Screening of Fusarium moniliforme as Potential Fungus for Integrated Biodelignification and Consolidated Bioprocessing of Napier Grass for Bioethanol Production

Abstract

A fungus capable of producing ethanol from various carbon substrates was screened for direct ethanol production from lignocellulose. Fusarium moniliforme BIOTECH 3170 produced ethanol from glucose, xylose, and cellobiose after three days with theoretical yields of 86.4%, 68.6%, and 45.4%, respectively. The coculture of glucose and xylose progressed sequentially at 79.2% of the theoretical yield, with both sugars completely consumed in five days. The solid-state consolidated bioprocessing of cellulose produced 25.2 g/L of ethanol after 20 days. After 28 days of the integrated biodelignification and consolidated bioprocessing of Napier grass at solid-state conditions, up to 10.5 g/L of ethanol was produced, corresponding to an ethanol yield of 0.032 g/g biomass. Given a sufficient carbon source, the screened fungus could produce up to 42.06 g/L ethanol. F. moniliforme BIOTECH 3170 demonstrated the characteristics of a fungus for potential ethanol production from cellulose, mixed sugars, and lignocellulosic materials.

1. Introduction

Lignocellulosic biomass is a renewable, cheap, and abundant alternative substrate to food-based feedstocks for bioethanol production. This class of biomass feedstock is one of the most extensively studied for the sustainable production of ethanol for biofuel applications. Often, however, many of these studies on lignocellulosic-bioethanol production involve the use of an energy- or cost-intensive three-step sequential process (in separate reaction vessels): (1) thermochemical delignification to deconstruct the recalcitrant lignocellulosic matrix of the biomass, resulting in the release of plant polysaccharides (mainly cellulose and hemicellulose) that are more accessible to enzymatic attack (i.e., by cellulases or hemicellulases), (2) enzymatic (or microbial) saccharification of the released plant polysaccharides into their corresponding monosaccharides, and (3) yeast ethanol fermentation of the monomeric saccharine products of enzymatic hydrolysis [1].

The high costs of the above-mentioned three-step conventional process have been attributed to the high-energy inputs needed to achieve intense temperature and pH conditions for biomass deconstruction in the thermochemical delignification (Step 1) and the high cost of commercial enzymes (cellulases and hemicellulases) (Step 2). Research on alternative approaches that can reduce the production cost of ethanol from lignocellulosic biomass has recently been an active area of study. One of these strategies involve the harnessing of the natural biocatalytic activities of certain species of fungi, most of which are filamentous, and have simultaneous ligninolytic, saccharogenic, and ethanologenic abilities.

Among several process configurations for the production of bioethanol from lignocellulose, consolidated bioprocessing (CBP) holds great potential, as it can considerably reduce production cost due to its simplified operation, reduced capital cost, and lower risk of contamination [2,3]. In CBP, hydrolytic enzyme production, polysaccharide hydrolysis, and fermentation all occur synchronously in a single vessel with a capable microbial system. Ascomycetes from the genera Fusarium [4,5,6], Neurospora [7,8,9], Monilia [10], and Basidiomycetes from the genus Phlebia [11,12,13], and Trametes [14,15] were able to produce ethanol directly from cellulose and from chemically pretreated biomass without the addition of exogenous hydrolytic enzymes. In these studies, however, the delignification performance of these microorganisms (sequentially or simultaneously) in consolidated bioprocessing was considered little or not at all.

Conventional chemical and physicochemical pretreatment methods for lignin degradation are costly, energy-intensive, and produce inhibitory byproducts [16]. Meanwhile, biological pretreatment utilizes the ability of microorganisms to degrade plant cell-wall lignin or alter its structure with the production of extracellular ligninolytic enzymes [1]. The biological degradation of plant cell-wall lignin is a characteristic feature of many filamentous fungi from the phyla Basidiomycota and Ascomycota. These fungi, which commonly thrive in wood and plant litter, produce multiple oxidative and hydrolytic biocatalysts, and play an important role in the global carbon cycle [17]. The integration of biodelignification and CBP can be a promising route for ethanol production from lignocellulose by further reducing costs associated with chemical pretreatment and detoxification. Thus far, only basidiomycetes Phlebia MG-60 [11,13], Trametes hirsuta [14] and Trametes versicolor [15] were able to produce ethanol directly from nonpretreated lignocellulose.

In this study, several basidiomycetous and ascomycetous fungi with interesting characteristics that are useful for lignocellulosic-ethanol production via integrated biodelignification and consolidated processing were screened for their ability to produce ethanol from simple, polymeric, and lignocellulosic substrates. Ethanol production from Napier grass by the screened fungus via integrated biodelignification and CBP was investigated. The maximal ethanol production capability of the screened fungus was also evaluated.

2. Results and Discussion

2.1. Screening of Ethanol-Producing Fungi

2.1.1. Preliminary Screening

Eighteen filamentous fungi from the Ascomycota and Basidiomycota phyla were screened in search for a potential fungus for integrated biodelignification and CBP for bioethanol production. Preliminary screening was conducted using glucose, xylose, and cellobiose where the fungi were assessed for their ethanol-producing capabilities. Among the 18 screened filamentous fungi, only three species, belonging to the Fusarium genus, were able to produce ethanol (Figure 1). Ethanol yields of over 80% were obtained for all three fungi from glucose after 3 days. When xylose was used as the carbon source, ethanol production was generally slower. F. moniliforme BIOTECH 3170 produced the highest theoretical yield at 68.6% after 5 days. When cellobiose was used as the carbon source, production was also relatively slower. The highest theoretical yield was 45.4%, obtained by F. culmorum after 5 days.

2.1.2. Final Screening

A final decisive screening was conducted on the ethanol-producing fungi in the preliminary screening using cellulose and Napier grass as substrates. The bonsolidated bioprocessing of cellulose was investigated at both solid-state and submerged culture conditions (Figure 2). In both conditions, only F. moniliforme BIOTECH 3170 was able to produce significant amounts of ethanol from cellulose. After 20 days of CBP, 25.2 ± 0.7 g/L of ethanol was produced at solid-state conditions with a theoretical yield of 6.34 ± 0.07%. The CBP of cellulose at submerged conditions produced lower ethanol concentration at 5.9 ± 0.3 g/L with a theoretical yield of 5.12 ± 0.19%. Higher ethanol concentration in the solid-state condition may be explained by the higher titers of cellulase around the vicinity of the substrate due to the lower liquid volume simulating its natural condition in the environment [17,18].

Performance parameters for the CBP of cellulose are summarized in

Table 1. Performance parameters for ethanol production from cellulose at solid-state and submerged culture conditions by various fungi after 20 days.

Culture Condition	Fungi	Cellulose Consumed	Ethanol Concentration	Ethanol Yield	Theoretical Yield	Conversion Efficiency
		(g)	(g/L)	(g/g Substrate)	(%)	(%)
Solid state	F. moniliforme BIOTECH 3170	0.55 ± 0.01	25.2 ± 0.7	0.072 ± 0.001	6.34 ± 0.07	46.3 ± 0.3
	F. oxysporum BIOTECH 3429	0.33 ± 0.00	0.0 ± 0.0	0.000 ± 0.000	0.00 ± 0.00	0.0 ± 0.1
	F. culmorum	0.27 ± 0.00	0.6 ± 0.0	0.002 ± 0.000	0.16 ± 0.00	2.4 ± 0.0
Submerged	F. moniliforme BIOTECH 3170	0.34 ± 0.03	5.9 ± 0.3	0.058 ± 0.002	5.12 ± 0.19	60.2 ± 3.6
	F. oxysporum BIOTECH 3429	0.22 ± 0.02	0.5 ± 0.3	0.005 ± 0.003	0.44 ± 0.26	7.7 ± 3.9
	F. culmorum	0.09 ± 0.05	0.5 ± 0.3	0.005 ± 0.002	0.41 ± 0.22	19.1 ± 1.2

. Relative to the consumed amount of cellulose, F. moniliforme BIOTECH 3170 was able to achieve conversion efficiencies of up to 46.3 ± 0.3% and 60.2 ± 3.6% for the solid-state and submerged conditions, respectively. When Napier grass was used as the substrate, F. moniliforme BIOTECH 3170 produced the highest ethanol concentration at 4.97 ± 0.67 g/L after 20 days of integrated biodelignification and CBP, whereas F. oxysporum BIOTECH 3429 and F. culmorum produced 1.24 ± 0.34 and 1.22 ± 0.38 g/L ethanol, respectively (Figure 3).

Table 2. Ethanol production performance from diverse carbon sources by various filamentous fungi.

Fungi	Strain	Glucose	Xylose	Cellobiose	Cellulose	Lignocellulose		Reference
		%TY	%TY	%TY	%TY (EC)	Feedstock a	BY (EC)
A. oryzae	NRRL 694	95.5	18.4		2.1 (0.6)			[19]
F. velutipes	Fv-1	87	1.0	83	0 (0)			[20,21]
F. clamydosporium	VTT-D-77055	82.2	43.1					[22]
F. culmorum	VTT-D-72012	54.8	47.0					[22]
F. culmorum		80.5	25.9	64.7	0.2 (0.6)	Napier grass *	0.001 (1.5)	Present work
F. graminearum	VTT-D-76013	82.2	23.5					[22]
F. moniliforme	BIOTECH 3170	86.4	68.6	45.4	6.3 (25.2)	Napier grass *	0.032 (10.5)	Present work
F. oxysporum	ATCC 10960	90.0	54.8					[22]
F. oxysporum	BIOTECH 3429	83.8	41.1	11.3	0.1 (0.3)	Napier grass *	0.001 (1.5)	Present work
F. oxysporum	BN	97.8	58.7			ILP rice straw	0.155 (9.3)	[6]
F. oxysporum	F3	80.2	48	82.7	89.2 (6.9)	AP wheat straw	0.160 (11.2)	[4]
					53.9 (14.5)	BM wheat straw	0.280 (8.4)
						Corn cob *	0.048 (1.9)
						Brewer’s spent grain	0.048 (3.6)
						Brewer’s spent grain	0.069 (5.2)
						AP Brewer spent grain	0.107 (8)
						AP Brewer spent grain	0.109 (8.2)
						AP wheat straw	0.210 (8.4)
F. oxysporum	MK956809				(1.52)	Milled olive waste	(2.47)	[23]
F. oxysporum	VTT-D-80134	97.8	86.1	64.5	0 (0)			[24]
F. oxysporum	VTT-D-80135	86.1	50.9					[22]
F. solani	VTT-D-77057	90.0	43.1					[22]
F. sporotrichioides	VTT-D-77058	86.1	15.7					[22]
F. sporotrichioides	VTT-D-80138	86.1	15.7					[22]
F. verticillioides		92.4	55.6			AP sugarcane bagasse	0.115 (4.6)	[5]
Monilia sp.		90.0	43.1		70 (17)			[10]
					60 (14)
M. corticolous	CCUG 0481	84.15	29.35					[25]
N. lepideus		74.4	66.5	66.9				[26]
N. crassa	NCIM 870	86.1			91 (9.9)	CEAP bagasse	0.260 (13)	[7]
					91 (9.9)	CEAP straw	0.240 (12)
					77 (16.9)	CAP Mesta Wood	0.220 (11)
					54 (11.9)	CAP Su Babul	0.200 (10)
					36 (9.9)	CAP Mesta Wood	0.350 (7)
					36 (9.9)	CAP Su Babul	0.300 (6)
Paecilomyces sp.	NF1	80.0	77.9	74.7				[27]
P. variotii	ATHUM 8891	80.9	90.7			Corb cob *	<0.030 (0.6)	[28]
						Brewer’s spent grain	<0.030 (1.2)
P. cinerea		80	17.6		52.9 (3)			[29]
Phlebia sp.	MG-60	86.9	65	70.6	28.3 (2.8)	AU hardwood Kraft Pulp	0.42 (8.4)	[11,12,13]
						Oakwood *	0.159 (7)
						Sugarcane bagasse	0.225
						AP sugarcane bagasse	0.34 (6.16)
						Newspaper	0.2 (4.2)
Phlebia acerina	SF 23754	93.9	78.3	92.3				[30]
Rhizomucor sp.	CCUG 61146	92.3	24.9					[31]
Rhizomucor sp.	CCUG 61147	89.4	22.0					[31]
R. javanicus	NRRL 13161	92.0	9.0	15.2	3.2 (0.9)			[19]
R. javanicus	NRRL 13162	85.3	6.7	14.1	1.8 (0.5)			[19]
R. oryzae	CCUG 22420	84.1	54.8					[25]
R. oryzae	CCUG 28958	80.2	31.3					[25]
R. oryzae	NRRL 13480	90.8	11.7	73.6	4.2 (1.2)			[19]
R. oryzae	NRRL 1501	98.2	7.4	68.4	4.6 (1.3)			[19]
R. oryzae	NRRL 2625	95.9	8.6	14.5	3.2 (0.9)			[19]
R. oryzae	NRRL 6201	99.4	22.3	26.0	3.2 (0.9)			[19]
S. commune		80.5	52.4	81.2	45.6 (0.09)			[32]
T. hirsuta		95.9	38.2	92.2		BM rice straw	0.17 (3.4)	[14]
						Rice straw *	0.15 (3)
T. versicolor	KT9427	90.0	86.1	96.1	41.4 (4.7)	Rice straw *	0.218 (4.8)	[15]

%TY = % theoretical yield; BY = biomass yield (g ethanol/g biomass); EC = ethanol concentration (g/L). ^a ILP = ionic-liquid-pretreated; AP = alkali-pretreated; BM = ball-milled; CEAP = cold-ethanolic-alkali-pretreated; CAP = cold-alkali-pretreated; DAP = dilute-acid-pretreated; AU = alkali-unbleached; * nonpretreated.

shows a list of filamentous fungi with theoretical yields of at least 80% from glucose, and their corresponding performance from other carbon substrates. While many filamentous fungi were able to convert glucose into ethanol at high yields, most fungi performed poorly on other substrates. Although F. oxysporum BN, F. moniliforme F3, F. verticillioides, Monilia sp., and N. crassa NCIM 870 produced ethanol from chemically pretreated biomass, only Phlebia sp. MG-60, T. hirsute, and T. versicolor KT9427 produced ethanol directly from nonpretreated lignocellulosic substrates. An ideal workhorse for effective bioethanol production from lignocelluloses must be able to convert both glucose and xylose into ethanol at high yields, and possess the capability to access and hydrolyze the polysaccharides into more usable forms. In this study, the ability of F. moniliforme BIOTECH 3170 to produce ethanol from glucose and xylose at high yields, and from cellulose at high titers is demonstrated. Its ability to directly produce ethanol from untreated Napier grass via integrated biodelignification and CBP at relatively higher titers than those in previous reports merits it as a potential workhorse to the described bioprocess.

2.2. Cofermentation of Glucose and Xylose

The fermentation behavior of F. moniliforme BIOTECH 3170 in a mixed-substrate culture using 10 g/L each of glucose and xylose is shown in Figure 4. Glucose was completely assimilated by F. moniliforme BIOTECH 3170 in 2 days, whereas xylose was used sparingly. Upon the complete consumption of glucose, xylose utilization became more apparent, although at a slower rate. After 5 days, both sugars were completely consumed, producing 8.09 ± 0.35 g/L ethanol with a theoretical yield of 79.2 ± 3.5%. The observed repression in the utilization of xylose in the presence of glucose, also known as carbon catabolite repression, was previously reported [5,22,33]. In the glucose–xylose cofermentation study by de Almeida [5], F. verticillioides completely utilized glucose while leaving 88% of the xylose unconsumed. A competition between glucose and xylose for the same transporter was suggested to be the reason for the delay in xylose assimilation [5]. This sequential assimilation of sugars due to the presence of a more preferred substrate may be averted by employing processes where hydrolysis and fermentation occur simultaneously, such as SFF and CBP. In such a configuration, sugars generated from saccharification are instantaneously assimilated and fermented by the microorganism, hence avoiding the accumulation of sugars.

2.3. Integrated Biodelignification and CBP of Napier Grass

Napier grass (Pennisetum purpureum), a high-yielding [34,35] perennial and tall herbaceous grass commonly found in tropical and subtropical countries [36], was used as the lignocellulosic substrate for ethanol production. The Napier grass used in this study contained 37.4 ± 0.1% glucan, 19.9 ± 0.3% xylan, and 17.2 ± 1.1% lignin. Biodelignification, being an oxidative process, was carried out in an aerobic environment for 7 days, whereas CBP was conducted in anaerobic conditions for 28 days. Changes in the composition of Napier grass, the formation of monosaccharides, and the production of ethanol were investigated during the bioprocess, as depicted in Figure 5 and

Table 3. Compositional changes and product formation during the integrated biodelignification and CBP of Napier grass.

Day	Oxygen Condition	Biomass (Solid)								Supernatant (Liquid)
		Residual Mass (g/2 g Napier Grass)				Percent Degradation (%)				Concentration (g/L) *
		Total Solid Biomass	Glucan	Xylan	Lignin	Total Solid Biomass	Glucan	Xylan	Lignin	Ethanol	Glucose	Xylose
0	Aerobic	1.87	0.75	0.40	0.34	0.0	0.0	0.0	0.0	0.00	n.d.	n.d.
7	Anaerobic	1.57	0.54	0.31	0.30	16.1	27.4	23.1	14.0	0.00	n.d.	n.d.
14	Anaerobic	1.42	0.47	0.27	0.28	24.1	36.9	32.6	18.6	6.33	n.d.	n.d.
21	Anaerobic	1.34	0.46	0.25	0.27	28.3	39.2	37.8	20.3	6.69	n.d.	n.d.
28	Anaerobic	1.31	0.45	0.23	0.28	30.1	40.3	42.1	19.9	10.54	n.d.	n.d.
35	Anaerobic	1.26	0.44	0.21	0.27	32.5	41.7	47.4	21.4	9.02	n.d.	n.d.

* n.d., not detected.

. After 7 days of aerobic biodelignification, 14.0% of the lignin was degraded, with the concurrent consumption of glucan and xylan at 27.4% and 23.1%, respectively. During anaerobic CBP, lignin degradation was marginal, while glucan and xylan continued to be used by the fungi. After 28 days of CBP, 7.4% of the lignin, and 14.3% and 24.2% of glucan and xylan, respectively, were further degraded. Ethanol production commenced only after the shift to anaerobic condition, producing up to 10.5 ± 2.4 g/L corresponding to an ethanol yield of 0.032 g/g Napier grass. Glucose and xylose were not detected during the integrated bioprocess.

In the study of Sutherland et al. [37] on lignin degradation by Fusaria, F. moniliforme 279, and F. moniliforme var. subglutinans M-1122 degraded 10.9% and 2.8%, respectively, of the lignin in wheat straw while consuming 30.2% and 24%, respectively, of the holocellulose content after 60 days of cultivation. Chang et al. [38] screened a lignin-selective F. moniliforme 812 that degraded 34.7% of the lignin of rice straw after 10 days while consuming only 2.1% of the holocellulose. Lignin degradation was attributed to the activities of lignin peroxidase and manganese peroxidase, which peaked after 10 days of aerobic solid-state culture. In this study, as much as 21.4% of the total lignin content of Napier grass was degraded by F. moniliforme BIOTECH 3170., predominantly during aerobic phase with, however, the co-consumption of holocellulose.

In the study of de Almedia [5] on submerged CBP of alkali-pretreated sugarcane bagasse by F. verticillioides, xylanase activity was 14-fold greater than that of endoglucanase. A final ethanol concentration of 4.6 g/L with a yield of 0.115 g/g biomass was achieved after 12 days of CBP. Similarly, neither residual glucose nor xylose was detected during CBP, implying immediate sugar assimilation by the fungus. Hydrolysis was suggested as the process bottleneck due to the suboptimal temperature for enzymatic hydrolysis during CBP, which was only carried out at 28 °C. Khuong et al. [13] studied the integrated biodelignification and CBP of sugarcane bagasse by the white-rot fungus Phlebia MG-60. After 4 weeks of aerobic delignification, glucan, xylan, and lignin contents were reduced by 10.2%, 25.0%, and 42.7%, respectively. After another 4 weeks of anaerobic CBP, ethanol was produced at 0.225 g/g biomass. In this study, F. moniliforme BIOTECH 3170 was able to produce up to 10.5 ± 2.4 g/L of ethanol from nonpretreated Napier grass in 4 weeks.

2.4. Maximum Ethanol Production

While yeasts produce ethanol at high titers, filamentous fungi are not very known for their ethanol production capability. The maximal ethanol production capability of F. moniliforme BIOTECH 3170 was investigated by feeding the fungus with a surplus of glucose for fermentation. As shown in Figure 6, up to 42.7 ± 1.7 g/L ethanol was produced from 150 g/L glucose, which corresponds to a theoretical yield of 55.7 ± 2.2%. The highest reported ethanol concentration produced by a filamentous fungus was 73 g/L by Paecilomyces sp. NF1 from 200 g/L xylose [27]. However, this fungus was unable to produce ethanol from cellulose without exogenous cellulase. Another high-ethanol producer, F. oxysporum VTT-D-80134, which produced 57 g/L ethanol from 150 g/L glucose, performed poorly with xylose, and failed to produce ethanol from cellulose [24]. The ability of F. moniliforme BIOTECH 3170 to produce up to 42.1 ± 1.7 g/L ethanol merits it as a potential workhorse for ethanol production at an industrial scale where the lower limit for an economical downstream distillation process is 40–50 g/L ethanol [39].

3. Materials and Methods

3.1. Fungal Strains and Inoculation

Fusarium oxysporum BIOTECH 3429, Fusarium moniliforme BIOTECH 3170 (NRRL 6393), Phanerochaete chrysosporium BIOTECH 3177, Volvariella volvaceae BIOTECH 3043, Agaricus bisporus BIOTECH 3081, Pleurotus sajor-caju BIOTECH 3031 (Philippine National Collection of Microorganisms, National Institute of Molecular Biology and Biotechnology, University of the Philippines Los Baños, Laguna, Philippines), Pleurotus florida, Pleurotus djamor, Auricularia sp., Hericium sp., Calocybe indica, Ganoderma lucidum (Mushroom Biotechnology Project, Rizal Technological University, Mandaluyong City, Philippines), Fusarium culmorum (isolated from the leaf blade of Napier grass (Pennisetum purpureum) from the University of the Philippines Los Baños, Laguna, Philippines), and five unidentified fungi (tissue cultured from fungal fruiting bodies isolated from decaying wood in the University of the Philippines Diliman, Quezon, Philippines) were used for screening. All fungi were maintained on potato dextrose agar (PDA) slants at 4 °C and subcultured every 1 to 2 months. Five 5 mm mycelial discs taken from the margin of an actively growing 7-day-old mycelium grown in PDA plates were used as inocula for all culture studies.

3.2. Carbon Substrate and Culture Medium

Monosaccharides D-glucose (Sigma) and D-xylose (Hi-media), and disaccharide D-cellobiose (Sigma) were used for preliminary screening. The α-cellulose (Sigma C8002) polysaccharide was used for consolidated bioprocessing. Napier grass (Pennisetum purpureum Schumach) was sourced from the Dairy Training Research Institute, College of Agriculture and Food Science, and the University of the Philippines Los Baños, Laguna, Philippines. The whole Napier grass plant, which included stalks and leaves, was milled using a laboratory Wiley Mill. The Mesh 20–80 fraction was obtained and subjected to Soxhlet extraction using ethanol as solvent to avoid possible interference in the compositional analysis [40]. The extractive-free Napier grass was bone dried at 105 °C for 12 to 16 h and stored in a desiccator until use.

A basal medium consisting of 10 g/L KH₂PO₄, 5 g/L yeast extract, 0.5 g/L MgSO₄·7H₂O, 0.5 g/L CaCl₂·2H₂O, 1 g/L Tween 80, and 1 mL/L of a trace element solution containing 5 g/L Fe₂SO₄·7H₂O, 5 g/L ZnSO₄·7H₂O, 5 g/L CuSO₄·5H₂O, 5 g/L MnSO₄·H₂O, and 5 g/L CoCl₂·6H₂O in deionized water (adjusted to pH 6.0) was used for all ethanol production experiments. For submerged fermentation studies, 1 mL of a resazurin indicator solution (10 g/L) was added per liter of the medium. For solid-state culture studies, a resazurin indicator strip (Oxoid BR0055) was attached to the inner wall of the flask. The resazurin indicator served as a visual cue for the aerobic (pink) or anaerobic (white) condition in the flask. The medium was autoclaved at 121 °C and 15 psi for 15 min prior to use.

3.3. Screening of Ethanol-Producing Fungi

3.3.1. Preliminary Screening

Preliminary screening was conducted via the fermentation of simple sugars in an anaerobic environment. Twenty milliliters of basal medium containing 20 g/L glucose, xylose, or cellobiose in 125 mL conical flasks was sterilized, inoculated, covered with butyl rubber stoppers, and incubated statically at 28 °C for a total of 5 days.

3.3.2. Final Screening

Ethanol-producing fungi from preliminary screening were considered for the final screening using cellulose and Napier grass. Cellulose CBP was conducted at solid-state and submerged conditions. The integrated biodelignification and CBP of Napier grass were performed at solid-state conditions only. Two grams of the carbon substrate was mixed with 6 and 100 mL of the basal medium in a 125 mL conical flask for the solid-state and submerged culture experiments, respectively. For cultures using Napier grass as the substrate, the basal medium was supplemented with 10 g/L of glucose as an initial carbon source. The substrate and basal media were sterilized separately, combined in a 125 mL conical flask, inoculated, covered, and incubated statically at 28 °C. Cotton plugs were used for first 3 days and subsequently replaced with butyl rubber stoppers for the next 17 days of incubation.

3.4. Cofermentation of Glucose and Xylose

Glucose and xylose mixture at 10 g each per liter of basal medium was fermented using the screened fungus to evaluate its fermentation behavior in a mixed-sugar environment. Twenty milliliters of the mixture in 125 mL conical flask was sterilized, inoculated, covered with butyl rubber stoppers, and incubated statically at 28 °C for five days.

3.5. Integrated Biodelignification and CBP of Napier Grass

Then, 2 g Napier grass and 6 mL of the basal medium (supplemented with 10 g/L glucose) were autoclaved separately, mixed in 125 mL conical flasks, inoculated, covered, and incubated statically at 28 °C. Cotton plugs were used for first 7 days to allow for aerobic biodelignification and subsequently replaced with butyl rubber stoppers for the next 28 days for anaerobic CBP.

3.6. Maximum Ethanol Production

Glucose at 50, 100, and 150 g per liter of basal medium was fermented using the screened fungus to evaluate its tolerance to ethanol. Twenty milliliters of the mixture in 125 mL conical flasks was autoclaved, inoculated, covered with butyl rubber stoppers and incubated statically at 28 °C for 15–25 days.

3.7. Analytical Methods

For the submerged culture experiments, the culture supernatant was analyzed directly for ethanol. For the solid-state culture experiments, 20 mL ultrapure water was used to extract the supernatant from the culture for ethanol analysis, whereas the residual solid lignocellulose was diligently washed and dried for compositional analysis. Residual Napier grass was analyzed for glucan, xylan, and lignin according to the determination of structural carbohydrates and lignin in biomass method by the National Renewable Energy Laboratory [40]. Ethanol analysis was performed with high-performance liquid chromatography (HPLC) using a Shimadzu CBA-20A system with RID-10A refractive index detector fitted with a BioRad Aminex HPX-87C column at 40 °C with 0.3 mL/min ultrapure water mobile phase or a Rezex Phenomenex RPM column at 80 °C with 0.6 mL/min ultrapure water mobile phase. Glucose and xylose analysis was carried out via HPLC using a Rezex Phenomenex RPM column at 80 °C with 0.6 mL/min ultrapure water mobile phase. All samples were filtered through a 0.45 μm filter prior to HPLC analysis.

3.8. Calculations

Biomass yield was calculated as follows:

$$BiomassYield=\frac{Ethanolconcentration\left({\mathrm{gL}}^{-1}\right)\times Supernatantvolume\left(\mathrm{L}\right)}{Weightofbiomass\left(\mathrm{g}\right)}$$

Theoretical yield is calculated as

$$TheoreticalYield \left(\%\right)=\frac{Ethanolconcentration\left({\mathrm{gL}}^{-1}\right)}{Sugarconcentration\left({\mathrm{gL}}^{-1}\right)\times Sugarstoichiometricconversion factor}\times 100\%$$

where the sugar stoichiometric conversion factors for glucose, xylose, cellobiose, and cellulose were 0.511, 0.511, 0.538, and 0.568, respectively.

Conversion efficiency was calculated as follows:

$$Conversion efficiency \left(\%\right)=\frac{Ethanol concentration \left({\mathrm{gL}}^{-1}\right) x Supernatant volume \left(\mathrm{L}\right)}{Mass of substrate consumed \left(\mathrm{g}\right)\times Sugar stoichiometric conversion factor}\times 100\%$$

3.9. Statistical Analysis

All culture experiments were performed in duplicate and are presented as mean ± standard deviation. Comparison of the data means between two time points was performed using the paired Student’s t-test at α = 0.05 while comparison of data means between two independent treatments was performed using the independent Student’s t-test at α = 0.05. Likewise, the comparison of the data means for more than two time points was performed using repeated-measures ANOVA at α = 0.05, while the comparison of the data means for more than two independent treatments was performed using ANOVA at α = 0.05. Tukey’s honest-significant-difference post hoc test was performed for multiple means showing significant differences. Statistical analyses were performed using the Data Analysis ToolPak of Microsoft Excel and Minitab 17.

4. Conclusions

A fungus capable of producing ethanol from various carbon substrates was screened. F. moniliforme BIOTECH 3170 produced ethanol from glucose, xylose, and a mixture of the two with high yields. The solid-state consolidated bioprocessing of cellulose produced ethanol at concentrations higher than those in many reported studies, albeit at lower yields, which is inherent to high-dry-matter cultures. Direct ethanol production from nonpretreated Napier grass was achieved via two-step aerobic and anaerobic integrated biodelignification and consolidated bioprocessing. These results warrant F. moniliforme BIOTECH 3170 as a potential microorganism for direct ethanol production from lignocelluloses.