A perspective on bioethanol production from biomass as alternative fuel for spark ignition engine

Abstract

The increasing consumption of fossil fuels has led to the development of alternative fuels for the future. Domestic biofuel production and the utilization of alternative fuels can decrease dependency on petroleum oil, reduce trade deficits, reduce air pollution and reduce carbon dioxide emission. Bioethanol is a renewable fuel produced by the fermentation of sugar which is derived from plants such as sugarcane or beet, maize, or cassava etc. However, bioethanol consumption in an engine is approximately 51% higher than gasoline since the energy per unit volume of ethanol is 34% lower than for gasoline. Bioethanol is an oxygenated fuel that contains 35% oxygen, which can reduce particulate matter and NO_x emissions caused by combustion of the fuel. Therefore, bioethanol–gasoline blends can significantly reduce petroleum use and GHG emission. In addition, utilization of lignocellulosic materials in bioethanol production is the most viable pathway from an environmental point of view. This paper reviews the current status and technologies involved in bioethanol production and the properties and engine performance from various biomass feedstocks which are the recommended sustainable alternative fuel in the future.

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A. H. Sebayang, H. H. Masjuki, Hwai Chyuan Ong, S. Dharma, A. S. Silitonga, T. M. I. Mahlia and H. B. Aditiya

The research on biofuels has been established and developed by group members from Centre for Energy Sciences, Faculty of Engineering, University of Malaya, Kuala Lumpur, Malaysia. Professor Dr. Masjuki Haji Hassan is the leader of Biofuel research in University of Malaya. He is secretary of Council of National Professors Engineering and Technology cluster. He is also the founding President of Malaysian Tribology Society (MyTRIBOS) and the Director of the Centre for Energy Sciences. Dr. Ong Hwai Chyuan, Mr Abdi Hanra Sebayang and Mr Surya Dharma are the academic staff and Researcher in Faculty of Engineering, University of Malaya and are also involved in biofuel research. The group collaborated with Professor Dr. T. M. I. Mahlia from Universiti Tenaga Nasional (UNITEN), Dr. Arridina Susan Silitonga from Medan State Polytechnic, North Sumatera, Indonesia and Mr Aditiya Harjon Bahar from University of Melbourne, Australia.

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1. Introduction

The energy sector plays a vital role in the world economy. Increasing fossil fuel prices, energy security issues and climate change have driven and forced energy development towards alternative and renewable energy sources. Due to the soaring interest in developing renewable and cleaner energy sources, biofuel production caught our attention in the early 2000s.¹ Biofuels are seen as part of the drive to move beyond the dominant fossil-fuel paradigm and the production has been increasing remarkably. This is due to many countries’ attempts to reduce oil imports, boost rural economies and improve air quality. Brazil and USA are the largest world’s ethanol producers and possess 70% of the world’s ethanol production.² The countries were able to export 17 and 20 billion liters from Brazil and USA respectively in 2006.³ Europe stands out with its sugarcane bioethanol program and outstanding results have been gained along the entire production chain from the development, increase in sugarcane variety, and engine manufacturing that can run any gasoline and bioethanol blend.⁴ North America and Sweden introduced vehicles that operate on a fuel blend of 85% ethanol with 15% gasoline.⁵ The world’s major bioethanol producers are shown in Fig. 1.³ The USA leads the world’s bioethanol production by almost 20 billion liters of bioethanol. Second is Brazil which produced almost 17 billion liters of bioethanol, with cheaper production cost of $0.18–0.20 per liter compared with USA ($0.33–0.47 per liter). The EU draws high production cost for every liter of bioethanol produced, which ranges between $0.50–0.97 per liter, although in comparison to China, EU produced less bioethanol by 3.4 billion liters, while China produced 3.85 billion liters. Expanding bioethanol production will increase the use of renewable energy resources, thus enabling independence from fossil fuels. Bioethanol has been receiving widespread interest at the international, national and regional levels. The research on ethanol production has been established with the potential of ethanol as a valuable replacement of gasoline in the transportation fuel market.

Fig. 1 The main producers and production costs of bioethanol.³

Ethanol can be obtained from the microbial conversion of lignocellulosic biomass through fermentation.⁶ The quality of the bioethanol depends on the feedstock. Therefore, many researchers have reviewed the designs and implementation technologies, from the simple conversion of various biomass, such as sugarcane, corn, cassava, rice straw and other agricultural wastes by fermentation, to the multi-stage conversion system of lignocellulosic biomass into bioethanol.^7,8 The fermentation method generally comprises of three steps: (1) the formation of a fermentable solution from the selected biomass, (2) the fermentation of this solution and (3) the separation and purification of the ethanol, usually by distillation.⁹ Favorability of bioethanol can be seen from its properties, including density, vaporization heat, flame temperature, fuel octane, and hydrogen and carbon content which affects the compression ratio.¹⁰ Using 10% of bioethanol in a gasoline blend has the benefits of: reducing CO emissions by 8–30%, reducing toxic emissions by 5–15%, slowing the depletion of the ozone layer and lowering pollution.¹¹ Currently, many countries have introduced 5–10% anhydrous ethanol blended with 95–90% gasoline (E10), and this mixture contains 3.5% oxygen compared to only 2.7% in pure gasoline.^10,12 The higher oxygen content in the blend can contribute to a better combustion and an increase thermal efficiency.^5,13 Furthermore, blending of bioethanol with gasoline affects its fuel properties, such as viscosity, lubricity, octane number, energy content, volatility and stability.¹⁴ However, a major drawback of bioethanol–gasoline fuel is that high blending of ethanol (40–60%) causes wear and corrosion of fuel pumps.⁵ The aim of this paper is to give a thorough overview of the current status of production, properties and engine performance of bioethanol as the substitute for gasoline fuel.

2. Feedstock for bioethanol production

The feedstock availability for bioethanol depends on the region, climate conditions as well as the soil properties.¹⁵ According to Balat et al.,¹⁶ the feedstock for bioethanol production can be categorized into three biomass types: (i) lignocellulosic biomass (e.g. straw, grasses and wood), (ii) starchy materials (e.g. corn, barley, corn, etc.) and (iii) sucrose-containing feedstocks (e.g. sugarcane, sugar beet, sweet sorghum, etc.). Algae is the feedstock with the incredibly high ethanol yield, which change the game on potential feedstock availability.^4,15,17 The potential for bioethanol production from various biomass feedstocks and their comparative production potential are given in Table 1.^18,19 A diverse classification of biofuel feedstocks and processes are currently being developed to meet sustainability and fuel quality standards, as well as the need of road, aviation and marine users. Biofuel can be marketed as a renewable and sustainable alternative fuel in the future. The classification of the generation for biofuel feedstocks is described in Fig. 2.²⁰ It is clearly seen that the main difference of the classifications is due to their feedstock type. First generation utilizes carbohydrate-source crops, which is in general obtainable in the forms of seeds, grains and sugars. Second generation uses lignocellulosic biomass, which is sourced from agricultural residues. In third generation, algae (micro and macro type) are utilized as the bioethanol production feedstock and show a higher reproducibility rate in a lower area requirement.

Table 1 Potential bioethanol production for various biomass feedstocks^18,19

Bioethanol feedstock	Bioethanol production potential (L per ton)
Sweet sorghum	60
Sugarcane	70
Sugar beet	110
Potato	110
Sweet potato	125
Cassava	180
Barley	250
Bagasse and other cellulose biomass	280
Wheat	340
Maize	360
Rice	430

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Fig. 2 The classification of the generation of biofuel feedstocks.²⁰

2.1 First generation biomass

First generation bioethanol is produced from sugar-rich biomass (consisting of linkages of monosaccharides of glucose and fructose) and starch-rich (polysaccharides of glucose) crops such as grain and corn. The contained sugars can be converted to ethanol only after hydrolysis to yield fermentable sugars. The technology is well-known but the high price is the undesirable side effect of this raw material. On the other hand, first generation have raised debate about using food products for fuel production.²¹ The details of producing bioethanol from various first generation sources are discussed below.

2.1.1 Sugarcane and sugar beet. Sugarcane (Saccharum officinarum) is from the genus Saccharum belonging to the poaceae family; it contains a large amount of organic nutrients and minerals to make free sugars as an ideal raw material for bioethanol production.²² It is the most important feedstock grown in tropical and subtropical countries that can be used as juice or molasses (byproduct of sugar mills) for bioethanol production.¹⁶ The sugar produced from sugarcane is readily converted into glucose (the simplest sugar monomer) and can be used as fermentation substrate under anaerobic conditions. Besides, glucose is converted to ethanol and carbon dioxide by glycolysis.²³ The typical content of fermentable sugar is 12–17%, in which 90% is sucrose and 10% is glucose and fructose.^22,24 Therefore, sugarcane contains a high sugar content and it is the main feedstock used for ethanol production in Brazil and India.¹⁵ In addition, the sugarcane crop is favorable since its growing season is only 3–5 months.²⁵

Sugar beet (Beta vulgaris), also called beet molasses, is a suitable feedstock for bioethanol production due to the high sugar content that can be extracted by fermentation without any modifications. Sugar beet contains 15–20% of dry matter, 85–90% of fermentable sugars and 10–15% free sugars.^4,22 The high free sugar content in sugar beet is usable in fermentation with an adjustment of the pH, and this feedstock gives a more profitable profile for bioethanol production.^26,27 The composition profile of sugarcane and sugar beet are listed in Table 2.^22,28

Table 2 The composition profile of sugarcane and sugar beet^22,28

Components	Concentration
	Sugarcane	Sugar beet
Solids (%)	13.7	17.3
Sugar (%)	12–17	16.5
Raffinose (%)	—	0.07
Monosaccharides (%)	0.63	0.15
Polysaccharides (%)	0.028	0.019
Lactate (%)	0.016	—
Acetate (%)	0.033	—
Sulphate (%)	0.039	0.02
Phosphate (%)	0.033	0.047
Nitrate (%)	—	0.015
Nitrite (%)	—	0.005
Aconitate (%)	0.09	—
K (%)	0	—
Na (%)	0.005	0.015
Cl (%)	—	0.003
Ca (%)	0.04	—
Mg (%)	0.028	—
Total-N (%)	—	0.105
Betaine-N (%)	—	0.046
Amino acid-N (%)	—	0.026
Ammonia-N (%)	—	0.006
Amide-N (%)	—	0.011

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2.1.2 Sweet sorghum. Sweet sorghum (Sorghum bicolor (L.) Moench) has been used as a potential bioethanol feedstock due to its high biomass yield and high concentration of readily fermentable sugars.^25,29 Sweet sorghum has a wide degree of adaptability for the tropical region, is well adapted to sub-tropical and temperate regions of the world, and is water efficient.,^25,30 Moreover, sorghum is efficient at photosynthesis based utilization of soil nutrients and its low water consumption means that it is tolerant to drought and flood, compared to sugarcane.^31,32 Sweet sorghum has fast lifecycle ranging from a 3–5 month growing season.^25,33 Sweet sorghum tolerates compacted subsoil at pH range of 5.0–8.5 with some degree of salinity and alkalinity and poor drainage, and its planting temperature is above 12 °C.^30,34 The productivity of sweet sorghum delivers 2000–8000 liters per ha of bioethanol, making sweet sorghum a suitable feedstock for bioethanol production.^8,26 The composition profile of sweet sorghum is shown in Table 3.³⁵

Table 3 The composition profile of sweet sorghum³⁵

Composition	Sweet sorghum
a One kg of the stalk was crushed at 2500 psi for 1 min in a press to extract the juice.
Juice^a (% total)	71.9
Sucrose (% juice)	7.6
Glucose (% juice)	2.6
Fructose (% juice)	1.6
Total sugars (% juice)	11.8
Fiber (% wt)	13.0
Cellulose (% wt)	44.6
Hemicellulose (% wt)	27.1
Lignin (% wt)	20.7
Ash (%)	0.4
Brix (% juice)	11–13

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2.1.3 Wheat, corn, and barley grain. Wheat (Triticum vulgare) is a very good raw material for bioethanol production due to the complete gelatinization of wheat starch at 65 °C.¹⁸ Utilization of wheat or unusable wheat leads to cost effectiveness and hence it is a good option for bioethanol production.³⁶ Moreover, 3.5 tons of wheat grain, or 3.03 tons dried wheat, can be harvested from one acre of good arable land.³⁷

Corn (Maize) is species of Zea and is an annual monoecious flowering hybrid of Zea mays.³⁸ Corn cultivars are widely adapted and can be grown from 0–55° latitude, from sea level to 12 000 ft and with a growing season of 42–400 days.³⁹ 29% of the world’s bioethanol production comes from corn grain.⁴⁰

Barley (Hordeum vulgare L.) is a very attractive feedstock for bioethanol production and is being used in Europe (62%), Asia (15%) and North America (14%).⁴¹ Barley grows well in many areas and it can become a financially cost effective bioethanol feedstock for many regions. Barley is typically utilized for brewing, animal feed, human food and other industrial uses.⁴² Barley is harvested sooner, and it still can give good maturity and high yield.^43,44 Furthermore, 70% of the barley composition is carbohydrate, which indicates good potential in bioethanol production.⁴³ The composition profile of wheat, corn and barley grain are presented in Table 4.^45–48

Table 4 The composition profile of wheat, corn and barley grain biomass^45–48

Composition	Wheat	Corn	Barley
Moisture (%)	12.5	15	11.1
Calories (cal per 100 g)	330	361	349
Protein (%)	12.3	10.2	2.4
Fat (%)	1.8	4.3	1.0
Ash (%)	1.7	1.2	0.9
Sugar (%)	3.6	5.3	5.3

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2.1.4 Cassava. Cassava (Manihot esculenta Crantz), also know as: manioc, yucca, tapioca, has a diameter and length of 5–10 cm and 15–35 cm respectively.⁴⁹ It is considered a tuberous root plant that is native to South America and cultivated around tropical and subtropical countries.^50,51 Cassava can grow at ambient temperatures of 25–30 °C with an optimum annual rainfall requirement of 760–1015 mm.²⁵ Cassava can tolerate prolonged drought since it has the ability to utilize nutrients from the stalk and the storage roots once moisture is restored.⁵² Cassava is harvested around 8–24 months after planting.⁴⁹ Cassava is one of the most important carbohydrate sources in the tropics since it holds a starch content of up to 90% dry weight.⁵⁰ The rich starch in cassava is related to the unique properties which make cassava suitable for food and non-food applications.⁵³ Therefore, cassava starch is one of the best fermentable substances for the production of ethanol. Cassava also contains protein 1% (fresh) and 1.41% (dry), 65% of moisture, 0.9% of ash and 0.03% of phosphorus.⁵⁴ Furthermore, cassava has a high carbohydrate content of 32–35%, which is considered productive in agro-economic applications. The composition profile of cassava biomass is presented in Table 5.^49,55

Table 5 The composition profile of cassava biomass (100 g basis)^49,55

Composition	Cassava
Calories (cal)	135
Peel (%)	10–20
Cork layer (%)	0.5–2.0
Edible portion (%)	80–90
Moisture (%)	62–66
Total solids (%)	38
Volatile solids (%)	99
Protein (g)	1.2
Total nitrogen (%)	0.22
Lipid (g)	0.20
Starch (g)	18–32
Fiber (g)	1.10
Carbohydrate (%)	35
Total carbon (%)	19
Ash (%)	0.9–1
Fat (mg)	0.1
Calcium (mg)	26
Phosphorus (mg)	32
Iron (mg)	1
Sodium (mg)	2
Potassium (mg)	394
Vitamin B2 (mg)	0.04
Vitamin C (mg)	34
Niacin (mg)	0.60
Cyanide (%)	—

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2.2 Second generation biomass (lignocellulosic biomass)

Second generation biomass are derived from lignocellulosic components of agricultural residues, such as straws, wood and other agricultural wastes.^56–59 This type of biomass is usable in second-generation biofuel (biomass to liquid) that has an improvement of CO₂ balance with the use of cheap, waste sources that do not compete with human food products.^15,56,60 The use of agricultural residues can provide benefit to the agricultural sector by providing additional potential income streams for farmers.⁶¹

The composition of lignocellulosic biomass is divided into three main components: lignin (10–20% dry wt), cellulose (30–50% dry wt) and hemicellulose (15–35% dry wt).^17,62 Hemicellulose (C₅H₈O₄)_n, is a polymer structure of hexose (D-glucose, D-mannose and D-galactose) and pentose sugars (D-xylose and L-arabinose).⁶³ It contains D-glucuronic, D-galacturonic and methylgalacturonic acids in sugar acids (uronic acids).⁶⁴ Hemicelluloses are available as xyloglucans or xylans depending on the types of plants.⁶⁵ Moreover, hemicelluloses are linked to cellulose as abundant carbonics in plants.⁶⁶ The dominant source of hemicellulose biomass are woody biomass, hardwoods and softwoods.^17,67,68 Cellulose (C₆H₁₀O₅)_n is a long-chain structural component of glucose monomers (D-glucose) linked to β-(1,4)-glycosidic bonds.⁶⁹ The cellulose molecules also consist of long chains of β-glucose monomers gathered into micro-fibril bundles.⁷⁰ Cellulose is insoluble in water, and a hydrolysis process is required to break down the polymer to free complex sugar molecules, also known as a saccharification process.⁷¹ The product of cellulose is a six-carbon sugar from woody and agricultural biomass.⁶⁷ Lignin C₉H₁₀O₃(OCH₃)0.9–1.7]_n is a structural compound of three types of monomers (p-coumaryl, synapyl alcohols and coniferyl) joined together by a set of linkages to create a matrix.⁷² The complex structures such as hydroxyl, methoxyl and carbonyl have a high polarity which responsible for the hardness of the bonds and consistency of the binding material which holds the cellulosic fibers.⁷³ Lignin is resistant to chemical and biological degradation, which affects the quality of ethanol in bioethanol production.⁶⁶ The major lignin sources are softwood barks, hardwood barks and herbaceous species such as bagasse, corncobs, peanut shells, rice hulls and straws.^7,41,74 The molecule structure of hemicellulose, cellulose and lignin are shown in Fig. 3.^75,76 The composition profile of the lignocellulosic biomass is presented in Table 6.^{41,69,77–81} Furthermore, extensive chemical ash compositions are summarized in Table 7.^82–85

Fig. 3 Lignocellulosic biomass (a) cellulose, (b) hemicellulose and (c) lignin.^75,76

Table 6 The composition profile of hemicellulose, cellulose and lignin from lignocellulosic biomass

Lignocellulosic biomass	Cellulose glucan (% wt)	Hemicellulose (% wt)				Lignin (% wt)		Ref.
		Xylan	Arabinan	Galactan	Mannan	Acid insoluble lignin	Acid soluble lignin
Barley straw	35.65	16.86	2.11	—	13.8	20.7	2.4	41
Corn stover	38.3	21.0	2.7	2.1	—	17.4	—	78
Wheat straw	30.2	18.7	2.8	0.8	—	17	—	79
Rice straw	31.1	18.7	3.6	—	—	13.3	—	69
Rye straw	30.9	21.5	—	—	—	22.1	3.2	69
Oat straw	39.4	27.1	—	—	—	17.5	—	77
Sunflower stalks	42.1	29.7	—	—	—	13.4	—	77
Sugarcane bagasse	43.1	31.1	—	—	—	11.4	—	69
Sweet sorghum bagasse	27.3	13.1	1.4	—	—	14.3	—	69
Olive tree pruning	25.0	11.1	2.4	1.5	0.8	16.2	2.2	81
Poplar	43.8	14.8	—	—	—	29.1	—	80
Spruce	43.8	6.3	—	—	14.5	28.3	0.53	69
Oak	45.2	20.3	—	—	4.2	21.0	3.3	69

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Table 7 The chemical composition of different biomass

Feedstock	Chemical composition (% wt)										Ref.
	SiO2	CaO	K2O	P2O5	Al2O3	MgO	Fe2O3	SO3	Na2O	TiO2
Wood and woody biomass
Eucalyptus bark	10.04	57.74	9.29	2.35	3.1	10.91	1.12	3.47	1.86	0.12	82
Poplar bark	1.86	77.31	8.93	2.48	0.62	2.36	2.36	0.74	4.84	0.12	82
Wood residue	53.15	11.66	4.85	1.37	12.64	3.06	3.06	1.99	4.47	0.57	84
Agricultural residue
Bamboo whole	9.92	4.46	53.38	20.33	0.67	6.57	0.67	3.68	0.31	0.01	85
Rice husks	94.48	0.97	2.29	0.54	0.21	0.19	0.22	0.92	0.16	0.02	84
Sugarcane bagasse	46.79	4.91	6.95	3.87	14.6	4.56	11.12	3.57	1.61	2.02	84
Sunflower husks	23.66	15.31	28.53	7.13	8.75	7.33	4.27	4.07	0.8	0.15	83
Other biomass residue
Mixed waste paper	28.62	7.63	0.16	0.2	53.53	2.4	0.82	1.73	0.54	4.37	84
Sewage sludge	33.28	13.04	1.6	15.88	12.91	2.49	15.7	2.05	2.25	0.8	83
Wood yard waste	60.1	23.92	2.98	1.98	3.08	2.17	1.98	2.46	1.01	0.32	84

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2.3 Third generation biomass

Currently, microalgae and seaweeds are gaining popularity as third generation biofuel (TGB) feedstocks as the wider expansion of bio-refineries leads to a shortage of energy crops that are intended for biofuel industries.¹⁷ The potential of TGB feedstock depends on the structure of various polysaccharides walled by cellulose-based cell walls, which can be further processed and fermented into biofuel.^6,17 TGB does not require as much land as the conventional agriculture, has a high productivity area, recycles carbon dioxide, is able to grow from different type of water source (e.g., seawater, brackish water and wastewater), and is compatible with the bio-refineries’ integrated bioethanol production system.⁸⁶ Besides that, TGB does not raise doubts about impact on food supply and land reservation security.⁸⁷

2.3.1 Microalgae. Microalgae are an abundant and carbon neutral renewable resource which can be considered as a TGB that can be used in bioethanol production.⁸⁸ There are various types of microalgae such as C. vulgaris, Chloroccum sp., N. gaditana, etc. that are potential feedstocks for biomass conversion into bioethanol.^87,89 Microalgae have a fast grow rate with large yields of biomass and are able to sustain hostile environments including insufficient nutrients and high salinity.^90,91 The cell walls of microalgae are cellulose, mannans, xylans, and sulfated glycans.⁹² Table 8 summarizes the composition profile of algae biomass sources.^86,91,93,94

Table 8 Composition profile of microalgae

Microalgae	Composition (% wt)									Ref.
	Carbohydrates	Xylose	Mannose	Glucose	Galactose	Lipids	Proteins	Starch	Others
G. verrucosa	42.67	—	—	24	—	—	—	—	8.83	86
C. vulgaris	44	—	—	20.52	—	35	32	12–17	—	94
C. infusionum	32.52	9.54	4.87	15.22	2.89	—	—	11.32	56.16	93
T. suecica	27.41	—	—	—	—	14.25	58.32	—	—	94
Chloroccum sp.	32.52	9.54	4.87	15.22	2.89	—	—	11.32	56.16	93
C. sorokiniana	18.2	13.8	—	70.8	—	—	42.8	—	0.78	91
N. gaditana	11.2	28.8	—	59.0	—	—	39.5	—	0.17	91
S. almeriensis	14.5	33.4	—	52.2	—	—	36.7	—	0.71	91

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2.3.2 Seaweeds. Seaweeds are considered third generation biomass which are a carbon neutral renewable resource compared to first and second generation biomass.⁹⁵ Seaweeds yield a high rate of biomass and show a fast growth with good productivity in comparison to the land crops.⁹⁶ The high yield of seaweeds is supported by the lesser energy requirements coupled with their ability to absorb nutrients throughout their surface areas. This makes it possible for seaweeds to save energy since there is no requirement to transport nutrients internally.⁹⁷ The constituents of seaweeds depend on species, habitat, maturity and environmental conditions, but generally they contain fatty acids, oxylipins, non-starch polysaccharides, dietary fiber, minerals and vitamins, terpenoids, carotenoids, phlorotannins, steroids and protein.^98,99 Seaweeds can be harvested naturally or by additional technological efforts, which vary the production cost and yield.¹⁰⁰ Seaweeds are able to use the unlimited supply of water and nutrients from the ocean and as a result yield about ±90% of the ±1.6 million tons of total dry seaweed. The growing process of seaweeds also dissolves carbon dioxide on the surface of water, and the required nutrients can also be absorbed from processing effluents.¹⁰¹ The composition profile of seaweeds is presented in Table 9.^{96,98,99,101–104}

Table 9 Composition profile of seaweeds

Seaweeds	Composition (% wt)						Ref.
	Carbohydrates	Fiber	Lipids	Protein	Moisture	Ash
Laminaria spp.	—	—	2	12	—	26	101
C. lentillifera	59.27	3.17	0.86	12.49	25.31	24.21	99
S. horneri	19.93	—	0.82	22.94	86.94	32	102
C. veravelensis	37.23	—	2.8	7.77	87.88	33.7	96
Sargassum spp.	41.81	9.84	0.75	10.25	11.16	26.19	103
T. triquetra	45.68	—	4.83	10.12	15.83	40.34	96
S. naozhouense	47.73	4.83	1.06	11.2	—	35.18	98
H. clathratus	82.26	2.7	2.18	6.39	59.63	6.47	104
U. reticulata	55.77	4.84	0.75	21.06	22.51	17.58	99

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3. Conversion process of biomass to bioethanol

3.1 Biomass feedstock handling

The handling of biomass feedstock is the preliminary step to prepare the feedstock, involving cleaning and preliminary cutting to reduce the physical size before the pretreatment process.¹⁰⁵ During feedstock handling, the storage of biomass feedstock is essential to maintain the quality of the supply for bioethanol production. Large storage places are used for biomass feedstock to avoid affecting the biomass through excessive humidity, rodents and microbial growth. Moreover, drying under the sun and thermal or mechanical drying techniques are the common methods before storage.¹⁰⁶ Fig. 4 shows the conversion process of biomass to bioethanol.¹⁰⁷

Fig. 4 Conversion process of biomass to bioethanol.¹⁰⁷

3.2 Pretreatment of biomass feedstock

In bioethanol production, biomass has complex structures that are required to be broken down into oligomeric subunits by pretreatment.⁶⁸ The purposes of pretreatment are: to remove hemicellulose, to remove or redistribute lignin (for lignocellulosic biomass); to increase the mean pore size and to provide better access for enzymes (during enzymatic hydrolysis).¹⁰⁸ The pretreatment methods can be largely classified into four methods as below.

3.2.1 Physical pretreatment. Physical pretreatment reduces the particle size of the feedstock to increase surface/volume ratio, which eases the subsequent processes in the production.^109,110 Physical pretreatment is divided into three techniques as follow:
• Mechanical process. Mechanical pretreatment reduces the feedstock’s size by breaking the physical structure. The typical process starts by washing then size reduction by grinding followed by extracting the essence and solid fraction separation.^111,112 In lignocellulosic material, size reduction is important to increase lignocellulose exposure in the hydrolysis process.⁶⁸ However, it has been reported that for particle size below ±40, mesh is not effective for the conversion to bioethanol.¹¹² In addition, the biomass is generally ground to about 3–8 mm particles in a more compact form with a higher density such as pellets or briquettes.¹¹³ Therefore, particle size is an important property to be optimized since it affects the power required for the equipment (e.g. knife mill, hammer mill) as well as the economic feasibility of the selected method.¹¹⁴ Zheng et al.¹¹⁵ stated that mechanical process such as attrition milling, ball milling, compression milling and steam treatment can be used to destruct lignin and give better access for enzymes to attack cellulose and hemicellulose in enzymatic hydrolysis.
• Extrusion process. Extrusion process is a combination of physical and chemical processes to prepare the biomass in bioethanol production.⁶⁸ This process practices heating, mixing, shearing and screw speed to shatter the lignocellulose structure and finally increase the accessibility of carbohydrates to enzymatic attack.¹¹⁶ The extrusion process can be improved by removing hemicelluloses and lignin by micro/nano fibrillation. This process produces five times higher yield compared to hot compressed water treatment, resulting in 77% delignification, and conversion of 69% and 38% of cellulose and hemicelluloses respectively into glucose, xylose and arabinose.^115,117 Zheng et al.¹¹⁵ stated that a twin-screw extruder can remove 80% of xylose with soluble lignin. The process was conducted to corncobs with 100 rpm screw speed, 100 °C barrel temperature, a mass flow rate of 4 kg h⁻¹, and produced 88% of glucose. Furthermore, the extrusion method was studied by employing sequential extrusion and clean fraction pretreatment, which resulted in a 90% glucose yield due to solvent involvement and is recognized as a promising method for lignocellulosic biomass pretreatment.¹¹⁸ Similarly, Karunanithy and Muthukumarappan¹¹⁹ reported that mixing, shearing and heating disturb the feedstock’s composition physically and chemically during the extrusion process while it passed through to the extruder barrel. The effect extrusion process for various feedstocks is presented in Table 10.^119–124

Table 10 The effect of extrusion process for various feedstocks

Extrusion process	Lignocellulose	Extruder	Extrusion conditions	Sugar yield	Ref.
Physical pretreatment	Corn stover	Single screw extruder	Screw speed: 75 rpm, temperature: 125 °C	Glucose: 75%, xylose: 49%, combined sugar: 61%	119
	Big bluestern	Single screw extruder	Screw speed: 100 rpm, temperature: 150 °C, moisture content: 20% wb, particle size: 8 mm	Glucose: 55.2%, xylose: 92.8%, combined sugar: 65.4%	122
Acid pretreatment	Rice straw	Twin screw extruder	Screw speed: 40 rpm, temperature: 120 °C H2SO4 concentration: 3% wt	Xylose: 83.7%	124
	Pine sawdust	Twin screw extruder	Screw speed: 110 rpm, temperature: 60 °C, head pressure: 780 psi, H2SO4 concentration 70% wt	Glucose: 44.4%	123
Alkali pretreatment	Corn stover	Twin screw extruder	Screw speed: 80 rpm, temperature: 140 °C, NaOH ratio: 0.04 g g^−1 biomass	Glucose: 86.6%, xylose: 50.5%	121
	Switchgrass	Single screw extruder	Screw speed: 155 rpm, temperature: 176 °C, particle size: 8 mm, NaOH concentration: 0.02 g g^−1 biomass	Glucose: 40.5%, xylose: 60%, combined sugar: 47.9%	120

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• Ultrasonic process. In ultrasonic pretreatment, ultrasonic wave breaks feedstock cells by mechanical shearing and formation of shock waves.¹²⁵ In lignocellulosic sludge type biomass, the ultrasonic process brings benefits including an improvement in biodegradability, efficient sludge disruption, lower processing time, and no additional chemical substances required.¹²⁶ Additionally, the ultrasonication process improves the feedstock digestibility by affecting the chemical, biological and physical properties of the feedstock.¹²⁷ The destruction of cells sets hydrolytic enzymes free and helps to increase the hydrolysis rate of biomass, and the irradiation of cellulose by ultrasonication increases the enzymatic hydrolysis rate to around 200%.^128,129 However, this process is well-known as an expensive method and requires a high energy input. In a study, Kidak et al.¹³⁰ reported the material disruption by ultrasonication gives more desirable results with higher ultrasonic power. They also found that the method requires less retention time since a longer ultrasonication period gives no additional improvement.

3.2.2 Chemical pretreatment. Chemical pretreatment is identified as structure disruption of the biomass through chemical reactions. Like mechanical pretreatment, chemical pretreatment removes lignin, reduces the crystallinity of cellulose and enhances the biodegradability of the biomass.¹³¹ Chemical pretreatment is widely used with other techniques. The common methods of chemical pretreatment are as follows:
• Acid process. Acid pretreatment utilizes acidic substances (typically H₂SO₄ or HCl) for lignocellulosic biomass due to its powerful ability to rupture the components into simpler form.^110,114 The low temperature for this process makes it a low cost pretreatment and it can loosen the cell wall matrix through hemicellulose degradation.⁶⁸ The process does not affect lignin, but cellulose microfibrils are sufficient to produce high yields of monomeric sugars for fermentation.^26,132 However, the utilization of acid pretreatment increases the corrosion of the equipment, hence it is important to overcome this issue especially in a large scale production with high acid reagents.^110,112 Therefore, many acid processes have been modified to improve all downstream processes as well as the bioethanol yield and prices.²⁶ Tang et al.¹³⁴ performed acid pretreatment on Eulaliopsis binata using dilute sulfuric acid, resulting in 21.02% total sugars with low inhibitor levels after pretreatment by 0.5% dilute sulfuric acid at 160 °C for 30 min and at a solid-to-liquor ratio of 1 : 5. Marzialetti et al.¹³⁵ performed acid hydrolysis on loblolly pine using various acids, including trifluoroacetic acid, HCl, H₂SO₄, HNO₃, and H₃PO₄. The research found that trifluoroacetic acid yielded the most sugar monomers of 70% from hemicellulose at 150 °C at pH 1.65, and this acid is claimed to be the mildest acid used in the study.
• Alkali process. Alkali process is the pretreatment process of feedstock catalyzed by alkali chemicals at room temperature for up to 24 h.^112,132 This method is applied in a simple operation and gives high conversion yields with only short span of time.¹¹⁰ It produces less sugar degradation, but the appearing inhibitors are expected to be eliminated in order to optimize the pretreatment conditions.¹³⁶ Therefore, this pretreatment method is claimed to be more effective for bioethanol production than acid pretreatment.¹³⁷ The suitable alkali reagents are NaOH and lime, which are employed to break intercellular bonds crosslinking hemicellulose and other components (lignin and cellulose).¹³³ Moreover, the alkali pretreatment can cause structure swelling, increasing porosity, decreasing polymerization degree and crystallinity, separation of lignin, hemicellulose, and cellulose and also disrupts the lignin structure.¹¹⁴ Many studies on alkali process have focused on pretreatment to improve substrate digestibility. Sun and Cheng¹³³ reported that alkali pretreatment is capable of lignocellulosic delignification and at the same time enhancing the reactivity of carbohydrates. It is also found that by utilizing sodium hydroxide in alkali pretreatment, it improves lignocellulose digestibility in typical lignocellulosic feedstocks, for instance wheat straw, thus it is appropriate for second generation biomass pretreatment.^138,139 Chang et al.¹⁴⁰ reported oxidative lime pretreatment with a feedstock of poplar wood. The study reported that 78% of lignin was removed with glucose improvement of 71% through enzymatic hydrolysis. Sun et al.¹⁴¹ conducted alkali pretreatment by different alkali solutions to release hemicellulose for wheat straw. It showed that 80% hemicellulose was released as well as 60% of lignin at 20 °C by 1.5% sodium hydroxide for 14 hours of pretreatment. Additionally, hemicellulose can produce high amounts of xylose compared to glucose and galactose at the subsequent hydrolysis process. Park et al.¹⁴² conducted bioethanol research by utilizing alkali pretreatment, resulting in 74% ethanol after 19 hours fermentation by a combination of Saccharomyces cerevisiae and Pichia stipitis as the fermentation process. Alkali reagents such as KOH, NaOH, hydrazine, anhydrous ammonia and Ca(OH)₂, are typically featured in alkali pretreatment of biomass. Moreover, alkali reagents can cause biomass swelling that exposes the internal surface area as well as a decrease in crystallinity and polymerization degree.^138,143
• Organosolv process. The organosolv process utilizes organic solvents to break the internal lignin–hemicellulose bonds at high temperature (100–250 °C) and provides more access to cellulose.^68,144 The typical suitable organic solvents for this pretreatment method are namely tetrahydrofurfuryl alcohol, ethylene glycol, methanol, acetone and ethanol, and they are noted for their ability to solubilize lignin as well as treating cellulose so that it is digestible for enzymatic hydrolysis.^68,145 The effect of organosolv pretreatment on enzymatic hydrolysis was studied by Mesa et al.¹⁴⁶ The pretreatment of sugarcane bagasse to produce glucose under a temperature of 175 °C for 40–60 min yielded 22.1 g glucose/100 g biomass. Moreover, pretreatment by the organosolv technique can also be combined with acid (by adding HCl, H₂SO₄, oxalic or salicylic acid) if the process is conducted between 185–210 °C to break the hemicellulose bonds.¹⁴⁷ However, it is suggested that the mentioned combined technique should separate lignin and hemicellulose components so that xylose yield can be focused at the most desirable yield amount.¹³² In addition, removal of solvents in the organosolv process is generally required due to production of inhibitors which limits the activity of enzymatic hydrolysis and fermentative microorganisms.¹³³ The advantage of organosolv pretreatment is the ability to produce highly digestible cellulose subtracts for all biomass with high value utilization and to remove lignin which troubles the cellulolytic enzymes for lower enzymes dosage.^68,112 However, this technique produces high amount of soluble phenols, furfural, 5-hydroxymethyl-2-furfural (HMF), which are obtained from lignin after pretreatment.^68,110 The high cost of solvents is the disadvantage of organosolv pretreatment method, but ethanol and methanol are most commonly used solvents in this method due to their low cost.¹³³
• Ozonolysis process. Ozonolysis pretreatment involves ozone as a strong oxidant with high delignification efficiency.¹⁴⁸ This process removes lignin, slightly attacks hemicellulose and hardly effect on the cellulose.¹¹⁰ Ozonolysis is pretreatment method for feedstock which is catalyzed at atmospheric conditions (normal pressure and room temperature) and it does not produce inhibitors that impact the hydrolysis and fermentation in bioethanol production.¹³⁶ Ozonolysis is environmentally sustainable as ozone is a substance that can be decomposed by high temperatures or a catalytic bed, hence less pollution is ejected into the environment.¹⁴⁷ Lignocellulosic materials, for instance rye straw, wheat straw and cotton straw, are seen suitable to be handled with this pretreatment method.¹³² Nevertheless, further research on ozonolysis for bioethanol production is essential in order to further develop the beneficial points and practicality as it is well known that the method is a costly process due to the high amount of required ozone for this pretreatment.^68,149

García-Cubero et al.¹⁵⁰ performed ozonolysis pretreatment to improve sugar yield from wheat and rye straws. It was found that enzymatic hydrolysis of the ozone pretreated biomass resulted in an improvement of 57 and 88.6% of the hydrolysis yield compared to the non-ozone pretreated hydrolysis yields of 16 and 29% from rye and wheat straw respectively. The study concluded that biomass type and moisture content are the substantial factors in ozonolysis. Ozonolysis has also been used in industrial wastewater treatment, and as the pretreatment method for wider various applications.¹⁵¹ As a result of its high delignification efficiency, ozonolysis is also utilized in pulp bleaching in the paper industry.^152,153

• Ionic liquids (ILs) process. Ionic liquid pretreatment (ILs) utilizes the organic cations and inorganic anions in salts to pretreat the biomass at room temperature.^110,145 In lignocellulosic biomass, ILs dissolve lignin or cellulose, and decreases cellulose crystallinity for a better hydrolysis process as the subsequent process after pretreatment.¹⁴⁵ Besides, the IL process has good traits including non flammability, low vapor pressure, and balanced chemical and thermal stability. It also tends to sustain its liquid form in a relatively wide temperature range.⁶⁸ This means that this pretreatment method supports the improvement of cellulose accessibility at room temperature with or without catalysts (acid or alkali), and it avoids the formation of inhibitors.^110,116 Remsing et al.,¹⁵⁴ found that in the IL method carbohydrate in the biomass is more attracted to the anion than the cation. This is due to the preference of chloride ions to interact the with hydroxyl protons of the carbohydrate sugar, and it occurs in a stoichiometric ratio of 1 : 1.

Francisco et al.¹⁵⁵ reported that pretreatment of lignocellulosic biomass has gained significant attention and it can dissolve carbohydrates and lignin simultaneously. The cation structure and degree of anion charge delocalization are two major factors that can affect the properties of ILs such as refractive index, density, viscosity and solubility respectively. Thus, the IL method holds a strong thermal stability as well as negligible vapor pressure and these properties are subject to change depending on the user’s desire and requirement. IL pretreatment may produce impurities, namely water, halides and other volatile substances formed from the biomass or the solvent, although they can be removed by evaporation process.¹⁵⁶ ILs are namely green solvents as they does not contain toxic or explosive substances, and can be recycled to reduce the amount used, making them more environmentally friendly.^68,157 However, other studies have reported drawbacks of this method, including the compounds harmfulness and issues in recovering ILs.^110,145 Furthermore, this technology is expensive and requires further investigation of the feasibility of ILs on large-scale applications, since it holds potential in bioethanol production industry.^68,158

3.2.3 Biological pretreatment. Microorganisms are employed to degrade the structure of biomass in biological pretreatment.^41,112 The typical microorganisms employed for this technique are white, brown and soft-rot fungi.¹¹² Several of white-rot fungi, namely Ceriporia lacerata, Phanerochaete chrysosporium, Pleurotus ostreatus, Cyathus stercoreus, etc., have been evaluated and resulted in a high delignification efficiency of various lignocellulosic biomasses.^68,159 Brown rot fungi targets the hemicellulose and cellulose components of the biomass, white fungi degrades cellulose and lignin components and soft rot fungi are useful to release cellulose from the degradation of lignocellulose complex.^8,160 Biological pretreatment is very well-known for its environmental friendliness, although it tends to work on a slow rate compared to other pretreatment methods.^161,162 The advantages this method are low process costs and low energy requirements, which are due to decreased mechanical assistance, no requirements of additional chemical substances or applied pressure, it does not cause corrosion to the equipment, and there is a low production rate of inhibitors.^6,68 However, the disadvantages of biological pretreatment is the extremely slow degradation rate to attain a high degree of lignin degradation (as long as few months). This causes inefficiency in the subsequent processes of hydrolysis and fermentation required to complete the entire bioethanol production.¹⁵⁹ Therefore, there is significant focus on the development of biological pretreatment due to this method having been described as one of the most economic techniques to pretreat biomass for bioethanol industry.^105,136

There are many reviews reporting biological pretreatment and its future perspectives.^163–166 There are few studies that have reported that Aspergillus terreus, Trichoderma spp., Cyathus stercoreus and Lentinus squarrosulus are microbes that can degrade lignocellulosic material (lignin and holocellulose) at 65–80% and 45–75% respectively, with the pretreatment condition of 25–35 °C for 3–22 days.^165,166 In a similar study, Zheng et al.¹¹⁵ reported the effectiveness of white-rot fungi in decomposing lignocellulosic biomass, in addition to enhancing the subsequent enzymatic hydrolysis. The study determined that Irpex lacteus (the employed white-rot fungi type) succeeded in degrading lignin by 43.8%, and the subsequent enzymatic hydrolysis was found to be higher by 7-fold.

3.2.4 Thermochemical pretreatment. The application of direct combustion is utilized in thermochemical pretreatment as one simple biomass pretreatment, and this pretreatment generally relies on the biomass properties, including volatile content, moisture content, fixed carbon content, impurities (S, N, Cl) concentration, and ash content, all of which influence the technical and economic factors of bioethanol production.^167,168 The pairing of a low cost feedstock with thermochemical pretreatment for bioethanol production can bring out future securities in terms of energy, economic and infrastructure.¹⁶⁹ Fig. 5 shows thermochemical processes and they can be used to convert lignocellulosic biomass to liquid, solid and gaseous fuels .¹⁷⁰ In addition, several thermochemical pretreatment methods that are commonly applied in bioethanol production are discussed below.

Fig. 5 Lignocellulosic biomass by thermochemical technology.¹⁷⁰

• Steam explosion (STEX). Steam explosion (STEX) is widely used in bioethanol production for lignocellulosic biomass thermochemical pretreatment.^68,133 This pretreatment used high temperature (160–260 °C) and pressure (0.69–4.83 MPa) and also a sudden depressurization.⁶⁸ The mechanism used in STEX can be aided by the natural catalyst, acetic acid, which is released by hemicellulose acetate and organic acids such as levulinic and formic acid. The STEX method can used for an auto hydrolysis process or a natural process.¹⁷¹ Some factors can influence STEX efficiency such as moisture content, residence time, temperature, catalyst concentration, and time of presoaking. Optimising these can improve the sugar release, a study reported that the STEX method is able to yield xylose-sugars in a range of 45–65%.^71,172 However, STEX can also generate some toxic compounds that could affect hydrolysis and fermentation steps, such as acetic acid, furfural and hydroxymethylfurfural.¹⁴³ Therefore, inhibitor removal is required for every STEX pretreatment as to the inhibitors can disturb the sustainability of enzymes (in hydrolysis) and yeast (in fermentation), which leads to production additional cost.¹⁷¹
• Liquid hot water (LHW). The working principle of the LHW process is a thermochemical pretreatment of lignocellulosic biomass through subcritical pressure water with the assistance of CO₂ to enhance the hydrolysis process.¹⁷³ The LHW pretreatment is conducted with hot compressed liquid water at temperatures of 160–240 °C and pressure above 5 MPa for 15 min.¹⁷⁴ LHW process is able to yield high xylose recovery of 88–98%, with no additional substances as catalyst.^175,176 The advantages of LHW process are as a simple pretreatment process with low capital and maintenance costs (reactor materials) due to a lack of corrosion in addition to releasing high sugar yields.^136,176 Additionally, lignin and hemicellulose are soluble in LHW pretreatment since it uses high amounts of water.¹¹⁶ Some studies have reported the suitability of LHW pretreatment in bioethanol production from various feedstocks, such as wheat straw, corn stover and sugarcane bagasse. However, the LHW process requires high water and energy consumption which makes this process not viable at the commercial scale.^164,175 Nevertheless, LHW pretreatment method draws attention of researchers, especially in lignocellulosic biomass pretreatment. Pérez et al.¹⁷⁷ performed LHW pretreatment of wheat straw, yielding sugar recovery of 43.6%, and the subsequent enzymatic hydrolysis yielded 79.8% of sugar recovery. Jiang et al.¹⁷⁸ employed LHW pretreatment on branch, stem and boll of cotton stalk. It was found that 4.34 and 4.42% of hemicellulose was removed from the branch and stem respectively, while 58.09% hemicellulose was removed from the boll shell. This study also reported bioethanol yields of 18.3, 16.27 and 21.08 g per 100 g of stem, branch and boll shell respectively. Imman et al.¹⁷⁹ added NaOH to the LHW pretreatment of rice straw, and claimed that sugar yield was higher with the NaOH presence (0.25–1.0%). Furthermore, hemicellulose and lignin were efficiently removed from the solid residue and a high pentose yield was obtained in the liquid phase with low formation of unwanted by-products.
• Ammonia fiber explosion (AFEX). AFEX utilizes ammonia to decrease the crystallinity of cellulose in a solid lignocellulosic biomass, as it readily disrupts carbohydrate–lignin bonds.⁸⁰ AFEX pretreatment can increases lignocellulosic digestibility and enhance the yield from enzymatic hydrolysis as the subsequent process.¹¹⁶ Moreover, AFEX can achieve a conversion of 90% from cellulose hemicellulose into fermentable sugars and this pretreatment can be employed on numerous varieties of lignocellulosic materials.¹¹⁰ In fact, AFEX pretreatment can be also employed in a bioethanol production method of saccharification and cofermentation, using recombinant S. cerevisiae strains as the fermentation agent to obtain high ethanol yields.⁶⁸ The AFEX method does not introduce inhibitors for subsequent processes in the production line, and cell walls extractives, for instance lignin phenolic fragments, remain on the surface of cellulose.¹³² The disadvantage of AFEX method is to recover the ammonia due to its high volatility which boosts up the overall production cost.^68,143 AFEX pretreatment method is also useful for woody biomass and herbaceous crops.^116,147 Numerous studies have been conducted to determine the optimal AFEX pretreatment for different biomass sources. Alizadeh et al.¹⁸⁰ pretreated switchgrass with AFEX at 100 °C and an ammonia to biomass ratio of 1 : 1, yielding 0.2 g ethanol per g dry biomass. Bals et al.¹⁸¹ observed that enzyme formulation by AFEX pretreatment produced high sugar yields. Using switchgrass as the biomass, 520 g sugar per kg biomass was released after enzymatic hydrolysis with AFEX pretreatment, while 410 g sugar per kg biomass is normally released.
• Wet oxidation (WO). Wet oxidation pretreatment uses a combination of water, oxygen pressure, elevated temperature and alkali hydrolysis, and it is proven as an efficient pretreatment for lignocellulosic biomass to produce high yield bioethanol.^68,137 Oxidation pretreatment operates at relatively low temperatures (170–200 °C), short reactor times (5–15 min) and pressures from 10–12 bar O₂.¹³² In fact, a higher oxygen content at temperatures above 170 °C reduces the energy demand as a result of exothermic process.⁶⁸ In wet oxidation process, lignin and hemicellulose, as well as cellulose, are solubilized and have their digestibility enhanced, respectively, due to the oxidative reaction and the formation of hydrolytic acid.¹⁴⁹ Wet oxidation produces lower inhibitors (furfural and HMF) than LHW or steam explosion methods.¹⁸² In fact, the addition of alkali substances (Na₂CO₃) in this method maintains the process’ pH, reduces toxic compounds, and neutralizes carboxylic acid.^110,137 However, this pretreatment method requires high oxygen and catalyst supply, thus driving up the production cost.¹⁸³ Arvaniti et al.¹⁸⁴ reported that the wet oxidation method can pretreat rape straw to produce bioethanol with a yield of 67% through simultaneous saccharification and fermentation (SSF).
• Microwave process (MWP). Microwave process (MWP) combines thermal and non-thermal effects in a watery environment. The microwave gives vibrations to the biomass molecules, which leads to intermolecular collisions that result in heat provision and acceleration of the process.¹⁶ In fact, microwaves give a higher heat effect compared to conduction or conventional pretreatment since there is a direct interaction between the object and the electromagnetic wave in a form of supercritical heat transfer.¹⁸⁵ MWP is also can be performed paired with alkali catalysts, namely NaOH, Ca(OH)₂, Na₂CO₃, and has been utilized to pretreat lignocellulosic materials including coastal berm, switchgrass, and rice straw and hulls.^186,187 The major positive capability of this method is able to start and stop the process instantly, in addition to requiring less energy to run.¹⁸⁸ However, the feasibility of the microwave process at the commercial scale is doubtful, and hence further study is strongly suggested to make the method more feasible in the future.¹⁸⁹ Chittibabu et al.¹⁹⁰ studied the effect of microwave pretreatment on banana pseudostem assisted with an alkali (NaOH), yielding sugar in as much as 84% after enzymatic hydrolysis with cellulase. In a similar study, Nikolić et al.¹⁹¹ employed MWP on cornmeal, and after fermentation by Saccharomyces cerevisiae var. ellipsoideus 13.4% ethanol was yielded.
• CO₂ explosion pretreatment. CO₂ explosion pretreatment is employed and conditioned as a phase of supercritical fluid, which a compressed gas above the critical point temperature and forming liquid-like substance.^68,192 CO₂ is injected to the biomass reactor in a very high pressure and is heated at high temperature up to 200 °C and is maintained for a desired period of time.^193,194 At this high pressure, the injected CO₂ then disrupts the biomass and dissolves in water, where carbonic acid is formed from this reaction and the acid supports the enhancement of the biomass hydrolysis. However, the CO₂ explosion method is not compatible with dry biomass due to the lack of moisture content.^193,195 CO₂ explosion pretreatment also can be paired with other pretreatment methods, for instance, Cha et al.¹⁹⁶ combined CO₂ explosion with ammonia explosion to pretreat rice straw, resulting in a yield of 93.6% glucose. Gu¹⁹⁷ reported CO₂ explosion process as a nontoxic green solvent. In this method, the CO₂ used to pretreat the biomass can be taken from the CO₂ produced from fermentation process and it can emit the CO₂ emission to the atmosphere. The advantages of employing CO₂ are the cheaper cost compare to ammonia in the explosion, lower production of inhibitor compounds compared to steam explosion, and a higher yield than enzymatic hydrolysis without pretreatment.¹¹⁰
• Sulfite pretreatment to overcome the recalcitrance of lignocellulose (SPORL). Sulfite pretreatment to overcome the recalcitrance of lignocellulose (SPORL) is a pretreatment method that can be used to react with the strong rigidity of lignocellulosic such as woody biomass or softwood. This can overcome the recalcitrance of lignocellulose which negatively affects the sugar and ethanol recovery in bioethanol production.^26,110 In this pretreatment, sulfite or bisulfite solution is reacted with the woody biomass at temperature, pH and pretreatment period of 160–190 °C, 2–5, and 10–30 min respectively.²⁶ The SPORL method enhances the sugar yield (57–88%) compared to acid pretreatment and reduces the inhibitor compounds (up to 65%) produced.¹⁸² Therefore, SPORL is seen as a promising pretreatment method for woody biomass as it affects the downstream processes, as well as enhancing the efficiency of the bioethanol production yield and its production cost.^110,147 Zhang et al.¹⁹⁸ reported that SPORL pretreatment improves the biomass digestibility, reducing lignin hydrophobicity by lignin sulfonation, and removes hemicellulose from the biomass. The pretreatment was applied within the range of temperature (163–197 °C), time (3–37 min), sulfuric acid dosage (0.8–4.2% on switchgrass) and sodium sulfite dosage (0.6–7.4% on switchgrass). The results showed that SPORL pretreatment improved the digestibility of switchgrass through sufficiently removing hemicellulose, partially dissolving lignin, and reducing hydrophobicity of lignin by sulfonation.

3.3 Hydrolysis

The hydrolysis process breaks down the complex sugar structures that are built up in the biomass, for instance, polysaccharides in starch and lignocellulose, into the simplest sugar monomers, such as xylose, glucose, etc. In general, hydrolysis is designed to provide suitable fermentable sugars for fermentation microbes in the fermentation process. This effort is necessary since particular microbes are only able to convert particular sugar monomers into ethanol.^66,112 Commonly, hydrolysis can be carried out in the following methods:

3.3.1 Dilute acid hydrolysis. Dilute acid is typically utilized to hydrolyze lignocellulosic biomass, since acid is capable of degrading the lignocellulosic structure into fermentable sugars. Dilute acid hydrolysis utilizes around 1% of the acid concentration to degrade the biomass and it yields around 50% of glucose as the end product.^199,200 Dilute acid hydrolysis of hemicellulose and cellulose has different approaches in order to identify composition of lignocellulosic. Balat et al.¹⁶ reported that hemicellulose can degraded by dilute acid and releases xylose monomers (5-carbon sugar) under a mild hydrolysis environment. Meanwhile, cellulose components require higher temperature for the dilute acid to strike the complex structure of cellulose, and this second approach yields glucose monomers (6-carbon sugar).¹⁶ Dilute acid is a practical hydrolysis method for a continuous-production type of bioethanol plant, but prior size reduction of the feedstock is required. Dilute acid is performed in an affordable hydrolysis period and produces a fair end-product. However, it is still a challenge for this method to produce a desirably high sugar yield.¹⁶⁷ Besides, inhibitory products are formed as by-products of the process, including acetate, furfural, hydroxybenzaldehyde (HBA), 5-hydroxymethyl furfural, etc., which are harmful for fermenting microorganisms.²⁰¹

3.3.2 Concentrated acid hydrolysis. Concentrated acid is used to hydrolyze typically lignocellulosic biomass in a fair temperature condition and it employs strong acid (concentration more than 10%). Concentrated acid hydrolysis brings out better sugar yield compared to dilute acid hydrolysis, and it is aimed for a better cost efficiency of hydrolysis process. Despite the high sugar recovery and the ability of the acid to be recovered and re-concentrated, concentrated acid hydrolysis requires extreme precautions, especially during handling the acid substance since concentrated acid is extremely hazardous to human health and the environment.^16,202 The drawback of the concentrated acid hydrolysis method is the tendency to corrode the production equipment, which potentially brings a large economic loss to the overall bioethanol production. This method is claimed to have a shorter processing time,²⁰³ although others have argued that it is done under a longer hydrolysis period compared to dilute acid hydrolysis.¹⁹⁹

3.3.3 Enzymatic hydrolysis. Enzymatic hydrolysis is a common method in bioethanol production which uses a specific type of enzyme based on the type of the biomass component. For instance, cellulase is used to hydrolyze cellulose and amylase to hydrolyze amylose. This hydrolysis method is a complex degradation process, especially for lignocellulosic material, aimed to release the simplest and fermentable sugar monomers. In lignocellulosic biomass, enzymatic hydrolysis works in three ways: (i) the biomass is deformed physically and chemically, (ii) degradation of biomass’s surface, releasing less-complex sugar formations into the solution, (iii) and the last is hydrolysis of the released less-complex sugars in the solution into sugar monomers.²⁰⁴

The advantage of enzymatic hydrolysis is that it can take place at only a mild hydrolysis environment (pH of around 4.8 and temperature of around 318–323 K). Enzymatic hydrolysis produces high yields and a better end-product compared to acid hydrolysis which does not promote any corrosion tendency to the processing equipment.²⁰⁵ The costly manufacturing of enzymes indeed drives the enzymatic hydrolysis cost and ultimately the overall bioethanol production cost. However, the growing advance of technologies and rapid development of enzyme manufacturing can reduce the enzyme price. This will affect the bioethanol industry as well for enzymes to become more viable and expandable.²⁰⁶ The production of enzymes, for instance cellulase, can be extracted from fungi and bacteria. The recorded fungi as cellulase and hemicellulose-source microbes include Trichoderma reesei, Trichoderma longibrachiatum, and Trichoderma viride.²⁰⁷ Meanwhile, the typical cellulase-producing bacteria include Acetivibrio, Bacteroides, Bacillus, Cellulomonas, Clostridium, Erwinia, Microbispora, Ruminococcus, Streptomyces and Thermomonospora.²⁰⁵

By employing a commercial cellulase, Tye et al.²⁰⁸ utilized kapok (Ceiba pentandra) fiber as a second generation biomass in an attempt to yield the most fermentable sugar. They observed that cellulase managed to alter the complex structure of the fiber and yielded 85.2% of reducing sugar with prior acid pretreatment. Tan and Lee⁹⁵ have performed hydrolysis on seaweed solid waste enzymatically to produce bioethanol. The authors observed that enzymatic hydrolysis produced a very high glucose content of 99.8% at very mild hydrolysis conditions of 50 °C and pH 4.8. The study also subsequently performed fermentation and yielded a desirable quality of ethanol of 55.9%. However, simultaneous saccharification and fermentation (SSF) was modified in order to obtain a high ethanol yield of 90% from seaweed waste. It is show that enzymatic hydrolysis is able to be enhanced with the correct additional process and paired with the suitable biomass. Simultaneous saccharification and fermentation (SSF) of seaweed waste can yield a higher ethanol quality of 90.0%. This shows that enzymatic hydrolysis is able to be enhanced with the correct additional process and paired with the suitable biomass.⁹⁵

3.4 Fermentation

Fermentation is a biomass conversion process into bioethanol by microorganisms (yeast and fungi or bacteria), which occurs by digesting fermentable sugars and producing ethyl alcohol and other byproducts.^26,112 Saccharomyces and Pichia are the most common yeast type employed in bioethanol production as well as bacteria Zymomonas and Escherichia and Aspergillus.⁶ The nature type of fermentation microorganisms are able to convert particular sugar monomers into ethanol, namely hexoses (C6), which are usually yielded in first and second generation production, and pentoses (C5), which are usually yielded in second generation production.²⁰⁹ Most of the studies used Saccharomyces cerevisiae yeast type. Wyman et al.²¹⁰ reported that Saccharomyces cerevisiae is able to theoretically produce 90% of ethanol from glucose sugar. Similar study by Abbi et al.²¹¹ stated that S. cerevisiae is able to ferment hexose sugar, while P. stipitis, P. tannophilus and C. shehatae yeast are typically used to ferment xylose into ethanol efficiently. Kluyveromyces marxianus yeast is able to withstand high temperatures between of 45–52 °C, and is capable of digesting a wide variety of sugar monomers, namely xylose, mannose, arabinose, and galactose.^66,212 In order to upgrade the ability of the conversion process, microbe engineering is one alternative which resulted from technological advancement. The benefits include a higher ethanol yield, enabling more substrate to be digested, and more resistance against inhibitors.

In a conventional path, fermentation is occurs last after the biomass is pretreated and completely hydrolyzed. However, as the technology advances, new methods to conduct fermentation are developed as the effort to gain better ethanol quality as well as better production efficiencies. This paper gathers the varieties in fermentation methods, and each of the methods is discussed as follow.

3.4.1 Simultaneous saccharification and fermentation (SSF). Simultaneous saccharification and fermentation (SSF) technique is a single fermentation reactor in order to minimize the production of inhibitors, combining both processes (saccharification and fermentation) to run simultaneously, and to reduce additional equipment cost.^114,213 In a single reactor, enzymatic hydrolysis of the biomass is run, and immediately the presented fermentation agent converts the released sugar monomers into ethanol. Moreover, the SSF technique can decrease the production period and enhance the efficiency of the production. Additionally, ethanol accumulation in the reactor does not inhibit the hydrolysis activity, making SSF a favorable method for bioethanol production.^15,144 However, this fermentation technique is limited by the respective optimum conditions for both hydrolysis and fermentation, which is quite challenging to control to meet both satisfactory conditions at the same time.²¹⁴ In common practice, hydrolysis is best carried out at around 50 °C, but fermentation requires warm to mild temperatures for the microbes to remain active, around 28–37 °C, hence to meet both desired conditions requires a special engineering effort. Fig. 6 shows the flowchart of simultaneous saccharification and fermentation process.²¹⁵

Fig. 6 The flowchart of the simultaneous saccharification and fermentation (SSF) process.²¹⁵

3.4.2 Separate hydrolysis and fermentation (SHF). The separate hydrolysis and fermentation (SHF) technique is a fermentation process that can be perform in different vessels, where each vessel carries out a specific task.⁸ SHF allows each both processes, hydrolysis and fermentation at their most optimum conditions and yielding the desired products to the maximum potential.¹⁵ For instance, this technique enables hydrolysis to be performed at the optimum temperature of 45–50 °C and fermentation to be performed at about 30 °C without any hassle.^8,15 SHF also allows fermentation of sugar based on its type. The hydrolysate flows into the first vessel to ferment the glucose content of the hydrolysate by glucose-fermenting microbes. The process is then continued by distilling the solution and flowing it to the second fermentation vessel to ferment the next sugar type by the specific fermentation microbes.²⁶ SHF technique also reduces the inhibition production since the removal is practical.¹¹⁵ SHF technique is considerably cost effective when it focuses on the substrates, substances employed and the high quality ethanol yield, although it may not be cost effective in the equipment installment.^8,216 Fig. 7 shows the flowchart of the separate hydrolysis and fermentation process.²¹⁵

Fig. 7 The flowchart of the separate hydrolysis and fermentation (SHF) process.²¹⁵

3.4.3 Simultaneous saccharification and co-fermentation (SSCF). This fermentation technique utilizes the integration principle in employing mixed microbes to ferment both more than one sugar type (e.g. pentoses and hexoses) resulting from the previous processes of pretreatment and hydrolysis.^8,10 The use of mixed microbes is limited to the respective ability of the microbes, where hexose-fermenting microbes usually grow faster than pentose-fermenting microbes, and this leads to a higher rate of ethanol conversion from hexose.⁶ SSCF holds several beneficial characteristics in bioethanol production, including a lower enzyme requirement, faster production rate and lesser cost projected since the process is performed in a single reactor.¹⁵ On the other hand, SSFC requires extra conscientious controlling since the employed mixed microbes may require different optimum temperatures, as well as between the hydrolysis and fermentation process since they are run in the same reactor.²¹⁷ C. shehatae, S. cerevisiae, E. coli KO11, E. coli FBR5 are the type of employed microbes to be mixed in SSCF technique to produce bioethanol from varieties of biomass, namely barley hull, wheat straw, etc.^1,43,218,219 Fig. 8 describes the flowchart of SSCF process.²¹⁵

Fig. 8 The flowchart of the simultaneous saccharification and co-fermentation (SSCF) process.²¹⁵

3.4.4 Consolidated bioprocessing (CBP). Consolidated bioprocessing is a technique in bioethanol production where enzyme production, enzymatic hydrolysis and biomass fermentation are all carried out within the same single reactor. Hence, this technique is usually called direct microbial conversion (DMC).¹³² In general, CBP employs a specific microorganism type that is capable of conducting such an array of tasks without additional flowing or removal processes to another reactor. In lignocellulosic biomass, for example, production of cellulase enzyme, hydrolysis of cellulose, and fermentation of the yielded sugars are performed by one type of microorganism. This method is a low cost conversion of cellulosic biomass into bioethanol with a high rate and acceptable yields.^17,26 Although others have reported that CBP consumes long processing time and an undesirable ethanol yield.^1,26 With CBP, external cellulose enzymes are unnecessary for the pretreatment and hydrolysis processes of bioethanol production.^26,220 The common microbes employed for this method include Thermoanaerobacter ethanolicus, Clostridium thermoshydrosulfuricum, Thermoanaerobacter mathranii, Thermoanaerobium brockii, Clostridium thermosaccharolyticum. These microbes have good abilities that are utilized in many inexpensive lignocellulosic biomass-to-ethanol conversion and are able to withstand extreme temperature.⁸ Cardona and Sánchez²²¹ reported the potential yeast strain of K. marxianus for the CBP method and it resulted in a good growth display of endoglucanase and β-glucosidase on the cell surface at temperature as high as 48 °C of which the ethanol was produced from the cellulosic material β-glucan with a yield of 0.47 g ethanol per gram of consumed carbohydrate. However, the CBP technique is not highly recommended since the typical microorganisms employed for CBP are intolerant of high ethanol yields, hence further microbial engineering is strongly recommended to upgrade the applicability of this technique.^222,223 Fig. 9 describes the flowchart of consolidated bioprocessing process.²¹⁵

Fig. 9 The flowchart of the consolidated bioprocessing (CBP) process.²¹⁵

3.5 Distillation and dehydration

Distillation in bioethanol production is required to separate the ethanol content out of the fermented mixture by utilizing the boiling point of ethanol (78.3 °C). In other words, in water–ethanol distillation, ethanol will be vaporized before water.^16,224 In a typical bioethanol refinery, the fermentation product is flowed to the distillation column to be distilled, and the product from this process is then flowed to the rectifier and concentrated to just below the azeotropic point, as seen in Fig. 10 which shows the water–ethanol azeotropic point.^109,225 Meanwhile, the remaining un-distilled fermented product is flowed to a stripping column to additionally remove the water content. Afterwards, the product of this process is combined with the ethanol produced from the previous process.²²⁶ As the result, bioethanol recovery produces quality about 99.6%.⁶⁷ The solid compounds of the product are separated using centrifuge.²²⁷ Distillation or bioethanol recovery process is an energy intensive process which requires high volumes of cooling water and produce some gallons of water used for every gallon of ethanol obtained.¹⁰⁸

Fig. 10 Water–ethanol equilibrium mixture and azeotropic point.^109,225

Dehydration is advantageous to remove the remaining water in the azeotropic phase.^107,228 The dehydration technique is quite similar to extractive distillation but this process utilizes an additional component which lowers the heterogeneous boiling azeotrope.²²⁹ Prior to the dehydration process, the ethanol is first distilled to achieve above 96%, then filtered through molecular sieves, which absorbs the water component from the mixture.¹⁰⁹ The result of this process is an upgraded ethanol product. The used molecular sieves can be recycled by heating the sieves to remove the absorbed water.¹¹⁴

Overall, the literature for bioethanol production from biomass feedstock are tabulated in Tables 11^230–237 and 12.^238–243

Table 11 Comparison of bioethanol production for first generation biomass feedstock

Biomass	Fermentation process	Results (ethanol concentration)	Ref.
Sugarcane (Saccharum officinarum)	Escherichia coli KO11 and Klebsiella oxytoca P2	39.4–42.1 g L^−1	230
Sweet sorghum (Sorghum bicolor)	Kluyveromyces marxianus DMKU 3-1042	7.43% w/v at 37 °C	234
Wheat (Triticum vulgare)	Pichia kudriavzevii	71.95 g L^−1 at 40 °C	233
Sugar beet (Beta vulgaris)	S. cerevisiae Ethanol Red and S. cerevisiae safdistill C-70	85.0–87.0 g L^−1	235
	S. cerevisiae IR-2, S. cerevisiae ATCC 26603, S. cerevisiae IFO 0309 and Z. mobilis IFO 13756	0.44 g g^−1 and the lowest productivity of 0.08 g h^−1 L^−1 by Z. mobilis	232
Sweet sorghum (Sorghum bicolor)	S. cerevisiae CFTR 01 and SG	9.0% w/v by Keller variety	231
	S. cerevisiae ATCC 7754 and Z. mobilis ATCC 29191	50.26 mL L^−1	237
Watermelon (Citrullus lanatus)	S. cerevisiae Ethanol Red	25.0% w/v at pH 3.0 and 35.0% w/v at pH 5.0	236

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Table 12 Comparison of pretreatment process for second generation biomass feedstock

Biomass	Pre-treatment	Hydrolysis	Results	Ref.
Sugarcane bagasse	Ball milling (4 h)	Acremonium cellulase at 5 FPU g^−1 substrate of cellulase and 20 U g^−1 substrate of xylanase from optimash BG at 45 °C, pH 5.0 for 72 h	Glucose of 89.2 ± 0.7%	238
			Xylose of 77.2 ± 0.7%
	H2SO4 of 1% vol at 60 °C for 24 h	In an autoclave at 121 °C for 40 min after removing the excess H2SO4 of 1% vol	Total sugar of 68.0 g L^−1	239
Wheat straw	Knife milling with 0.7–1.0 mm and washed with water and dried	H2SO4 of 1.85% vol at 90 °C with for 18 h which 20 : 1 of liquid to solid ration. Suspension centrifuged and the residue is washed with hot water	Glucose of 1.70 ± 0.30 g L^−1	240
			Xylose of 12.80 ± 0.25 g L^−1
Rice straw	Chopped to 5–6 mm	H2SO4 of 4.4% vol at 1 : 10 solid to liquid ration in boiling water bath for 1 h. The process was filtered and pH was adjusted to 5.5	Total sugar of 20 g L^−1	241
	Chopped and steam exploded at 3.5 MPa, 275 °C for 2 min	Saccharification used cytolase, novozyme at 50 °C for 120 h	Xylose of 5–10 g L^−1	242
Maize straw	NaOH 2% wt at 80 °C for 1 h	Hydrolysis by cellulase of trichoderma reesei ZU-02 and cellobiose of Aspergillus niger ZU-07	Xylose of 23.6 g L^−1	243
			Glucose of 56.7 g L^−1
			Arabinose of 5.7 g L^−1

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4. Fuel properties bioethanol

The quality of combustion engine fuel can be identified through the chemical and physical properties of the fuel.²⁴⁴ The standardized properties for bioethanol are listed under ASTM D4806 standard, and the comparison with gasoline is given respectively in Tables 13^245–247 and 14.^247–249

Table 13 Bioethanol properties based on ASTM D4806 (ref. 245–247)

Property	Specification	ASTM test method
Ethanol min, (% vol)	92.1	D5501
Methanol, max, (% vol)	0.5	—
Solvent-washed gum max, (mg/100 mL)	5.0	D381
Water content, max, (% vol)	1.0	E203
Denaturant content, min, (% vol)	1.96	—
Denaturant content, max, (% vol),	4.76	—
Inorganic chloride content, max, (mg L^−1)	40	D512
Copper content max, (mg kg^−1)	0.1	D1688
Acidity (as acetic acid CH3COOH) max, (mg L^−1)	0.007	D1613
pHe	6.5–9.0	D6423
Appearance-visibly free of suspended or precipitated contaminants (clear & bright)

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Table 14 Properties of bioethanol and gasoline^247–249

Property	Ethanol	Gasoline
Chemical formula	C2H5OH	C4 to C12
Molecular weight (g mol^−1)	46.07	100–105
Carbon (% wt)	52.2	85–88
Hydrogen (% wt)	13.1	12–15
Oxygen (% wt)	34.7	0
Specific gravity	0.794	0.7–0.78
Density @ 15 °C, (kg m^−3)	785–809.9	750–765
Boiling temperature, (°C)	78	27–225
Reid vapor pressure @ 37.8 °C, (kPa)	17	53–60
Research octane no.	108	90–100
Motor octane no.	92	81–90
(R + M)/2	100	86–94
Cetane no.	—	5–20
Fuel in water, (%vol)	100	0
Water in fuel, (% vol)	100	0
Freezing point, (°C)	−114	−40
Kinematic viscosity (mm^2 s^−1)	1.2–1.5	0.5–0.6
Flash point, closed cup, (°C)	12	−42
Auto ignition temperature, (°C)	423	257
Lower heating value (MJ kg^−1)	26.9	44
Higher heating value (MJ kg^−1)	29.7	47.3
Vapor Flammability limits, (% vol)	3.5–15	0.6–8
Specific heat, liquid (kJ kg^−1 K^−1)	1.7	2.4
Specific heat, vapor (kJ kg^−1 K^−1)	1.93	2.5
Stoichiometric air/fuel ratio	9	14.5–14.7
Fuel in vaporized stoichiometric mixture, (% vol)	6.5	2

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The detailed discussion of some of the properties related to bioethanol and gasoline are discussed below.

4.1 Ethanol yield

Ethanol yield is the parameter to evaluate the performance of the feedstock in bioethanol production.²⁵⁰ Ethanol yield can be described as theoretical and actual/experimental ethanol yield. Theoretical ethanol yield is obtained through stoichiometry calculation of the fermentable sugars, and is usually used as comparison factor. Meanwhile, actual ethanol yield is obtained from the actual production process of sugars fermentation, which is derived from the employed biomass. Actual ethanol yield depends on the conditions of fermentation, especially on the employed fermentation agents.²⁵¹ Therefore, the feedstock and microorganism selection were employed in the ethanol production in order to produce high yield and good ethanol quality. The concentration of the produced ethanol can be detected by checking their corresponding refractive indexes obtained from the refractometer readings.²⁵² The standard of ethanol yield or content for bioethanol is ASTM D4806.²⁵³

4.2 Acidity or alkalinity

The acidity or alkalinity of bioethanol is tested by reacting it with phenolphthalein to test whether the solution is alkali or acid, and the acidity is expressed as a percentage by mass of acetic acid.²⁵⁴ The acidity equation is given as below:²⁵⁴where: V = the volume of the NaOH solution (in mL), ρ = the density of the sample at 20 °C (in g mL⁻¹), 0.0006 = the mass of CH₃COOH corresponding to 1 mL of NaOH (in g).

The acidity or alkalinity of bioethanol describes the concentration of acid-type compounds that are present in the tested solution, and is usually regarded as total acid number.²⁵⁵ High acid content can cause the formation of gums and lacquers on metal surfaces, increased viscosity that impairs oil circulation, and also cause engine corrosion.²⁵⁶ ASTM Standard D1613 is the standard to measure the acidity or alkalinity of bioethanol. According to this standard, acidity or alkalinity of bioethanol has maximum value of 0.007 mg L⁻¹.²⁵⁷

4.3 Water content

The stability of ethanol depends on the chemical composition of the bioethanol, water content and temperature.²⁵⁸ Water content in ethanol is generally related to the two types of purities: (i) anhydrous, where water content obtained is less than 1%; (ii) hydrous, where water content possession is ranged 5–10% in the ethanol.²⁴⁸ Water contamination in ethanol is possible since ethanol has a hygroscopic nature, which absorbs water from the atmosphere when stored in open container.²⁵⁹ The water content in the bioethanol can be measured in accordance to ASTM E203, where this standard describes the maximum specification of 1.000% vol s.²⁴⁶

4.4 Denatured content

Denatured ethanol contains certain additives that can make it inconsumable and toxic, which is developed to prohibit recreational consumption. The additives also give a bad taste and foul smell to the ethanol.²⁴⁷ Foul-tasting or toxic substances are added for about 10% of the produced ethanol after distillation to accomplish this parameter. Denatured ethanol also can be blended with gasoline to improve the octane number of gasoline.²⁴⁸ The denatured ethanol is set to a minimum value of 1.960% vol and maximum value of 4.760% vol according to ASTM D 4806.²⁴⁶

4.5 pHe

The pHe is the pH of a denatured fuel ethanol, and it is quite difficult to measure this property since high quality denatured ethanol is not an aqueous solution.²⁶⁰ Typically, the pHe test is conducted after the denaturing process and corrosion inhibitors addition.²⁶¹ The ASTM D6423 method describes the pHe test, which covers rehydration specification of the probe between readings and repeatability for same operator, same apparatus and identical test material.²⁴⁷ The range of pHe of bioethanol is 6.5–9.0 with 90% confidence factor and reproducibility of 0.52.^259,260

4.6 Octane number

A measurement of fuel resistivity towards denotation and self-ignition is described as octane number.²⁶² Octane number can be rated as motor octane number (MON) or research octane number (RON), which both concludes the behavior of particular fuel combustion in an engine during high or steady load condition.^105,263 As an excellent anti-detonating additive, bioethanol can improve the octane number of gasoline-base fuel.^263,264 The octane number of bioethanol is specified by ASTM D2700, which are MON is 92; RON is 108; and bioethanol–gasoline blend ranges of MON is 81–90 and RON is 90–100.^248,249

The comparison properties of bioethanol and bioethanol gasoline blends are summarized in Tables 15^{251,253,265–269} and 16.^{253,269–274}

Table 15 The comparison properties of bioethanol from various biomass feedstock

Properties	Unit	Sugar molasses^269	Sugarcane^266	Farmstead^253	Cassava tubers^265	Cassava flours^251	Palm juice^268	Sweet sorghum syrup^267
Boiling point	°C	78.5	78.4	—	78.5	—	—	78
Heat of combustion	MJ L^−1	23.625	—	—	—	—	—	—
Heat of vaporization	kJ mol^−1	33.74	—	—	—	—	—	—
Lower heating value	kJ cm^−3	—	—	—	—	—	—	21.09
Octane rating	—	106–108	—	—	—	—	—	—
Stoichiometric air/fuel ratio	—	9/1	—	—	—	—	—	—
Concentration	—	99.6–99.8	—	—	—	98	96	—
pH	—	—	—	—	5–6	6.78	3–4	5.0
Acidity	mg L^−1	≤30	—	—	—	—	≤36	—
Water content	% vol	≤0.3	0.378	6.94	—	—	—	—
Viscosity at 40 °C	mm^2 s^−1	—	—	—	—	2.2	—	—
Density @ 20 °C	kg m^−3	789.0	791	816.2 at 15 °C	791.0	—	795.0 at 25 °C	785.0
Point of humidity	—	<6.5	—	—	—	—	—	—
Flammability limits	°C	—	—	—	—	—	—	13–42
Flash point	°C	—	13–14	24.5	—	23	—	13
Vapor pressure	kPa	—	—	14.5	—	—	—	17
Auto ignition temperature	°C	—	—	—	—	—	—	366
Distillation	% vol	—	—	—	—	78–100	—	—

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Table 16 The properties of bioethanol gasoline blends

Property	Unit	Test method	Gasoline	Farmstead^253		Sugar molasses^269					Cassava^270
				E58.9	E60	E10	E15	E20	E25	E30	E5	E10	E15
Density @ 20 °C	kg m^−3	ASTM D1298	745.4	917.0	908.0	739.6	749.5	754.1	757.1	761.3	740.8	743.9	746.9
Flash point	°C	ASTM D93	—	24.5	24.5	—	—	29.2	30.0	29.2	—	—	—
Heating value	MJ kg^−1	ASTM D240	42.54	—	—	—	—	—	—	—	43.45	42.51	41.58
Auto ignition temperature	°C	—	246.0	—	—	—	—	—	—	—	—	—	—
Research octane number	—	ASTM D2699	91.3	—	—	97.1	98.6	100.4	99.5	102.5	—	—	—
Motor octane number	—	ASTM D2700	84.0	—	—	—	—	—	—	—	—	—	—
Distillation		ASTM D86
(a) Initial boiling point	% vol		38.8	—	—	—	—	—	—	—	—	—	—
(b) 10% evaporated	% vol		68.5	—	—	—	—	—	—	—	—	—	—
(c) 50% evaporated	% vol		109.6	—	—	—	——	—	—	—	—	—	—
(d) 99% evaporated	% vol		161.5	—	—	—	—	—	—	—	—	—	—

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Property	Unit	Test method	Gasoline	Cassava^273
				E10	E20	E30	E40	E50	E60	E70	E80	E90	E100
Density @ 20 °C	kg m^−3	ASTM D1298	745.4	750.8	760.5	778.2	779.2	780.5	781.2	782.3	783.4	784.0	789.0
Flash point	°C	ASTM D93	—	−40	−20	−15	−13.5	−5.0	−1.0	0.0	5.0	8.5	12.5
Heating value	MJ kg^−1	ASTM D240	42.54	—	—	—	—	—	—	—	—	—	—
Auto ignition temperature	°C	—	246.0	260	279	281	294	320	345	350	362	360	365
Research octane number	—	ASTM D2699	91.3	93	94	95	97	99	100	103	104	106	129
Motor octane number	—	ASTM D2700	84.0	—	—	—	—	—	—	—	—	—	—
Distillation		ASTM D86
(a) Initial boiling point	% vol		38.8	—	—	—	—	—	—	—	—	—	—
(b) 10% evaporated	% vol		68.5	—	—	—	—	—	—	—	—	—	—
(c) 50% evaporated	% vol		109.6	—	—	—	—	—	—	—	—	—	—
(d) 99% evaporated	% vol		161.5	—	—	—	—	—	—	—	—	—	—

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Property	Unit	Test method	Gasoline	Potato waste^271				Cassava^272				Sugarcane^274
				E5	E10	E15	E20	E6	E10	E15	E20	E20
Density @ 20 °C	kg m^−3	ASTM D1298	745.4	—	—	—	—	754	757.6	758.6	759.7	759.7
Flash point	°C	ASTM D93	—	—	—	—	—	—	—	—	—	—
Heating value	MJ kg^−1	ASTM D240	42.54	—	—	—	—	41.41	40.8	40.08	39.47	39.47
Auto ignition temperature	°C	—	246.0	—	—	—	—	—	—	—	—	—
Research octane number	—	ASTM D2699	91.3	89.7	92.3	94	99.4	92.8	93.0	92.4	93.0	93
Motor octane number	—	ASTM D2700	84.0	—	—	—	—	84	83.8	82.8	83.4	83.4
Distillation		ASTM D86
(a) Initial boiling point	% vol		38.8	40.9	38.9	44	40.8	42.6	47.1	43.2	40.5	40.5
(b) 10% evaporated	% vol		68.5	54.3	53.1	57.2	55.4	61.3	65.1	62.7	62.4	62.4
(c) 50% evaporated	% vol		109.6	93.5	71.9	71.4	71.6	111.4	98.3	87.6	79.2	79.2
(d) 99% evaporated	% vol		161.5	184.1	175.1	182.4	176.6	206.1	191.6	203.6	201.4	201.4

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5. Engine performance and emissions using bioethanol and the blends

As one of renewable energy resources obtained from biomass, bioethanol has been tested intensively in internal combustion engines. Bioethanol is a viable alternative to unleaded gasoline fuel as automotive fuel, and it can be used without modified engines.^271,275,276 The application of bioethanol in spark-ignited engines has been studied and assessed through its combustion performance parameters, including brake thermal efficiency, brake specific fuel consumption, and engine power, which brings the bioethanol viability as a biofuel closer to the common practice. Additionally, gasoline bioethanol blends which substitute as fuel can enhance the blend property qualities and combustion performance in engines. Ghazikhani et al.²⁷⁰ analyzed the performance of two stroke SI engine using a gasoline–ethanol blend, which resulted in a CO emission reduction by 32% from an ethanol concentration of 15% in the blend. Similarly, Schifter et al.²⁷² observed that for a 20% ethanol blend in a single cylinder engine, the CO emission was reduced by 52%. In a four-stroke SI engine, Najafi et al.²⁷¹ investigated the performance of the engine fueled by gasoline–ethanol blends, and they observed that E20 enhanced the combustion efficiency by 30% and CO emitted was at 45.42%, compared to pure gasoline fuel. Using a higher ethanol ratio in the blend, Koç, et al.²⁷⁷ studied a single-cylinder SI engine combustion performance powered by blends E50 and E85. The authors reported that brake specific fuel consumption (BSFC) was improved to 20.3% and 45.6%, and NO_x emission was decreased to 14.29% and 11.22% for E50 and E85 respectively. The benefit of blending fossil fuels with bioethanol is also eligible for diesel engines. As reported by Gomasta and Mahla,²⁷⁵ the engine performance efficiency was increased to 13% for ethanol–diesel blends, and hydrocarbon (HC) emissions were reduced to 35% at full load for 20% ethanol ratio in the blend compared to pure diesel fuel. The summary of engine performance and emissions productions using ethanol blends are presented in Table 17.^{270–272,275,277–279}

Table 17 The summary of engine performance and emissions using ethanol blends^a

Engine	Fuel type	Test condition	Engine performance analysis			Emissions analysis				Ref.
			BSFC	BTE	BP	CO2	CO	HC	NOx
a BSFC = brake specific fuel consumption; BTE = brake thermal efficiency; BP = brake power; C = cylinder; ↓ = decrease; ↑ = increase; E0 = 100% vol gasoline; E5 = 5 vol% ethanol–95 vol% gasoline; gasoline; DME1 = 1 vol% dimethyl ether–99 vol% ethanol; E69.5 + 0.5 = 69.5 vol% ethanol–0.5 vol% cycloheptanol–30 gasoline; SI = spark ignition; CI = compression ignition.
1C, two stroke, SI	E0, E5, E10, E15	2500–4500 rpm	BSFC↓	—	—	6.3%↓	32%↓	6%↓	38%↓	270
1C, four stroke, SI	E0, E6, E10, E15, E20	2000 rpm	BSFC↑	—	—	—	52%↓	19%↓	60%↑	272
4C, four stroke, SI	E0, E5, E10, E15, E20	1000–5000 rpm	BSFC↓	30%↑	BP↑	10.41%	45.42%	31.69%↓	45.55%↑	271
1C, four stroke, SI	E0, E50, E85	1500–5000 rpm	Min. 20.3% and max. 45.6%↑	—	BP↑	—	1%↓	24%↓	—	277
4C, four stroke, SI	DME0, DME1, DME2	1400 rpm	—	Max 10%, ↑	—	—	—	702 ppm↓	1054 ppm↑	278
1C, four stroke, CI	E0, E5, E10, E15, E20	1500 rpm	BSFC↓	13%↓	—	—	38%↓	35%↓	—	275
4C, four stroke, SI	E69.5 + 0.5, E64.6 + 0.4, E59.7 + 0.3, E49.8 + 0.2	2000–2800 rpm	—	31.89%↑	—	8%↓	0.08%↓	—	—	279

---

6. Conclusion

Bioethanol is gaining interest for its ability to fit in the current energy crisis. Bioethanol is produced diversely according to the feedstock type, although in general it follows a full sequence of pretreatment, hydrolysis, fermentation and distillation. In first generation bioethanol production, sugar-rich food crops are treated, hydrolyzed and fermented to produce ethanol. Besides that, agricultural crop residues can be utilized for second generation production. Moreover, the lack of source availability of first and second generation led researchers to explore new feedstocks in bioethanol production, utilizing micro and macro algae due to their favorable rapid growth rate. This advances the technology to give more available methods and techniques, for instance pretreatment processes can be performed by tens of different techniques. The increased variety improves the ability to select the most suitable routes that match with one’s desired requirements and outcomes from the available sources. Furthermore, the suitability of gasoline bioethanol blends as an alternative fuel in spark-ignited engine is characterized by the fairness of its fuel properties. The properties of gasoline bioethanol blends such as an ethanol purity ratio of 99%, pHe of 6.5–9.0, water content of 1.0%, density of 785–809.9 were ruled according to ASTM standard. The gasoline bioethanol blends within these properties are suitable as an alternative fuel in spark-ignited engine.

This review discusses the available methods for the production of bioethanol as an alternative fuel with tremendous potential. It also suggests that bioethanol production should not only be focused on the feedstock potential and the selection of production route, but also it encourages other practical aspects when it comes to actual realization, especially the environmental impact. The feedstock exploration for bioethanol production is expandible, which means there is a lot of room for improvement and a large number of combinations of possible production paths to be sorted and arranged for suitable feedstocks. Bioethanol is indeed considered as the most influential variable for the transportation sector, since with its benefits bioethanol will bring independence from the non-renewable and more heavily polluting fossil fuels. Finally, bioethanol is one solution for the energy and environmental crisis and it is suggested to have a good synergy from both technological developments and supporting energy policies to solve energy concerns.

Acknowledgements

The authors gratefully acknowledge the financial support provided by the Ministry of Education of Malaysia and University of Malaya, Kuala Lumpur, Malaysia, under the High Impact Research Grant (Project title: Clean diesel technology for military and civilian transport vehicles, Grant no.: UM.C/HIR/MOE/ENG/07 D000007-16001), University Malaya Research Grant Scheme (Grant no.: RP022A-13AET), Fundamental Research Grant Scheme (Grant no.: FP009-2014A), SATU (Grant no.: RU021A-2015) and Postgraduate Research Grant (Grant no.: PPP-PG014-2015A).

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