From Microalgae to Bioenergy | Encyclopedia MDPI

From Microalgae to Bioenergy: History

View Latest Version

Please note this is an old version of this entry, which may differ significantly from the current revision.

Subjects: Engineering, Environmental

Contributor:

Sheetal Kishor Parakh

Zinong Tian

Jonathan Zhi En Wong

Yen Wah Tong

Among the available renewable resources, microalgae biomass, a third-generation feedstock, is promising for energy production due to its rich biochemical composition, metabolic elasticity, and ability to produce numerous bioenergy products, including biomethane, biohydrogen, and bioethanol.

anaerobic digestion
biomethane
biohydrogen
biophotolysis

1. Introduction

Energy has played a crucial role in economic and social development [1]. However, over 80% of our energy demand is fulfilled by non-renewable and less environment-friendly fossil fuels such as coal, natural gas, and oil [2]. These fuels are the primary source of carbon dioxide emissions, responsible for over 90% of global carbon emissions [3]. This dependency on fossil fuels has created two crises: fossil fuel depletion and global climate change. Despite this, energy demand continues to rise and is projected to increase by 47% in the next 30 years, particularly in developing countries [4]. It is, thus, imperative to find alternative renewable and clean energy sources to ensure a sustainable future. Bioenergy, the oldest known form of energy derived from biomass, is a promising option for meeting growing energy demand sustainably [5]. Biomass can be replenished in a relatively shorter time when compared with fossil fuels, making bioenergy a renewable energy source [6]. Bioenergy is carbon-neutral, releasing only the carbon dioxide that biomass consumes during its growth. Compared to bioenergy, fossil fuels release new carbon dioxide into the atmosphere that was sequestered and stored under the earth’s crust for millions of years [7]. Another advantage of bioenergy is its local production, reducing the need for long-distance energy transportation [8]. Local bioenergy production reduces the environmental impact of energy production and stimulates the local economy by providing energy security and economic opportunities [9].

Bioenergy is a broad term that refers to energy derived from various biomass sources, including food/non-food crops, agricultural/forest residue, and even algae [10]. Biomass can be divided into three generations based on its type. First-generation biomass includes traditional food and energy crops, such as corn, sugarcane, maize, soybean, and palm. Second-generation biomass comprises non-food crops and waste, such as woody/grassy plants and forest product residues [11]. Currently, a majority of biofuel is derived from first-generation biomasses, such as sugar (36.3 billion liters/year), maize (61.8 billion liters/year), palm oil (18.3 billion liters/year), and soybean oil (13.6 billion liters/year) [12]. However, using first-generation feedstocks for bioenergy production presents a food vs. fuel dilemma and challenges the food security [13]. Although second-generation feedstocks do not include food crops, these still compete with food production systems for resources such as arable land, freshwater, and fertilizers [14], making them unsustainable for fuel production. Third-generation feedstock, algae, has the potential to meet future bioenergy demands without compromising resources used for food production [15].

Microalgae are highly efficient photosynthetic organisms that rapidly grow throughout the year in various habitats, including aquatic (fresh or marine) and terrestrial ecosystems [16]. Microalgae exhibit relatively higher growth rates than terrestrial plants, completing an entire growth cycle in just a few days [17]. Moreover, microalgae can be cultivated on non-arable land using wastewater or seawater as a source of nutrients [18]. High biomass yield, minimal resource requirements, and a consistent biomass supply make microalgae a leading candidate for bioenergy production. Initially, the interest in using microalgae for bioenergy production was primarily due to its high lipid content (up to 60–70%), which could be converted into biodiesel using transesterification processes [19]. However, microalgae biomass can also be converted into other forms of bioenergy using biochemical and thermochemical processes. Biochemical conversion mainly includes anaerobic digestion [20], alcoholic fermentation [21], and fermentation/biophotolysis [22] processes for biomethane, bioethanol, and biohydrogen production, respectively. Thermochemical conversion includes pyrolysis [23], gasification [24], and hydrothermal processes [25] for bio-oil/biochar/syngas, syngas, and bio-oil/hydrochar/biogas production, respectively. Both biochemical and thermochemical processes are efficient pathways to recover energy from microalgae biomass since these methods utilize the whole biomass and do not depend on extracting specific macromolecules [26]. However, biochemical processes have a competitive advantage over thermochemical processes due to their ability to process wet biomass, operate at ambient processing conditions (temperature and pressure), and exhibit high selectivity toward the desired product, making them more sustainable and environment-friendly in the long run [27].

2. Microalgae Biomass and Its Components

Algae are efficient, sunlight-driven green cell factories that convert carbon dioxide into various biomolecules, including lipids, carbohydrates, and proteins [31]. The cellular content of these biomolecules varies significantly between different algae species [32]. Algae can be classified into two broad categories: microalgae and macroalgae. Microalgae are unicellular and smaller (up to a few millimeters), whereas macroalgae are large (up to a few meters) and multicellular. There are more than 50,000 microalgae species in nature, of which 4000 species have been identified, and only a few species (<50) have been commercialized [33]. Therefore, microalgae biomass has immense untapped and unexplored potential. Table 1 displays the biochemical composition of commonly used microalgae species.

Table 1. Biochemical composition of some microalgae species (% w/w, on a dry mass basis).

Microalgae Species	Lipid	Protein	Carbohydrate	References
Botryococcus braunii	25–75	1.5	4–55	[16]
Chlorella emersonii	23–63	36	41	[19]
Chlorella protothecoides	40–60	10–28	11–15	[19]
Chlamydomonas reinhardtii	15–18	9.2	59.7	[16]
Chlorella sorokiniana	26.2	45.5	23.7	[34]
Chlorella vulgaris	41–58	51–58	12–17	[19]
Dunaliella salina	6–25	57	32	[16]
Euglena gracilis	4–20	39–61	14–18	[16]
Isochrysis galbana	11	27	34	[35]
Nannochloropsis gaditana	23.3	48.3	9.3	[20]
Nannochloropsis granulata	24–28	27–36	18–34	[35]
Neochloris oleoabundans	35–65	10–27	17–27	[19]
Porphyridium cruentum	9–14	28–39	40–57	[20]
Scenedesmus dimorphus	16–40	8–18	21–52	[19]
Scenedesmus obliquus	30–50	10–45	20–40	[19]
Tetraselmis chuii	12	31–46	12	[35]

All biomolecules in microalgae cells play crucial roles in growth, reproduction, metabolism, and other cellular functions [36]. Lipids, essential components of cell membranes, energy storage, and cell signaling molecules, can be categorized into two groups in microalgae: polar lipids and non-polar lipids [37]. Polar lipids, such as phospholipids and glycolipids, are structural lipids that help maintain cell shape and structure. Polar lipids constitute 41–92% of the total lipids in microalgae biomass. Non-polar lipids, or neutral lipids, such as sterols and free fatty acids (FFA), usually function as energy storage molecules and make up 5–51% of the total microalgal lipids [38]. Neutral lipids, stored as triacylglycerols (TAGs), are preferred for biodiesel production due to their lower degree of unsaturation and the fact that industrial-scale transesterification is designed to process acylglycerols and has limited efficacy on other lipid types [39]. This makes microalgae species with high concentrations of neutral lipids (TAGs) promising candidates for biodiesel production. Besides lipids, carbohydrates (polysaccharides or oligosaccharides) are energy-storage molecules and structural elements in all living cells [40]. Structural carbohydrates are primarily present in the cell wall, whereas storage components can accumulate inside or outside the chloroplast [19]. Some carbohydrates may also be excreted as exopolysaccharides [41]. Carbohydrates in microalgae cells consist mainly of cellulose and starch and are free of lignin [42], making them a suitable candidate for bioethanol production through fermentation or biomethane production through anaerobic digestion. In addition to carbohydrates and lipids, proteins play essential roles in living organisms. Proteins serve as the major structural components of cells and transport nutrients and other molecules in and out of cells. They also act as enzymes or catalysts for various cellular biochemical reactions [43]. However, the high protein content in microalgae biomass may lead to a low carbon/nitrogen (C/N) ratio, limiting the biomass conversion to bioenergy [44]. To address the issue of low C/N ratio, various physiochemical approaches have been developed (Table 2) to lower protein content and enhance the lipid and carbohydrate content of microalgae biomass. Nevertheless, as shown in Table 2, high lipid or carbohydrate accumulation in microalgae does not always coincide with high biomass production, posing challenges for using microalgae as a bioenergy feedstock.

Table 2. Different approaches to enhance lipid and carbohydrate content in the microalgae biomass as well as biomass yield and their outcomes (↑: increase, ↓: decrease, and -: not available).

Approach		Outcomes		References
Approach	Lipid	Carbohydrate	Biomass	References
Nutrient stress
Nitrogen deprivation/starvation	↑	↑	↓	[19,45]
Phosphorus deprivation/starvation	↑/↓	-/↓	↓	[19,45]
Trace metal availability	↑/↓	↑	↑/↓	[19,46]
Carbon availability	↑	-	↑	[19,47]
pH stress
Acidic	↑/↓	↑/↓	↑/↓	[19,48]
Alkaline	↑/↓	↑/↓	↑/↓	[19,48]
Temperature stress
High temperature	↑/↓	-	↑/↓	[49]
Low temperature	↑/↓	-	↑/↓	[49]
Light stress
Low Light	↑/↓	↑/↓	↓	[50]
High Light	↑/↓	↑/↓	↑/↓	[50]
Saline stress	↑	↑	↑/↓	[19,51]

With the advent of metabolic engineering and its suite of genome-editing tools, some researchers are now genetically modifying microalgae cells by overexpressing or knocking out specific genes to alter metabolic pathways and improving the productivity of the compound of interest [52]. For instance, knocking out the phospholipase A₂ gene from Chlamydomonas reinhardtii using the CRISPR-Cas9 system resulted in a 64% increase in lipid productivity without compensating for the biomass growth rate [53]. In another study, the individual expression of three genes (glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAT), and diacylglycerol acyltransferase (DGAT)) in Neochloris oleoabundans resulted in a 52% increase in lipid content without significantly affecting the microalgae growth [54]. Additionally, the cloning and transforming of specific genes from other species of microorganisms like S. cerevisiae can help improve biomolecule biosynthesis pathways in microalgae cells [55]. Due to the potential for manipulating microalgae to produce high yields of energy-rich compounds like lipids and carbohydrates, it is considered one of the most important feedstocks for bioenergy production [56]. Figure 1 presents an overview of the conversion technologies that can transform microalgae biomass into bioenergy.

Figure 1. Different biomass conversion pathways to produce bioenergy from microalgae.

3. Biochemical Conversion

The essential part of the biochemical conversion process is to use microorganisms or enzymes to convert biomass into bioenergy [27]. Biochemical conversion technologies can handle biomass with high water content (>50%) [57], making it suitable for processing wet algae biomass. Since drying microalgae biomass is an energy-intensive step [58], biochemical conversion technologies are promising for transforming microalgae into bioenergy. The classical biochemical conversion processes include anaerobic digestion for biomethane production, alcoholic fermentation for bioethanol production, and biological hydrogen production.

3.1. Anaerobic Digestion

Anaerobic digestion (AD) is a complex process in which microorganisms degrade organic biomass under anaerobic conditions and convert it into biogas, majorly comprising methane and other products, such as carbon dioxide, trace amounts of hydrogen, and ammonia [59]. Biogas produced from the AD process can be directly combusted in gas boilers to generate heat or electricity or upgraded into natural gas-quality biomethane and injected into gas grids [60]. AD process consists of four steps, hydrolysis, acidogenesis, acetogenesis, and methanogenesis, where each step is led by a unique functional group of microorganisms [61]. In the first stage of AD (hydrolysis), existing macromolecules in the biomass, such as proteins, lipids, and carbohydrates, are broken down into simpler molecules, such as amino acids, long-chain fatty acids, and simple sugars, respectively. Hydrolysis is achieved by hydrolytic bacteria, mainly belonging to the phyla Firmicutes and Bacteroidetes, secreting a mixture of hydrolytic enzymes comprising cellulase, xylanase, pectinase, amylase, lipase, and protease [62]. In the second stage of AD (acidogenesis), hydrolyzed products obtained at the end of the hydrolysis stage are converted into volatile fatty acids, such as acetates, propionate, butyrate, valerate, and isobutyrate, using facultative and obligate anaerobic bacteria species, majorly belonging to the phyla Firmicutes, Bacteroidetes, Chloroflexi, Proteobacteria, and Atribacteria. Alcohol and other inorganic compounds, such as hydrogen, carbon dioxide, ammonia, and hydrogen sulfide, are also produced during the acidogenesis stage [63]. During the third stage of AD (acetogenesis), the acidogenesis products, such as propionate, butyrate, isobutyrate, valerate, and isovalerate, are further broken down into acetate as well as hydrogen and carbon dioxide using obligate anaerobic bacteria species, majorly belonging to phyla Firmicutes, Synergistota, and Myxococcota [64]. In the final step of AD (methanogenesis), methanogens (a specialized group of archaea belonging to the phyla Euryarchaeota, Bathyarchaeota, and Verstaraeteachaeota) convert acetic acid and hydrogen into methane and carbon dioxide [65]. Tuning these four metabolic stages in the AD process can influence final product yields [61]. AD is a well-established commercial technology that is currently being applied to a wide range of organic substrates (sewage waste [66], food waste [67], high-strength organic wastewater [68], and agricultural or forest residue [69]).

Research on anaerobic digestion (AD) of microalgal biomass began in 1957 when microalgae (Chlorella and Scenedesmus) were cultivated for wastewater treatment and subjected to AD for biomethane production [70]. However, the resulting biomethane yields were significantly lower due to two main factors: the rigidity of microalgae cell walls and the low carbon-to-nitrogen (C/N) ratio in microalgae biomass. Most microalgae cell walls are rigid, which limits anaerobic microorganisms’ access to biodegradable microalgal organic matter [28]. The rigidity of microalgae cell walls can be attributed to components such as hemicellulose, cellulose, glycoprotein structures, and certain carbohydrates (e.g., glucose, xylose, rhamnose, and galactose) [71]. Some types of microalgae species also contain algaenan in their outer cell walls. Algaenan is a heteropolymer compound, highly resistant to acidic and basic environments [72]. Several studies have reported intact microalgae cells in the AD effluent after a hydraulic retention time of 30–180 days [73,74], highlighting the recalcitrant nature of microalgal cell walls and their resistance to bacterial degradation during the AD process. Since insufficient biodegradation leads to lower biomethane production, a pretreatment step is necessary to disrupt the microalgae cell walls. Secondly, a C/N ratio of 20–30 (especially 25) is recommended for the optimal functioning of the AD process [75]. If the ratio falls below 20, high amounts of ammonia-nitrogen are released in the anaerobic digester due to the imbalance between microbial carbon and nitrogen requirements. Ammonia buildup can inhibit methanogen growth, leading to volatile fatty acid (VFA) accumulation and process failure [76]. However, the C/N ratio in microalgae biomass is usually between 4–8 [33]. To address the low C/N ratio in microalgae, several researchers have suggested co-digesting microalgae biomass with other biomass streams with a high C/N content [77]. Various biomass pretreatment and co-digestion technologies have been developed in the past decade to improve biomethane production from microalgae biomass.

3.2. Biohydrogen Production

Biohydrogen is considered the fuel of the future due to its higher heating value (142 kJ/g) and ability to produce energy without emitting carbon dioxide [124]. It can be used in fuel cells to generate electricity and as a fuel for automobiles, providing a carbon-neutral solution to current energy crises [125]. To tap into the potential of biohydrogen as a future fuel, microalgae have emerged as a promising source for its production. Microalgae can produce biohydrogen through two different pathways: bio-photolysis and fermentation. During biophotolysis, microalgae cells act as a biocatalyst to directly produce biohydrogen, whereas in fermentation, microalgae biomass serves as a feedstock for biohydrogen-producing microorganisms [126].

3.3. Alcoholic Fermentation

Bioethanol is the most extensively used renewable fuel for transportation and can be produced from microalgae using three pathways: dark fermentation (DF), photo fermentation (PF), and traditional fermentation (TF) [181]. Although DF and PF are similar process terminologies used to depict alcoholic fermentation and biohydrogen production pathways, the mechanisms involved in these pathways are different. Some examples of DF, PF, and TF for bioethanol production are listed in Table 8 and Table 9. In the DF process, microalgae species, such as Chlamydomonas reinhardtii, Chlamydomonas moewusii, Chlorococcum littorale, Chlorogonium elongatum, and Chlorella fusca, ferment intracellular polysaccharides into bioethanol under dark and anaerobic conditions [182,183,184]. Pyruvate, an intermediate compound, is generated through hydrolysis and glycolysis of intracellular polysaccharides (starch). Subsequently, pyruvate is converted into various end products, including acetate, ethanol, formate, glycerol, lactate, biohydrogen, and carbon dioxide, depending on the type of microalgae species and surrounding environmental conditions [185]. Studies on microalgae DF for bioethanol production reported low bioethanol yields of less than 2% w/w, as shown in Table 8. This could be attributed to the complex network of metabolic pathways involved in microalgal DF and difficulties associated with understanding and selectively manipulating those metabolic pathways to enhance bioethanol production. Therefore, the practical application of the DF pathway for bioethanol production has not received much attention.

Compared to the DF pathway, the PF pathway results in a more specific and efficient bioethanol production [186]. The PF pathway comprises two steps: photosynthesis and fermentation. During the first step of photosynthesis, inorganic carbon (carbon dioxide) is fixed into organic carbon (phosphoglycerate) through the Calvin cycle and later converted to pyruvate. In the second step, pyruvate is fermented into ethanol with the help of two key enzymes, pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhII). pdc catalyzes the conversion of pyruvate into acetaldehyde and carbon dioxide through a nonoxidative decarboxylation reaction, whereas adhII oxidizes the resulting acetaldehyde into ethanol [187]. However, these enzymes are naturally missing or expressed in insufficient quantities in microalgae. Therefore, genetic engineering is used to heterologously express pdc and adhII genes in microalgae, preferably cyanobacteria, to enable direct bioethanol production [188]. Cyanobacteria have relatively well-characterized genetic backgrounds, demonstrate a high tolerance to foreign gene introduction, and exhibit amenability to genetic modifications [189]. Foreign DNA (Deoxyribonucleic acid) can be introduced into cyanobacteria under controlled conditions through shuttle vectors or by directly integrating it into the chromosome via targeted homologous recombination. A research study by Deng and Coleman [190] was the first to report the cyanobacteria Synechococcus elongatus PCC7942 strain as the platform of bioethanol production. The authors constructed a new S. elongatus strain using a shuttle vector pCB4, cloned from the coding sequences of pdc and adhII genes obtained from the bacterium Zymomonas mobilis. Following four weeks of culture, the transformed S. elongatus strain produced a bioethanol titer of 0.23 g/L. Later, Dexter and Fu [191] used the same two genes from Z. mobilis and integrated them into the chromosome of Synechocystis sp. PCC 6803 using a double homologous recombination system and produced a bioethanol titer of 0.46 g/L in six days of cultivation. Since then, several genetic engineering efforts have been made to improve the bioethanol yield from cyanobacteria while maintaining cell growth (Table 8). However, PF pathways produce much lower bioethanol yields (<6 g/L), making the ethanol separation process (distillation) too costly for large-scale applications. Poor bioethanol yields could be attributed to co-factor imbalance [192], low ethanol tolerance levels [193], competition for carbon usage between biomass synthesis and target product formation [194], and inefficient carbon fixation mechanisms [195,196]. However, there is still room for optimizing the bioethanol yield from cyanobacteria through alternate gene expression approaches.

Table 8. Dark fermentation and photo fermentation for microalgae-based bioethanol production.

Dark Fermentation
Microalgae Species	Operating Conditions	Starch Content (% of Dry Cell Weight)	% Starch Decomposed	Bioethanol Yield (% of Dry Cell Weight)	References
Chlamydomonas reinhardtii UTEX 2247	Incubation under dark and anaerobic conditions at 25 °C for 46 h Slurry concentration: 15% w/w	45	NA	1	[182]
Chlamydomonas sp. YA-SH-1	Incubation under dark and anaerobic conditions at 30–35 °C for 44 h Slurry concentration: 15–25% w/w	30	NA	1.3	[183]
Chlorococcum littorale	Incubation under dark and anaerobic conditions at 30 °C for 24 h Slurry concentration: 1.4% w/w	15	46	1.6	[184]
Photo Fermentation
Cyanobacteria	Genes expressed (source of genes) and their expression mechanism	Promotor used	Gene deletion (Effect)	Bioethanol titer (g/L) and days of cultivation (d)	Reference
Synechococcus elongatus PCC7942	pdc (Zymomonas mobilis), Shuttle vector; adhII (Zymomonas mobilis), Shuttle vector	rbcLS	NA	0.23 in 28 d	[190]
Synechocystis sp. PCC 6803	pdc (Zymomonas mobilis), Homologous recombination; adhII (Zymomonas mobilis), Homologous recombination	psbA2	NA	0.46 in 6 d	[191]
Synechocystis sp. PCC6803	pdc (Zymomonas mobilis), Homologous recombination; adh, slr1192 (Endogenous overexpression), Homologous recombination	Prbc	phaA and phaB (Disrupting PHB biosynthesis pathway)	5.5 in 26 d	[197]
Synechocystis sp. PCC6803	pdc (Zymomonas mobilis), Homologous recombination; adhII (Zymomonas mobilis), Homologous recombination	nblA	glgC (Disrupting glycogen biosynthesis pathway) and phaC + phaE (Disrupting PHB biosynthesis pathway)	3 in 3 d	[194]
Synechocystis sp. PCC6803	zwf (Endogenous overexpression to enhance NADPH production) Homologous recombination; pdc (Zymomonas mobilis), Homologous recombination; yqhD, NADPH-dependent adh (Escherichia coli), Homologous recombination	Pcpc560	NA	0.59 in 14 d	[192]
Synechocystis sp. PCC6803	pdc (Zymomonas mobilis), Shuttle vector; adh, slr1192 (Endogenous), Shuttle vector	PnrsB	NA	0.45 in 7 d	[196]
	pdc (Zymomonas mobilis), Shuttle vector; adh, slr1192 (Endogenous), Shuttle vector; rbcSC, slr0009-slr0011-slr0012-FLAG with RuBisCO-encoding genes (Endogenous), Shuttle vector	PnrsB (for pdc and adh) and psbA2 (for rbcSC, 70glpX, tktA, and fbaA)		0.7 in 7 d
	pdc (Zymomonas mobilis), Shuttle vector; adh, slr1192 (Endogenous), Shuttle vector; 70glpX with FBP/SBPase-encoding genes (Synechococcus PCC 7002), Shuttle vector			0.75 in 7 d
	pdc (Zymomonas mobilis), Shuttle vector; adh, slr1192 (Endogenous), Shuttle vector; tktA, sll1070 with TK-encoding genes (Endogenous), Shuttle vector			0.6 in 7 d
	pdc (Zymomonas mobilis), Shuttle vector; adh, slr1192 (Endogenous), Shuttle vector; fbaA, sll0018 with FBA-encoding genes (Endogenous), Shuttle vector			0.75 in 7 d
Synechocystis sp. PCC6803	pdc (Zymomonas mobilis), Shuttle vector; adh, slr1192 (Endogenous), Shuttle vector; fbaA, sll0018 with FBA-encoding genes (Endogenous), Shuttle vector; tktA, sll1070 with TK-encoding genes (Endogenous), Shuttle vector	PnrsB (for pdc and adh) and psbA2 (for tktA and fbaA)	NA	1.2 in 20 d	[195]
Synechocystis sp. PCC6803 (Fe₂O₃-treated culture)	pdc (Saccharomyces cerevisiae), Shuttle vector; adh (Endogenous), Shuttle vector	psbA1	NA	4.9 in 25 d	[198]
Synechocystis sp. PCC6803 (MgO-treated culture)		psbA1	NA	5.1 in 25 d	[198]

Note: NA—not applicable, NADPH—nicotinamide adenine dinucleotide phosphate, pdc—pyruvate decarboxylase, adh—alcohol dehydrogenase, phaA—polyhydroxyalkanoate-specific β-ketothiolase, phaB—polyhydroxyalkanoate-specific acetoacetyl-CoA reductase, PHB—polyhydroxybutyrate, glgC—glucose-1-phosphate adenylyltransferase, phaC—polyhydroxyalkanoate synthase, phaE—polyhydroxyalkanoate polymerase subunit, zwf—glucose 6-phosphate dehydrogenase, RuBisCO (rbcSC)—ribulose-1,5-bisphosphate carboxylase/oxygenase, FBA (fbaA)—Fructose-1,6-bisphosphate aldolase, FBP/SBPase (70glpX)—fructose-1,6-/sedoheptulose-1,7-bisphosphatase, TK (tktA)—transketolase.

Traditional fermentation has been the most widely studied method of bioethanol production, as it typically yields higher bioethanol quantities (21–88% w/w (% of dry cell weight) and 5–43 g/L (based on the working volume)) compared to DF (<2% w/w) and PF (<6 g/L) (Table 8 and Table 9). In the traditional fermentation process, the carbohydrate content of microalgae biomass is used as a feedstock by ethanologenic microorganisms, such as yeast (Saccharomyces cerevisiae) and bacteria (Zymomonas mobilis). However, S. cerevisiae is more commonly used for bioethanol fermentation due to its tolerance towards low pH and high ethanol concentrations [199]. As described earlier in Section 3.1, microalgae biomass pretreatment is crucial before fermentation for easy access to intracellular microalgal compounds. Various biomass pretreatment methods are discussed in detail in Section 3.1.1, and the effects on bioethanol production have been summarized in Table 9. Shokrkar et al. [200] compared the effect of acidic and enzymatic pretreatments on the bioethanol production performance of a mixed microalgae culture and reported a 1.3 times higher bioethanol production when microalgae culture was pretreated with enzymes. De Farias Silva et al. [201] observed no significant variations between the acidic and enzymatic pretreatments for Chlorella vulgaris and Scenedesmus obliquus biomass. Another study demonstrated an alternate approach for bioethanol production without any acidic or enzymatic pretreatments [202]. The authors combined the extraction and fermentation process in which a lysozyme and calcium chloride mixture was used to extract glycogen from Arthrospira platensis. Extracted glycogen was simultaneously degraded to glucose with the help of a recombinant Saccharomyces cerevisiae culture, which produced alpha-amylase and glucoamylase. However, such an approach can only work for cyanobacteria, which lack robust cell wall structure. The examples listed in Table 9 confirm that the effectiveness of microalgae pretreatment varies depending on the species.

The traditional fermentation process can be divided into two groups: separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) [203]. During SHF, hydrolysis and fermentation processes are conducted separately in different reactors, whereas, during SSF, hydrolysis and fermentation processes proceed simultaneously in the same reactor. The main advantage of the SHF process is the possibility to separately optimize the operating conditions of the hydrolysis and fermentation processes. El-Mekkawi et al. [21] used the response surface method (RSM) during SHF to optimize the process variables, such as algal biomass, yeast loading, and fermentation time, achieving a higher bioethanol concentration of 19 g/L. Other advantages of the SHF process are the potential use of cheaper chemicals, shorter residence time, and easy operation. However, its high capital cost is moving the research direction toward SSF. Kim et al. [204] compared the effect of SHF and SSF on bioethanol production from Phorphydium cruentum and observed that SSF produced slightly better bioethanol yields (74–80%) over SHF (70–78%). Similarly, Megawati et al. [205] observed a slightly better bioethanol production result with SSF (48.5%) compared to SHF (46%). Although most of the bioethanol production studies have used a single strain of yeast to study the fermentation process, some studies have applied a co-fermentation approach in which two or more different strains of yeast are used with a capacity to simultaneously degrade pentose and hexose sugar [201]. However, using a combination of different yeast strains would still require careful and thorough investigation.

Table 9. Pretreatment of microalgae biomass for bioethanol production through traditional fermentation.

Microalgae Species	Pretreatment	Fermentation Microorganisms	Fermentation Operating Conditions	Bioethanol Concentration (g/L) and Yield (% of Dry Cell Weight)	References
Separate hydrolysis and fermentation
Carotenoid-free Chromochloris zofingiensis SAG 211-14	Autoclave (120 °C for 0.34 h) followed by two-stage enzymatic pretreatment with α-Amylase (90 °C, 2 h, 4.5 pH) and glucoamylase (60 °C, 22 h, 6.5 pH)	Saccharomyces cerevisiae CCUG 53310	Initial pH: 4.8 Inoculum size: NA Temperature: 37 °C Time:	25 ± 2%	[206]
Carotenoid-free Chromochloris zofingiensis SAG 211-14	Autoclave (120 °C for 0.34 h) followed by three-stage enzymatic pretreatment with Cellic Ctec2 and Cellic Htec2 (45 °C, 48 h, 5 pH), α-Amylase (90 °C, 2 h, 5 pH) and glucoamylase (60 °C, 22 h, 5 pH)	Saccharomyces cerevisiae CCUG 53310	Initial pH: 4.8 Inoculum size: NA Temperature: 37 °C Time:	62 ± 2%	[206]
Carotenoid-free Haematococcus pluvialis SAG 192.80	Autoclave (120 °C for 0.34 h) followed by two-stage enzymatic pretreatment with α-Amylase (90 °C, 2 h, 4.5 pH) and glucoamylase (60 °C, 22 h, 6.5 pH)	Saccharomyces cerevisiae CCUG 53310	Initial pH: 4.8 Inoculum size: NA Temperature: 37 °C Time:	35 ± 0.3%	[207]
Carotenoid-free Haematococcus pluvialis SAG 192.80	Autoclave (120 °C for 0.34 h) followed by three-stage enzymatic pretreatment with Cellic Ctec2 and Cellic Htec2 (45 °C, 49 h, 5 pH), α-Amylase (90 °C, 2 h, 5 pH) and glucoamylase (60 °C, 22 h, 5 pH)	Saccharomyces cerevisiae CCUG 53310	Initial pH: 4.8 Inoculum size: NA Temperature: 37 °C Time:	88.1 ± 0.5%	[207]
Chlorella vulgaris	1 N HCl, 90 °C, 1 h	Saccharomyces cerevisiae	Initial pH: 5 Inoculum size: 3% v/v Temperature: 30 °C Time:	46%	[205]
Chlorella vulgaris FSP-E	2% H₂SO₄, 121 °C, 0.34 h	Saccharomyces cerevisiae FAY-1	Initial pH: NA Inoculum size: NA Temperature: 30 °C	21% (43 g/L)	[208]
Mixed microalgae consortium	0.5 M H₂SO₄ and 2.5% (w/v) MgSO₄ at 121 °C, 0.67 h	Saccharomyces cerevisiae ATCC 7921	Initial pH: 6.5 Inoculum size: 3% v/v Temperature: 30 °C	5 g/L	[200]
Mixed microalgae consortium	Three-stage enzymatic pretreatment with β-glucosidase/cellulase (65 °C, 3 h), α-amylase (95 °C, 3 h) and amyloglucosidase (55 °C, 3 h)	Saccharomyces cerevisiae ATCC 7921	Initial pH: 6.5 Inoculum size: 3% v/v Temperature: 30 °C	6.4 g/L	[200]
Arthrospira platensis NIES-39	1 g/L lysozyme and 100 mM CaCl₂	Saccharomyces cerevisiae strain BY4741 AASS/GASS	Initial pH: 5.2–5.4 Inoculum size: 5% v/v Temperature: 38–40 °C	32%	[202]
Wastewater-grown microalgae biomass dominated by Microcystis	0.5 N H₂SO₄, 120 °C, 4 h	Immobilized Saccharomyces cereviciae ATCC 4126	Initial pH: 4.5 Inoculum size: 15% v/v Temperature: 30 °C	19 g/L	[21]
Porphyridium cruentum KMMCC-1061	One-stage enzymatic hydrolysis with pectinase and cellulase (37 °C, 7 h, 4.8 pH)	Saccharomyces cerevisiae KCTC 7906	Initial pH: 4.5 Inoculum size: 0.1% w/v Temperature: 37 °C	70–78% (based on initial glucose content)	[204]
Simultaneous Saccharification and Fermentation
Chlorella vulgaris	Two-stage enzymatic pretreatment with α-Amylase (90 °C, 6 pH) and glucoamylase (80 °C, 5 h, 6 pH)	Saccharomyces cerevisiae	Initial pH: 5 Inoculum size: 3% v/v Temperature: 30 °C	49%	[205]
Porphyridium cruentum KMMCC-1061	One-stage enzymatic hydrolysis with pectinase and cellulase (37 °C, 10 h, 4.8 pH)	Saccharomyces cerevisiae KCTC 7906	Initial pH: 4.5 Inoculum size: 0.1% w/v Temperature: 37 °C	74–80% (based on initial glucose content)	[204]
Co-fermentation
Dried and milled Chlorella vulgaris biomass powder Neoalgae^® (Micro seaweed products B-52501749).	3% H₂SO₄, 120 °C, 0.5 h	75% Saccharomyces cerevisiae Cameo S.p.A. and 25% Pichia stipitis ATCC 58, 785	Initial pH: 5–6 Inoculum size: 7.5 g/L Temperature: 30 °C	49 ± 5%	[201]
	One-stage enzymatic hydrolysis with Viscozyme^® L, AMG 300 L, and Pectinex Ultra SP-L (50 °C, 4 h, 5 pH)			49 ± 0.5%
Scenedesmus obliquus SAG 276.7	3% H₂SO₄, 120 °C, 0.5 h			87 ± 6%
Scenedesmus obliquus SAG 276.7	Ultrasonication followed by One-stage enzymatic hydrolysis with Viscozyme^® L, AMG 300 L, and Pectinex Ultra SP-L (50 °C, 8 h, 5 pH)			41 ± 1.5%

Note: NA—Not available.

This entry is adapted from the peer-reviewed paper 10.3390/fermentation9060529

© Text is available under the terms and conditions of the Creative Commons Attribution (CC BY) license; additional terms may apply. By using this site, you agree to the Terms and Conditions and Privacy Policy.