 5.8S rRNA, ITS2 and partial 28S rRNA gene was amplified, sequenced, matched against NCBI data base (http://blast.ncbi.nlm.nih.gov) and the results were used to construct a phylogenetic tree (ESI Fig. A1†). As per the phylogenetic tree, yeast isolate DBTIOC S24 is closely related (>99% similarity) to S. cerevisiae. Hence, it was identified and designated as S. cerevisiae DBTIOC S24 (NCBI GenBank Accession Number: KT375337). In view of screening results, DBTIOC S24 strain was selected for further evaluation and characterization. The biochemical properties for substrate utilization were positive for fructose, dextrose, galactose, rafinose, trehalose, mannose, inulin, salicin, melezitose and esculin (Table 2).

Table 2 Substrate utilization characteristics of S. cerevisiae DBTIOC S24 isolate

Substrate	Results^a	Substrate	Results^a	Substrate	Results^a
a (+) assimilation; (++) fermentation; (−) no growth.
Lactose	+	Inulin	++	Rhamnose	−
Xylose	−	Na-gluconate	−	Cellobiose	+
Maltose	−	Glycerol	−	Melezitose	++
Fructose	++	Salicin	++	α-Methyl D-mannoside	−
Dextrose	++	Dulcitol	−	Xylitol	−
Galactose	++	Inositol	−	ortho-Nitrophenyl-β-galactoside	−
Rafinose	++	Sorbitol	+	Esculin	++
Trehalose	++	Manitol	+	D-Arabinose	−
Melibiose	−	Adonitol	−	Citrate	+
Sucrose	++	Arabitol	−	Malonate	−
L-Arabinose	−	Erythritol	−	Sorbose	−
Mannose	++	α-Methyl D-glucoside	−	Control (negative)	−

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3.2 Fermentation performance of DBTIOC S24 at various temperatures, pH and initial glucose concentration

Tolerance for high temperatures, inhibitors, pH, ethanol concentration and high sugar concentrations are the main barriers for any yeast during fermentation. Thus, the characterization of yeast tolerance to these stresses is essential for efficient lignocellulosic ethanol fermentation.^20,21 As the results show in Fig. 2A, S. cerevisiae DBTIOC S24 isolate consumed glucose completely and produced a maximum 83.12 g L⁻¹ ethanol at 30 °C. Higher glucose consumption rate was observed in the initial 24 h in all the individual temperature sets. At 42 °C, ethanol yield was more than 84.94% but glucose consumption rate and ethanol productivity decreased, which could be due to the combined inhibitory effects of the produced ethanol and the higher temperature. Reduced growth and lower ethanol production at high temperature during fermentation is reported elsewhere.^13,22 At 25 °C, a minimum 77% ethanol yield was observed after 24 h while, 84–90% ethanol yield were recorded in the rest of the temperature sets, is noteworthy.

Fig. 2 Effect of temperature, pH, and glucose concentration on fermentation using S. cerevisiae DBTIOC S24; (A) at different temperatures (25–42 °C) with initial glucose concentration 180 g L⁻¹, pH 5.5; (B) at different pH (3–7) with an initial glucose concentration of 180 g L⁻¹, temperature 30 °C and (C) at different initial glucose concentrations (60–180 g L⁻¹) with pH 5.5, 30 °C. Glucose consumption (solid bar) and ethanol concentration (unfilled bar).

Further, to evaluate the fermentation efficiency of yeast at various pH, DBTIOC S24 isolate was inoculated in YFM (180 g L⁻¹ glucose) with different initial pH (3–7) and incubated at 30 °C. Fig. 2B represents the glucose and ethanol content after 24, 48 and 72 h. Complete glucose was consumed within 24 h in broth medium in the initial pH 5–7 range while, at pH 3–4, almost complete glucose consumption was possible only after 48 h. The maximum production of ethanol (85.17 g L⁻¹) was achieved within 24 h when the initial pH was 6. The ethanol yield was observed in range of 81–94% in all pH variation sets with a maximum at pH 6. The highest ethanol productivity 3.55 g L⁻¹ h⁻¹ was obtained at pH 6 followed by pH 5 (3.23 g L⁻¹ h⁻¹). Therefore, DBTIOC S24 shows activity in a much wider range of pH. Optimum pH for maximum ethanol fermentation was reported from pH 4.5–5.5 using S. cerevisiae.^20,23 Principally, it depends upon ΔpH i.e. pH difference of inside and outside pH of yeast cells.²⁴

With the cost effective and low negative impact on the environment, lignocellulosic ethanol production can be a lucrative option using high gravity fermentation. Therefore, it is worthwhile to examine the effect of different glucose concentrations (60, 120, 180 g L⁻¹) on the fermentation efficiency of DBTIOC S24 isolate. As shown in Fig. 2C, complete glucose was consumed in all the sets with in 24 h at both the temperatures except the one with 180 g L⁻¹ initial sugar concentration at 42 °C. At 30 °C, 78.53 g L⁻¹, 54.08 g L⁻¹ and 27.89 g L⁻¹ ethanol were produced after 24 h with initial glucose concentration 180 g L⁻¹, 120 g L⁻¹ and 60 g L⁻¹, respectively. Remarkably, ethanol yield were recorded in the range of 86–93% and 82–86% at 30 °C and 42 °C, respectively. Considering the above observations, DBTIOC S24 isolate can be used for efficient ethanol production at wide range of temperature and pH using high gravity fermentation with cost effective down streaming.

3.3 Synergistic effect of lignocellulosic inhibitor cocktails on ethanol fermentation

Ethanol fermentation in the presence of inhibitors can cause the slowing or cessation of microbial cell growth, reduced ethanol productivity and low ethanol yield which is a challenge. In order to study the dose dependent response against the key lignocellulosic inhibitor cocktails, the effect of acetic acid (0–4.5 g L⁻¹), formic acid (0–1.0 g L⁻¹), 5-HMF (0–3.0 g L⁻¹) and furfural (0–2.0 g L⁻¹) on fermentation efficiency of DBTIOC S24 isolate was investigated. However, by adopting acid pretreatment or steam explosion and employing severe conditions, the inhibitors concentration was much lower than the concentration range evaluated in our study.^7,25 As indicated in Table 1, fifteen different combinations of inhibitors cocktails with and without pH adjustment (5.0 ± 0.2) were inoculated and incubated for 46 h. Without adjusting the initial pH, maximum 2.17 g L⁻¹ biomass and 76.81 g L⁻¹ ethanol were produced in the absence of inhibitors while, ethanol concentration and productivity decreased in the presence of inhibitor cocktails. Ethanol concentration and productivity drastically decrease with an increase in weak acid concentration to above 0.66 g L⁻¹. The effects of weak acids are strongly pH dependent. At pH value below the pK_a-value of the acid, the undissociated form of weak acids predominates. Undissociated acids enter inside the cell through passive diffusion and get dissociated due to higher internal pH. Hydrogen ions are pumped out through an ATP coupled reaction and take potassium ions to maintain an ionic stasis. Although low levels of acids activate the glycolytic rate by stimulating ATP production, higher levels become inhibitory due to the acidification of the cytosol after depletion of the available ATP, resulting in inhibitory to several glycolytic enzymes.^24,26 The inhibitory effect of these compounds can be greatly overcome by adjusting the initial pH of the medium to 5.0 ± 0.2. After adjusting the initial pH to 5.0, the tolerance level of S. cerevisiae DBTIOC S24 isolate also increased. Nevertheless, the maximum 78.89 g L⁻¹ ethanol was produced in the presence of inhibitors followed by in the absence of inhibitors (77.04 g L⁻¹). At higher pH values, a reason for reduced inhibition due to the smaller ΔpH and less cellular stress is because of the decreased intake concentration of undissociated acid.^24,27 Other than weak acids, furans (furfural and 5-HMF) also play a significant role to inhibit yeast growth and ethanol productivity. The inhibitory effect of furfural on growth and fermentation was enhanced by increasing the furfural concentration. These results agreed with those obtained by Palmqvist et al.²⁸ using S. cerevisiae in the presence of furfural (4 g L⁻¹). Unlike the fufural, toxic effect of 5-HMF was not significant (up to 2 g L⁻¹) compared to furfural and acetic acid in cocktails. After adjusting the pH, the toxic effect of the inhibitors occurs after certain concentrations. Remarkably, the inhibitor effect was much less as compared to the yeast strains reported in the literature.¹³ Bellido et al.²⁹ also observed that an increase in acetic acid and furfural concentration led to a reduction in sugar consumption rates and ethanol concentration with increasing concentration while 5-HMF did not exert a significant effect. Nevertheless, the DBTIOC S24 isolated shows a much better tolerance to inhibitor cocktails and the above findings also suggested the role of pH to overcome the inhibitory effect on the ethanol fermentation efficiency of yeast. This strain may serve as a potential candidate for economically viable lignocellulosic ethanol production at the industrial scale.

3.4 Ethanol fermentation in the presence of ligno-cellulosic inhibitors in a bioreactor

To validate the versatility of S. cerevisiae DBTIOC S24 isolate, synthetic medium was amended with high sugar concentration and multiple inhibitors to create a high gravity multi-stress fermentation environment in the bioreactor. Fig. 3 indicates dry cell weight (DCW), glucose concentration, ethanol concentration and ethanol yield at different times and temperatures. From Fig. 3, it is clearly depicted that 37 °C was the optimum temperature for growth. A maximum 5.54 g L⁻¹ yeast was grown within 20 h in control followed by 5.52 g L⁻¹ yeast grown within 42 h in the presence of inhibitors at 37 °C. This could be due to the increased lag phase of yeast isolate in inhibitor amended medium. In terms of ethanol fermentation, maximum 91.05 g L⁻¹ ethanol (22 h) was produced at 30 °C followed by 81.07 g L⁻¹ at 37 °C (20 h) and 80.68 g L⁻¹ at 42 °C (42 h) in control conditions. The ethanol yields were varied from 88–96% (0.45–0.49 g g⁻¹). Sree et al.³⁰ reported 0.48 g g⁻¹ (at 30 °C) and 0.36 g g⁻¹ (40 °C) ethanol yield employing S. cerevisiae. Ortiz-Muniz et al.³¹ reported maximum 0.41 g g⁻¹ ethanol yield of S. cerevisiae ITV-01 at 30 °C. In presence of inhibitors, maximum 78.02 g L⁻¹ ethanol (82%) was produced with in 42 h at 37 °C followed by 75.33 g L⁻¹ at 30 °C (28 h) and 73.30 g L⁻¹ ethanol at 42 °C (64 h). Since, inhibitors were incapable to significantly affect the fermentation efficiency of S. cerevisiae DBTIOC S24 at higher temperature, therefore it may be useful to save cooling cost and reduce contamination chances at the industrial scale.

Fig. 3 Fermentation profile of S. cerevisiae DBTIOC S24 at different temperatures i.e. 30 °C (A–B), 37 °C (C–D) and 42 °C (E–F) using 180 g L⁻¹ initial glucose concentration at pH 5.0 ± 0.2 in absence (A, C and E) and presence (B, D and F) of inhibitors (acetic acid 2.5 g L⁻¹; formic acid 0.25 g L⁻¹; 5-HMF 0.75 g L⁻¹ and furfural 0.6 g L⁻¹) DCW: dry cell weight.

3.5 Ethanol fermentation via SHF

In this section, S. cerevisiae DBTIOC S24 isolate was evaluated for efficient ethanol fermentation using lignocellulosic hydrolysate because actual hydrolysate differs from the synthetic cocktails due to unidentified inhibitors which show a synergistic effect on yeast fermentation efficiency.⁹ The fermentation performance of DBTIOC S24 isolate was evaluated via SHF at 30 °C and 42 °C using pretreated rice straw on account of the exothermic nature of the fermentation process and a fair chance to increase in temperature in the industrial scale bioreactor due to negligible heat loss to the environment.

The SHF process was performed using dilute acid pretreated rice straw at 20% total solid loading using SacchariSEB C6L enzyme which resulted in a rice straw hydrolysate slurry containing glucose, 80.5 g L⁻¹; xylose, 30.2 g L⁻¹; acetic acid, 1.86 g L⁻¹; 5-HMF, 0.52 g L⁻¹; furfural, 0.21 g L⁻¹. The enzymatically hydrolyzed slurry was used as such for fermentation at 30 °C and 42 °C. Fig. 4A illustrates glucose and ethanol concentration along with ethanol yields at different time intervals. At 30 °C, maximum glucose is exhausted in the initial 6 h and the produced maximum 35.30 g L⁻¹ ethanol which corresponds to a 91% ethanol fermentation efficiency while, 73.5% ethanol yield was calculated after considering the initial glucose and cellulose content of the pretreated slurry. The furfural was completely metabolized in the initial 3 h whereas, 5-HMF conversion was comparatively slower and remained at 0.26 g L⁻¹ after 6 h. During the fermentation process, two main inhibitors compound relevant to lignocellulosic biomass i.e., furfural and 5-HMF are metabolized by yeast to their corresponding less inhibitory alcohol form.^21,32 At 42 °C, maximum 33.93 g L⁻¹ ethanol was produced within 29 hours with 83.73% fermentation efficiency achieved considering the initial glucose in the hydrolysate. While, considering both initial glucose and cellulose content, 70.6% efficiency was recorded. Ethanol productivity at 42 °C was calculated as 1.17 g L⁻¹ h⁻¹ which was lower than ethanol productivity at 30 °C (5.88 g L⁻¹ h⁻¹). This could be due to the combined adverse effect of ethanol and temperature on yeast physiology mainly because of changes in the cell membrane permeability, transport system, damage to the cell wall etc.²² Similar to SHF at 30 °C, furfural was completely metabolized in the initial 3 h and 5-HMF remained at 0.46 g L⁻¹ after 21 h at 42 °C. In order to evaluate the glucose consumption and fermentation efficiency of S. cerevisiae DBTIOC S24 in the absence of inhibitors (as control), YFM with 80 g L⁻¹ glucose was inoculated with yeast isolate (Fig. 4B). At 30 °C, glucose was almost completely consumed after 5 h with a maximum 36.17 g L⁻¹ ethanol content. At 42 °C, complete glucose consumption took 7 h with a maximum 31.72 g L⁻¹ ethanol content. The above observations indicate that DBTIOC S24 isolate has the capability to efficiently ferment non-sterilized and non-detoxified actual hydrolysate at a wide range of temperature (30–42 °C).

Fig. 4 Ethanol fermentation profile of S. cerevisiae DBTIOC S24: (A) using rice enzymatic hydrolysate in SHF and (B) using fermentation media with glucose (80 g L⁻¹) as a control, at 30 °C (solid line) and 42 °C (dotted line). The hydrolysate was used without filtration, sterilization or detoxification.

3.6 Ethanol fermentation via SSF

To evaluate fermentation efficiency of DBTIOC S24 isolate via SSF, dilute acid pretreated rice straw biomass was carried out in a 5 L bioreactor using 20 FPU (SacchariSEB C6L) per g of solid biomass. SSF with 20% solid loading was executed by an initial 3 h pre-saccharification at 50 °C followed by 45 h at 42 °C. The initial pre-saccharification step allows the enzyme to work at an optimal temperature resulting in increased liquefaction, which allows the biomass to easily ferment. Fig. 5A illustrates the glucose and ethanol concentrations along with the ethanol yield during SSF. The ethanol productivity during the first 18 h of SSF was 1.72 g L⁻¹ h⁻¹ with an ethanol concentration of 30.95 g L⁻¹. After 45 hours, a maximum 38.22 g L⁻¹ ethanol was obtained with 0.85 g L⁻¹ h⁻¹ productivity, which corresponds to an overall ethanol yield 80.65%.

Fig. 5 Fermentation profile of S. cerevisiae DBTIOC S24 in SSF of dilute acid pretreated rice straw slurry at (A) 20% and (B) 25% solid loading. 20 FPU enzyme per g biomass was added in starting (BPS: before pre-saccharification i.e., 3 h).

In order to increase the ethanol titer, another SSF experiment was performed at a higher solid loading (25%) under similar conditions except for the elimination of the pre-saccharification step (Fig. 5B). Maximum 49.45 g L⁻¹ ethanol with 83.70% yield was produced after 72 h. Ethanol concentration was much higher than the threshold concentration of ethanol (40 g L⁻¹) for distillation, making the process cost effective.³¹ Maximum ethanol productivity was observed after 16 h (2.12 g L⁻¹ h⁻¹) which, was subsequently decreased with increased fermentation time. On the basis of the above observations, there was no significant effect on ethanol production after elimination of the pre-saccharification step. Saini et al.¹⁸ reported that SSF without pre-saccharification led to more ethanol production even at a faster rate. Feasible high solid loading and elimination of the pre-saccharification step employing thermotolerant yeast would be an added advantage to reduce fermentation time and enhance the ethanol titer along with the productivity.¹⁸ Jung et al.³⁴ reported 70.7% ethanol yield of S. cerevisiae using rice straw biomass. The isolate used in the present study has a better ethanol fermentation efficacy than the other reported strain viz. 56.3% yield using S. cerevisiae³⁵ and 77.7% yield using Kluveromyces marxianus³³ from lignocellulosic biomass. Various new commercial cellulotic enzyme preparations contain lytic polysaccharide monoxoygenases (LPMOs) and it has a great influence on enzymatic hydrolysis by oxidative cleavage of crystalline cellulose/hemicelluloses. LPMOs needs oxygen or electron donors for their activity. Lignin has been speculated to be the electron supplier for the activity of LPMOs.³⁶ In the present study, pretreated biomass contains 28.9% lignin which is speculated to act as an electron donor for LPMOs without hampering the efficiency of the SSF process. The above results indicate that thermo and inhibitor tolerance yeast isolate is a lucrative option for the SSF process using whole dilute acid pretreated slurry without detoxification. SSF ethanol yields were higher (80.65%) in comparison to SHF (73.5%); similar results have also been reported previously.¹⁹ This could be due to several factors such as no substrate feedback inhibition of enzyme, less inhibition of the enzyme by inhibitors as DBTIOC S24 yeast bio-detoxifies the hydrolysate to lower concentration; and the high temperature reduces the chance of contamination.

4. Conclusion

A new thermo and inhibitor tolerant yeast strain was isolated and identified as S. cerevisiae DBTIOC S24. The yeast was active over a wide range of temperature (25–42 °C) and pH (3–7) with high ethanol fermentation efficiency and productivity. The tolerance of this strain to high concentrations of lignocellulosic inhibitors differentiates it from similar thermotolerant strains reported in the literature. Therefore, S. cerevisiae DBTIOC S24 shows a high potential for industrial scale fermentation via both SHF and SSF. To the best of our knowledge, this is the first report employing a thermotolerant S. cerevisiae isolate to produce 49.45 g L⁻¹ ethanol with 83.7% yield at 42 °C using unsterilized and non-detoxified rice straw hydrolysate via SSF. In turn, the above isolate has the capability to produce lignocellulosic ethanol production with a reduction in the fermentation time and process cost.

Acknowledgements

Authors acknowledge Indian Oil Corporation R & D centre and Department of Biotechnology, Govt. of India for providing financial support.

References

1. IEA, World Energy Investment Outlook: Executive summary, International Energy Agency, Paris, 2014.
2. A. Midilli and I. Dincer, Int. J. Hydrogen Energy, 2008, 33, 4209–4222.
3. R. Sims, M. Taylor, J. Saddler and W. Mabee, International Energy Agency, 2008, 16–20.
4. S. Kim and B. E. Dale, Biomass Bioenergy, 2004, 26, 361–375.
5. Y. Zhang, X. Chen, Y. Gu and X. Zhou, Appl. Energy, 2015, 160, 39–48.
6. H. J. Young and H. K. Kyoung, in Pretreatment of Biomass: Processes and Technologies, Academic Press, 2014, pp. 27–50.
7. R. Landaeta, G. Aroca, F. Acevedo, J. A. Teixeira and S. I. Mussatto, Appl. Energy, 2013, 102, 124–130.
8. S. Tian, J. Zhu and X. Yang, Appl. Energy, 2011, 88, 1792–1796.
9. A. K. Chandel, S. S. Silva and O. V. Singh, in Biofuel Production – Recent Developments and Prospects, InTech, Rijeka, 2011, pp. 225–246.
10. S. Kumar, P. Dheeran, S. P. Singh, I. M. Mishra and D. K. Adhikari, Am. J. Microbiol. Res., 2013, 1, 39–44.
11. W. R. Abdel-Fattah, M. Fadil, P. Nigam and I. M. Banat, Biotechnol. Bioeng., 2000, 68, 531–535.
12. V. Wallace-Salinas and M. F. Gorwa-Grauslund, Biotechnol. Biofuels, 2013, 6, 151.
13. L. Favaro, M. Basaglia, A. Trento, E. Van Rensburg, M. García-Aparicio, W. H. Van Zyl and S. Casella, Biotechnol. Biofuels, 2013, 6, 168.
14. S. Mohana, B. K. Acharya and D. Madamwar, J. Hazard. Mater., 2009, 163, 12–25.
15. D. Pant and A. Adholeya, Bioresour. Technol., 2007, 98, 2321–2334.
16. T. J. White, T. Bruns, S. J. W. T. Lee and J. W. Taylor, PCR protocols: a guide to methods and applications, 1990, vol. 18, pp. 315–322.
17. K. Tamura, D. Peterson, N. Peterson, G. Stecher, M. Nei and S. Kumar, Mol. Biol. Evol., 2011, 28, 2731–2739.
18. J. K. Saini, R. Agrawal, A. Satlewal, R. Saini, R. Gupta, A. Mathur and D. Tuli, RSC Adv., 2015, 5, 37485–37494.
19. R. R. Singhania, J. K. Saini, R. Saini, M. Adsul, A. Mathur, R. Gupta and D. K. Tuli, Bioresour. Technol., 2014, 169, 490–495.
20. A. Tahir, M. Aftab and T. Farasat, J. Appl. Pharmacol., 2010, 3, 72–78.
21. L. J. Jönsson, B. Alriksson and N. O. Nilvebrant, Biotechnol. Biofuels, 2013, 6, 16.
22. D. A. Costa, C. J. de Souza, P. S. Costa, M. Q. Rodrigues, A. F. dos Santos, M. R. Lopes, H. L. A. Genier, W. B. Silveira and L. G. Fietto, Appl. Microbiol. Biotechnol., 2014, 98, 3829–3840.
23. M. Hashem, A. N. A. Zohri and M. M. Ali, Afr. J. Microbiol. Res., 2013, 7, 4550–4561.
24. K. C. Thomas, S. H. Hynes and W. M. Ingledew, Appl. Environ. Microbiol., 2002, 68, 1616–1623.
25. A. K. Chandel, F. A. Antunes, P. V. de Arruda, T. S. Milessi, S. S. da Silva and M. D. G. de Almeida Felipe, in D-Xylitol, Springer, Berlin Heidelberg, 2012, pp. 39–61.
26. J. R. Almeida, D. Runquist, V. Sànchez Nogué, G. Lidén and M. F. Gorwa-Grauslund, Biotechnol. J., 2011, 6, 286–299.
27. M. E. Pampulha and M. C. Loureiro-Dias, Appl. Microbiol. Biotechnol., 1989, 31, 547–550.
28. E. Palmqvist, J. S. Almeida and B. Hahn-Hägerdal, Biotechnol. Bioeng., 1999, 62, 447–454.
29. C. Bellido, S. Bolado, M. Coca, S. Lucas, G. González-Benito and M. T. García-Cubero, Bioresour. Technol., 2011, 102, 10868–10874.
30. N. K. Sree, M. Sridhar, K. Suresh, I. M. Banat and L. V. Rao, Bioresour. Technol., 2000, 72, 43–46.
31. B. Ortiz-Muniz, O. Carvajal-Zarrabal, B. Torrestiana-Sanchez and M. G. Aguilar-Uscanga, J. Chem. Technol. Biotechnol., 2010, 85, 1361–1367.
32. M. J. Taherzadeh, L. Gustafsson, C. Niklasson and G. Lidén, Appl. Microbiol. Biotechnol., 2000, 53, 701–708.
33. A. Wingren, M. Galbe and G. Zacchi, Biotechnol. Prog., 2003, 19, 1109–1117.
34. Y. H. Jung, H. M. Park, I. J. Kim, Y. C. Park, J. H. Seo and K. H. Kim, RSC Adv., 2014, 4, 55318–55327.
35. Y. Z. Wang, Q. Liao, F. L. Lv, X. Zhu, Y. Ran and C. J. Hou, RSC Adv., 2015, 5, 55328–55335.
36. U. F. Rodriguez-Zuniga, D. Cannella, R. de Campos Giordano, R. D. L. C. Giordano, H. Jørgensen and C. Felby, Green Chem., 2015, 17, 2896–2903.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra00007j

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