Review Article | Volume: 9, Issue: 6, November, 2021

Microalgal biorefinery: Challenge and strategy in bioprocessing of microalgae carbohydrate for fine chemicals and biofuel

Tan Kean Meng Razif Harun Ramizah Kamaludin Mohd Asyraf Kassim   

Open Access   

Published:  Nov 10, 2021

DOI: 10.7324/JABB.2021.9601
Abstract

Microalgal carbohydrate is one of the major macromolecule metabolites, which has recently gained great attention as an alternative feedstock for wide-range sustainable biobased products. These biopolymers can act as a chemical platform for the production of biofuels through a biochemical conversion process. However, low microalgal carbohydrate productivity at a large-scale production has become a major problem for economical biofuel production. Several strategies have been proposed and the approach only increased carbohydrate content but reduced the microalgal biomass production, resulting in low microalgal carbohydrate productivity. Besides, the inappropriate pretreatments and fermentation approaches specifically with high-energy techniques could cause an increase in the cost of biofuel production. This present review gives a comprehensive discussion on microalgal carbohydrate enhancement strategies via cultivation techniques including the influence of environmental stress on the microalgal biomass and carbohydrate productivity. This paper also reviews the state of art on downstream processing of microalgal biomass prior to the hydrolysis and fermentation process. The different fine chemicals such as bioethanol, biobutanol, and biohydrogen production from microalgal carbohydrate are also discussed. The information from this review provides a framework for bioconversion of microalgal carbohydrate for biofuel and fine chemicals. This production could be beneficial for potential industrial implementation.


Keyword:     Carbohydrate microalgal biomass pretreatment hydrolysis solvent


Citation:

Tan KM, Harun R, Kamaludin R, Kassim MA. Microalgal biorefinery: Challenge and strategy in bioprocessing of microalgae carbohydrate for fine chemicals and biofuel. J Appl Biol Biotech; 2021; 9(06):1–17.

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

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1. INTRODUCTION

Recently, the interest in the production of fine chemicals and biofuel from algal biomass for partially replacing chemicals from petroleum-based feedstock has gained considerable worldwide attention. The utilization of microalgae biomass also exhibited more advantages over other types of renewable feedstock such as rice straw, plant trunk, leaf, and others. Microalgae contain less lignin and have a simple structure that is less recalcitrant compared to other types of renewable biomass. The other renewable biomass consists of complex biomass structure such as thick lignin, cellulose, and hemicellulose which could increase the difficulties of biofuel and fine chemical production [1]. Another advantage of these microalgae is that these microorganisms are microscopic photosynthetic organisms that use sunlight and carbon dioxide (CO2) as key regulators to conduct photosynthesis for their growth which can be integrated for CO2 biosequestration application. In addition, these microorganisms exhibit higher nutrient uptake, which accumulate in cell vacuoles and show a fast growth rate. This makes harvesting time between 1 and 10 days compared to terrestrial plants that require more than 3 months before the biomass can be harvested [24]. Moreover, the biomass produced during cultivation contains valuable chemical compounds including lipids, carbohydrates, and proteins. Generally, different types of microalgae strains will synthesize different biochemical metabolites, depending on the cell strains and cultivation condition. Table 1 summarizes the chemical composition distribution in different microalgae strains. Generally, all of these chemical compounds can be converted into other value-added products, such as animal feeds, bulk chemicals, and other bioactive compounds especially for the pharmaceutical industry (Fig. 1).

Table 1: Chemical composition of different algal species on a dry matter basis (%).

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The potential of high value-added products synthesized from microalgal biomass is totally dependent on the chemical composition within the microalgae. For instance, microalgae with a high lipid content can be converted into biodiesel. Extracted carbohydrates from microalgal cells can be converted into bioethanol or biobutanol. Furthermore, the low lignin properties in microalgae exhibit an exceptional potential to be used as liquid biofuel feedstock due to the lower harsh pretreatment process. However, to date, researchers are still facing several obstacles that impede the development of the microalgal biorefinery process. The obstacles include a long cultivation period of microalgae to reach the mature state or stationary phase. Low microalgal biomass production, insufficient macromolecule accumulation, immature harvesting technology, pretreatment, and low product yield after the fermentation process could affect the feasibility of the biorefinery process. This comprehensive review discussed current trends and challenges of cultivation conditions and their downstream processing including pretreatment processes, hydrolysis methods, and fermentation conditions that could contribute to the formation of high value-added products. A critical approach was suggested at the end of the review. The focus was more on the strategy for improving carbohydrate accumulation in microalgal biomass. This was to ensure economic feasibility in the biorefinery process for the production of biofuels and fine chemicals.


2. MICROALGAL CARBOHYDRATE BIOREFINERY

Microalgal biorefinery is the process of synthesizing biofuel and fine chemicals using technology transformation from a single raw material. Chew et al. [12] agreed that this microalgal biorefinery is one of the current approaches that separate biochemical compounds in microalgal biomass into different fractions without damaging other fractions. Generally, the microalgal biomass harvested from cultivation contains three main chemical compounds: lipids, proteins, and carbohydrates [13]. As shown in Figure 2, it is known that each of these compounds has a significant value for a wide range of industries. All these biochemical compounds could be produced through different biochemical pathways.


3. MICROALGAL CARBOHYDRATE

The growth of microalgae and carbohydrate accumulation are typically related to the photosynthesis reaction in microalgal cells [14]. During photosynthesis, microalgae require CO2, sunlight, and oxygen (O2) with the presence of water (H2O). These elements are needed to produce carbohydrate (C6H12O6) and biomass as the final photosynthesis products. The overall photosynthesis reaction described by Meyer [15] is shown in Equation (1):

6CO2 + 12 H2O → C6H12O6 + 6O2 + 6H2O (1)

Figure 1: Various value-added produced from microalgae biomass.

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Microalgal carbohydrates are one of the main products produced from microalgal photosynthesis and the carbohydrates can be in various forms. This macromolecule may be in various forms of monomers (i.e., monosaccharides) or polymers (i.e., di-, oligo-, and polysaccharides), depending on the microalgae species. Typically, the polysaccharides found in cyanobacteria and green or red algae are glycogen-type and starch-type polysaccharides, while β-glucan polysaccharides are mainly present in brown algae and diatoms [16]. These formations of polysaccharides in microalgae are of significance for the main structure of the cell wall and energy storage component and act as a food supply for the microalgae cell.

4. CARBOHYDRATE METABOLISM

The accumulation of carbohydrates in microalgae cells involves several main metabolite pathways. Among the pathways involved are glycolysis/gluconeogenesis, citrate cycle, pentose phosphate pathway (PPP), pentose and glucuronate interconversion, starch and sucrose metabolism have been proved that can enhance the carbohydrate biosynthesis in microalgae. All these pathways are important in terms of the contribution to carbohydrate accumulation in microalgal biomass. Figure 3 describes the formation of carbohydrates involving the targeted genes or enzymes and metabolites in microalgae. The enzymes such as α-amylase, isoamylase, pullulanase, β-amylase, and glucoamylase were among the important enzymes that have been reported that play an important role in the carbohydrate biosynthesis in microalgae [17].

Figure 2: Microalgal technology and conversion process for the microalgal biorefinery.

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Figure 3: Carbohydrate biosynthesis pathway in microalgae.

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According to the study by Kuchitsu et al. [18], the starch biosynthesis in Chlamydomonas reinhardtii was largely affected by the starch synthesis pathway. This can particularly be seen in the especially adenosine diphosphate-glucose-starch (ADP-glucose-starch) synthase enzyme under CO2-rich conditions. The importance of this synthase enzyme was further discussed by Patron and Keeling [19], who reported that this enzyme could enhance the synthesis of plastidic starch, which occurred in the plastid of green microalgae. Subsequently, the study also indicated that the formation of carbohydrates through the starch synthesis pathway was intercorrelated with the glycogen synthesis pathway. Other studies have also indicated the importance of the Embden-Meyerhof pathway and the PPP in converting glucose into disaccharides and polysaccharides, mainly sucrose and starch [20]. Both pathways are notable in microalgal carbohydrate metabolism which has been shown to be responsible for glucose accumulation under light and dark conditions, respectively.


5. STRATEGY TO IMPROVE MICROALGAL CARBOHYDRATE ACCUMULATION

Carbohydrate biosynthesis in microalgal cells can be influenced by several factors. One of the most common factors that contributed to microalgal carbohydrate production is the surrounding cultivation conditions (i.e., abiotic factors). It is essential to determine the optimum cultivation conditions, favorable or merely tolerable for the growth of microalgal species. Cultivation under unfavorable conditions could either increase or decrease the microalgal growth and carbohydrate content [21,22]. The most common cultivation conditions such as the surrounding temperatures, light intensity, pH, and CO2 concentration have reported that these cultivation parameters can significantly influence the microalgal growth and carbohydrate accumulation in microalgal biomass during cultivation [23]. These stress conditions applied toward microalgae have been proven as suitable approaches to enhance microalgal carbohydrate biosynthesis within an appropriate cultivation period and the detailed discussion is explained in the subsequent sections (Fig. 4).

5.1. Cultivation Parameters

5.1.1. Effect of the interaction between pH and CO2 concentration

Initial pH values are an essential parameter that can affect microalgal metabolism and carbohydrate formation in microalgal cells. The optimum pH value range for microalgae is generally between pH 6 and 9 and generally depending on the type of microalgal species. Cultivation of microalgae under the upper pH limit will suppress cell growth by reducing the affinity of microalgae toward the free CO2. This will subsequently lead to a decrease in microalgae photosynthesis rate and growth rate [24,25], whereas the lower limit of pH will induce the acidic environment which could alter the nutrient uptake or stimulate metal toxicity, thus affecting the microalgae growth [26]. The maintaining of the surrounding pH is important to achieve a continued active photosynthesis process under natural daylight [27]. However, the fluctuation of pH values can happen essentially during cultivation especially in the presence of CO2. This phenomenon is due to the presence of CO2 in the cultivation medium that could react with H2O and produce carbonic acid (H2CO3) [28]. Subsequently, H2CO3 decreases the pH value of the cultivation medium to a certain level. This may influence the nutrient uptake and enzyme kinetics involved in microalgal metabolism [29,30].

It was known that the addition of CO2 to the cultivation medium could enhance the microalgae biomass growth and affect the biomass compositions especially carbohydrate and lipid content in the microalgae cell [31]. Maintaining the pH value in the cultivation medium in a photobioreactor using the CO2 manipulation could increase the microalgae productivity and lipid accumulation in the microalgae [32]. The interaction between pH and CO2 concentration has been proved on Nannochloropsis sp. MASCC 11 which exhibited excellent growth and lipid production up to 108.2 and 782.7 mg l−1 on pH 6.00 and 5% CO2 concentration, respectively [33]. The supplementation of CO2 through aeration toward the microalgae culture did not only involve the lipid fraction, while it also involved the carbohydrate fraction in the microalgae biomass through the carbohydrate metabolism pathway. The previous study showed that the increase in the CO2 concentration up to 25% CO2 concentration (v/v) along the microalgae cultivation was increased gradually on the growth rate and carbohydrate content in the Scenedesmus bajacalifornicus BBKLP -07 strain. This increment of both growth rate and carbohydrate content was recorded as 0.16 ± 0.0012 d−1 and 26.19%, respectively, compared to the control treatment (0.04% CO2) [34]. This was due to the fact that CO2 could act as an inorganic carbon source for triggering the microalgae growth and carbon flux flow for carbohydrate synthesis through the photosynthesis process [35].

Figure 4: The summary diagram impact of different stress factors on the microalgal carbohydrate.

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Providing the CO2 toward the microalgae during cultivation has been proved to be a good strategy to improve the microalgae growth rate and biochemical accumulation inside the microalgae body [36,37]. However, further increasing the CO2 supplement toward microalgae will lead to a decrease in the cultivation pH value. This phenomenon will result in an adverse effect on the microalgae growth and subsequently affect its biochemical composition accumulation. It can be observed that the cultivation of the Chlorella sp. under 30% CO2 concentration (v/v) could reduce the specific growth rate of microalgae up to 76% compared under normal condition 0.04% CO2 (v/v) [38]. The increment of CO2 concentration beyond the microalgae susceptible limit not only limits the specific microalgae growth but also affects the carbohydrate accumulation in the cell biomass. There was an obvious decrease in carbohydrate composition in Chlorella sp. microalgae by 72.22% when the cultivation was conducted under 15% of CO2 (v/v) concentration.

Under the high CO2 concentration (low pH condition), the microalgae were required to achieve a high-energy demand to drive the proton gradient across the membrane, to stabilize the intracellular pH condition. Then, this energy loss will decrease the photosynthesis rate and affect the carbohydrate accumulation process in microalgae cells [39]. The capability of microalgae in response to the high CO2 concentration is species-dependent [40]. The microalgae that can tolerate the CO2 concentration in between 2% and 5% (v/v) are categorized as CO2-sensitive microalgae, while those that can tolerate between 5% and 20% (v/v) are categorized as CO2-tolerant microalgae. From an industrial perspective, the microalgae that possess the most tolerant and robust characteristics are important to ensure sustainable microalgae bioprocessing and technologies. The robust microalgae strains that can grow under a wide range of pH and high CO2 concentrations are economically feasible for continuous production with low maintenance under large-scale production. Based on the summary above, it clearly indicated that microalgae have a great potential to become one of the CO2 capture and utilization agents. These microalgae can be cultivated in an integrated system with flue gas and the biomass produced can be used as feedstock for microalgae-based products in a biorefinery.

5.1.2. Effect of temperature and light intensity

Another factor that could significantly affect microalgal growth rate and carbohydrate accumulation is the combined effect of light intensity and surrounding temperature. Generally, microalgae cultivation under outdoor conditions could be significantly affected by the light intensity and the surrounding temperature. It is known that light intensity influences the photoadaptation/photoacclimation and photoinhibition processes in microalgal cells. The majority of microalgae are light-saturated under light intensities of 200–400 μmol m−2 s−1. Under the low light intensity, the microalgae will exhibit a slow growth rate due to the inhibition of light harvesting pigments, chlorophyll a and b in that stage. However, the increase of the illuminated light intensity toward the microalgae over the certain threshold value will generate more heat which will raise the temperature. This will cause a decline in damage to the cell and the biochemical composition of microalgae [41].

Generally, the characteristic behavior of the microalgae toward light intensity and environmental temperature could be categorized as heat-sensitive and heat tolerance microalgae [42]. To date, most of the microalgae strains that are isolated nowadays could survive a wide range of light intensities and temperatures through acclimation or adaptation strategies within microalgae metabolism. This combination effect has been observed on Tetradesmus obliquus which performs well on 36°C with 434.75 mol m−2 s−1. The T. obliquus obtained the maximum biomass production up to 115 mg l−1 d−1 under this condition [43]. However, other strains like Chlorella vulgaris exhibited the maximum cell growth up to 1.13 ± 0.04 day−1 when the cultivation was performed under light exposure of 100 μmol m−2 s−1 and temperature of 25 ± 0.5°C [44]. The optimum light intensity and temperature were observed differently from each microalgae strain. Further increased light intensity and surrounding temperature could lead to the effect of photoinhibition. This can be observed when the microalgae were exposed to an intensity of 94.50 μmol m−2 s−1 toward the heat-sensitive microalgae. Low microalgal biomass production was obtained due to the degradation of the D1 protein in the photosynthetic system II in the microalgae [45].

Apart from that, the effect of light intensity and surrounding temperature was also reported that could influence the carbohydrate content in microalgal biomass [46]. It is worth mentioning that, during microalgal photosynthesis, carbohydrates are produced as the final product to be used as an energy source during respiration. Leading to this, a previous study indicated that the optimum light intensity and temperature as 150 μmol m−2 s−1 and 26°C could increase carbohydrate content in Pavlova lutheri up to 66% compared to the normal condition [47]. Nevertheless, it was found that a further increase of light intensity beyond 400 μmol m−2 s−1 appeared to reduce the carbohydrate content to 8% in this microalgae strain. The excessive exposure of light and surrounding temperature on microalgae most probably leads to the degradation of microalgal carbohydrates of which these biomolecules will be transformed into lipids in order to protect microalgal cells from photoinhibition [48].

Therefore, as per the discussion above, it is clearly indicated that the effect of light intensity and surrounding temperature toward microalgae is species-dependent. It is important to select the suitable light intensity and temperature to obtain the maximum biomass production and carbohydrate content in microalgae especially those cultivated under outdoor conditions. On the other hand, selecting the robust strain that is able to withstand a wide range of temperature and light intensity fluctuation with little or nonsignificant effect on growth and carbohydrate productivity is important especially for continuous outdoor cultivation.


6. DOWNSTREAM PROCESSING OF MICROALGAL BIOMASS

In order to produce biofuels and fine chemicals from microalgal carbohydrate through the biochemical pathway, several major steps such as biomass handling, pretreatment, hydrolysis, and fermentation are the important steps to ensure the economic feasibility of the end products’ formation (Fig. 5). The details of each process are described in the next subtopics.

6.1. Microalgal Biomass Pretreatment

Microalgae are eukaryotic microorganisms that have a complex polymer cell wall structure. The structure of a complex polymer cell wall mainly consists of noncellulosic polysaccharides, such as rhamnose, galactose, glucuronic acid, and glucosamine, whereas glucose is only a minor component [49]. These components play an important role in the formation of dynamic and rigid microalgae cell structure that allows microalgae to sustain or maintain cell bodies in harsh conditions. The plasticity properties enable microalgae ells to expand into different shapes [16]. In order to extract polysaccharides or carbohydrates from intracellular microalgal cells for biofuels and fine chemical production, pretreatment is a vital step for the valorisation of breaking down this rigid microalgal cell wall structure prior to fermentation. Pretreatment provides accessibility for enzymatic hydrolysis and improves digestibility of polysaccharides or carbohydrates available in the biomass [50]. To date, several microalgal cell disruption and pretreatment methods have been introduced by previous researchers. These methods can be categorized into three techniques: physical, chemical, and biological (Table 2) [51,52].

A physical pretreatment normally involved the mechanical, heat, or both combination to reduce the particle sizes; chemical pretreatment usually used the acid or alkaline to break the hydrogen or ester bond in the cell membrane, whereas the biological pretreatment involved the utilization of microbes or enzymes to disintegrate the biomass and release the sugars for subsequent hydrolysis and fermentation process (Table 3).

To date, there are no specific pretreatment methods that can be applied for all types of microalgal strains. This limitation is due to the fact that some pretreatment methods are species-specific and significantly contributed by the cell structure and composition. An effective pretreatment method should be considered with the aspects of cost-effective, time-saving, energy-efficient, and simple to upscale for industrial application.

6.2. Enzymatic Hydrolysis

As mentioned earlier, hydrolysis is a major step involved in biofuel and fine chemical production via the bioconversion process. This ensures that all microalgae carbohydrates available in microalgal biomass cells are converted into monomer sugars prior to the fermentation process. The utilization of an enzyme for hydrolysis depended significantly on the microalgae cell wall composition, biochemical distribution, and microalgae structure. All these factors are varying from each of the microalgae species.

The most common enzymes used in this process are cellulase and amylase [66,67]. The cellulase and hemicellulase have been reported by previous studies and are also considered as the common enzymes used to extract sugar from microalgal biomass [68]. These two enzymes are used to hydrolyze the intracellular cellulose and hemicellulose cell wall structure in microalgal biomass and produce simple sugar for fermentation. Investigations on the enzymatic hydrolysis of microalgal biomass, such as Chlorella sp., Scenedesmus sp., Nannochloropsis gaditana, and Tetraselmis suecica, for enhancing fermentation products have been indicated in earlier research [6971]. Studies have indicated that the hydrolyzed microalgal biomass could significantly increase the yield of final fermentation products.

Figure 5: Process for liquid fuel and chemicals from the fermentation of microalgal carbohydrate.

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Table 2: Types of pretreatment, advantages and limitations used to pretreat the microalgal biomass.

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Table 3: The process condition and sugar production based on different microalgal biomass.

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Theoretically, the efficiency of enzymatic hydrolysis of microalgal biomass depends significantly on various hydrolysis parameters, including temperature, pH, enzyme loading, and biomass concentration [67,72]. Selecting the optimum hydrolysis process is important to achieve the maximum sugar recovery from microalgae biomass. A study found that the maximum reducing sugars were obtained at optimum conditions for the hydrolysis of C. reinhardtii using 0.2% glucoamylase at 55°C and pH 4.5. Enzymatic hydrolysis of microalgal biomass beyond optimal conditions will result in low reducing sugar concentrations. In another study, the optimum enzymatic hydrolysis condition produced maximum sugars at 64% hydrolysis yield from C. humicola from the hydrolysis process using 10 g l−1 biomass at 40°C and an initial pH of 4.8 [73]. Such parameter conditions have been identified as one of the major bottlenecks ensuring the feasibility of sugar production from microalgal biomass. Thus, further research and development are needed to improve enzymatic hydrolysis under high biomass concentrations.

6.3. Fermentation of Microalgal Carbohydrate

6.3.1. Bioethanol

Liquid biofuels have commonly derived from microalgal biomass is bioethanol. This product is produced from anaerobic fermentation using microorganisms, such as yeast or bacteria as the biocatalyst. Theoretically, the maximum yield of carbohydrate fermentation is 0.51 kg ethanol and 0.49 kg CO2 per kg of sugar. In general, the simplified reaction equation for ethanol production is as follows:

C6H12O6 → 2CH3CH2OH + 2CO2 (2)

Glucose  Ethanol  Carbon dioxide

The study on the potential of microalgal carbohydrate as bioethanol feedstock using Saccharomyces cerevisiae has been widely reported [7476]. The fermentation of acid-treated C. vulgaris FSP-E containing 51% carbohydrate per dry weight biomass produced 11.7 g l−1 of bioethanol that corresponded to 87.6% ethanol yield [74]. Similar observations were reported on the fermentation of C. reinhardtii biomass has been reported that fermented from this biomass produced 235 mg of ethanol from the fermentation of 1 g of biomass through separate hydrolysis and fermentation (SHF) analysis [72]. Another study indicated that the fermentation of nitric acid (HNO3)-treated microalgae Spirulina platensis produced 16.32% ethanol yield [74].

The fermentation of lipid-extracted microalgal biomass residual has also been explored as an alternative approach to reduce the accumulation of fermented by-products. According to Harun et al. [76], the fermentation of lipid-extracted Chlorococcum sp. produced 3.83 g ethanol/10 g microalgal biomass. This study showed that extracted lipid was converted into biodiesel, and the method provided a new approach for the microalgal biorefinery concept.

Successful ethanol production from microalgal carbohydrates could also be attributed to the fermentation mode used during the conversion process. Various fermentation modes have been proposed for ethanol fermentation, including SHF; simultaneous saccharification and fermentation (SSF); simultaneous saccharification cofermentation; and separate hydrolysis and cofermentation.

Danquah et al. [77] compared the fermentation of microalgal Chlorococcum sp. biomass in different fermentation modes and concluded that SHF gave the highest bioethanol yield. In contrast, a different observation was reported regarding the fermentation of Chlamydomonas mexicana in SHF and SSF modes. A higher ethanol yield of 10.5 g l−1 was obtained from the fermentation of microalgal biomass generated from combined sonication and enzymatic hydrolysis in the SSF approach [75]. Increments in ethanol production based on the SSF approach could be explained as a method to provide better hydrolysis efficiency based on the cellulose activity for sugar production toward the biomass [78]. Table 4 shows the influence of the types of pretreatments, conditions, and yields of bioethanol production based on different microalgae strains. Based on this discussion, it can be concluded that not only does bioethanol production depend solely on the microalgal carbohydrate content, but also other factors such as pretreatment and fermentation modes are equally important to ensure the economic feasibility of the bioethanol production.

6.3.2. Biobutanol

Apart from bioethanol, biobutanol can also be used as an alternative biofuel for transportation. Chemically, biobutanol or butyl alcohol is a colorless liquid with four-carbon alcohols, molecular formula of C4H9OH. This chemical compound has a distinct odor and is completely miscible with organic solvents and partly miscible with H2O. Currently, butanol is used in various applications, which can be found in many chemical additives, solvents for perfume, and the manufacturing of antibiotics and as a chemical platform for other chemical syntheses [85]. Existing studies have indicated that this chemical compound can be used and blended up to 85% with gasoline with and without engine modification [86].

Biological biobutanol production via acetone-butanol-ethanol (ABE) fermentation using different ranges of feedstock including cellulosic and noncellulosic materials has been investigated globally. Biobutanol via the fermentation process has exhibited more advantages over the chemical reaction route. However, the production of biobutanol at the commercial stage faces several problems or challenges [87]. For example, ABE fermentation using cellulosic materials, such as empty fruit bunch (EFB), corn, sugarcane, and straw has been found to be relatively expensive. This may be due to the complicated technology used to overcome feedstock recalcitrance resulting from the presence of lignin in the biomass. Major issues associated with ABE fermentation at the commercial scale are food versus fuel found to be significant for sustainable economic production globally.

The production of biobutanol via fermentation using microalgal carbohydrates is a promising approach to overcome those issues, as this feedstock is simple and contains low lignin. It is also considered renewable and sustainable and contains a high concentration of carbohydrates. The potential of microalgae biomass as feedstock is highly positive and ensures feasible biobutanol production. Generally, ABE fermentation is performed using Clostridium sp. bacteria as the biocatalyst that will convert carbohydrates or starches from microalgal biomass into acetone, butanol, and ethanol under anaerobic conditions in a molar ratio of 3:6:1 as per (Eq. (3)).

Table 4: The types of pretreatment, fermentation condition, and bioethanol yield based on different microalgae strains.

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(C6H10O5)10 + 9H2O → 3C3H6O + 6C4H10O + C2C6O + 24 CO2 + 16H2 + Biomass (3)

Starch       Acetone  Butanol  Ethanol

Early research on biobutanol production from various types of feedstock, such as palm kernel cakes, empty EFB, rice straw, and macroalgae have been noted [8892]. To the best of our knowledge, there is little information on the production of biobutanol from microalgal biomass. A previous study on ABE fermentation of different types of microalgal biomass, such as Arthrospira platensis, Nannochloropsis sp., Dunaliella tertiolecta, Galdieria partita, C. vulgaris, Cosmarium sp., and Nostoc sp., has indicated that the production of biobutanol significantly depends on the carbohydrate composition and type of microalgal strain [93]. Other studies on the fermentation of whole microalgal cells cultivated in wastewater showed great potential for biobutanol production with butanol and ABE concentrations of 3.74 and 5.23 g l−1, respectively. These results were acquired from 3.5 g of microalgal biomass [94]. Later studies revealed that ABE fermentation of acid-hydrolyzed C. vulgaris biomass using Clostridium acetobutylicum strain produced approximately 3.37 and 5.14 g l−1 of butanol and ABE, respectively [95]. A similar observation was also reported by Wang et al. [96] who demonstrated that the initial biomass concentration significantly affected ABE fermentation and solvent production. According to the study on ABE fermentation using different initial biomass concentrations of 20, 40, 60, and 80 g l−1, further increased initial biomass concentrations would significantly improve biobutanol production. The maximum butanol production from the ABE fermentation of mutant C. vulgaris was achieved when the fermentation was carried out using 80 g l−1 of biomass. Kassim et al. [97] also investigated the potential of biobutanol production from alkali-pretreated marine microalgae T. suecica. It was found that a total of 0.14 g l−1 of butanol was produced from ABE fermentation using Clostridium saccharoperbutylacetonicum N1-4. Low biobutanol production from this study might be due to the low biomass concentration used during fermentation. Low biobutanol production from this study could be attributed to the fermentation using a low biomass concentration; however, it is insufficient to trigger the metabolic solventogenesis pathway for ABE production. This resulted in a low solvent concentration as nearly all the sugar in the hydrolysate had been converted into an organic acid during the fermentation process. Generally, ABE fermentation involves two major fermentation stages: acetogenesis and solventogenesis. Acetogenesis is a biological process that converts the available carbon source into organic acids such as butyrate and acetate, while solventogenesis is a biochemical production of solvents such as acetone and butanol. According to these studies, it indicated that suitable amount of initial biomass plays an important role to undergo the solventogenesis process during the fermentation process. On the other hand, it was also reported that biobutanol production from microalgae can be affected by the pretreatment process. A pretreatment process affecting the ABE fermentation process was indicated by a previous study [98]. This study compared production of biobutanol from two types of microalgae biomass samples extracted using different solvents namely ionic liquid extracted algae (ILEA) as well as hexane extracted algae (HEA) and resulted that the highest butanol production was achieved from the fermentation of HEA sample with a butanol concentration of 8.05 g l-1. The overall ABE productivity gave 0.35 g l−1 hours−1 for ILEA and 0.32 g l−1 hours−1 for HEA.

6.3.3 Biohydrogen

The production of biohydrogen from microalgal carbohydrates is one of the renewable processes to produce alternative bioenergy from microalgae biomass. Biohydrogen is a very favorable renewable fuel for its high-energy content (i.e., 118.7 kJ g−1) as it yields an output four times higher than bioethanol and methane. Generally, biohydrogen is a product from the fermentation of organic carbon from various types of microorganisms, including microalgae, bacteria, and cyanobacteria. These microorganisms produce hydrogen by releasing electrons during its metabolic reaction as follows:

Figure 6: Dark fermentation pathway for biohydrogen production (adapted from Monlau et al. [107]).

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2e + 2H2O → H2 + 2H2O (4)

The generation of biohydrogen can be carried out biologically through biophotolysis of H2O, photoreduction, and fermentation [99]. Biohydrogen from microalgal carbohydrates is typically obtained from either dark or an autofermentation process. In dark fermentation, the process is performed by hydrolyzing complex organic polymers via hydrogen-producing bacteria, such as Clostridium butylicium without the presence of H2O, O2, and sunlight. At the end of the process, other high value-added chemicals, such as butyric acid, lactic acid, and acetic acid are produced. On the other hand, photofermentation is a conversion of organic polymers into biohydrogen and CO2. This is performed with the presence of sunlight as an energy source. Theoretically, 4 mol of hydrogen could be produced from each mol of glucose, which corresponds to 33% energy yield of the reaction.

C6H12O6 + 2H2O → 2CH3COO + 2H + 2CO2 + 4H2 (5)

Various microalgal species including Chlorella sp., Scenedesmus sp., Nannochloropsis sp., and S. platensis as biohydrogen feedstock have been extensively considered [100102]. Microalgal carbohydrate content and its pretreatment process are imperative in order to ensure the efficiency of biohydrogen production.

Batch fermentation of lipid-extracted Scenedesmus sp. has been generated from different pretreatment processes including acid, base, chloroform, and a heat produced maximum biohydrogen concentration of 36 g VS l−1. This occurs when the fermentation was conducted using heat-treated microalgal biomass [103]. Studies have also indicated that utilizing a lipid-extracted sample could give dual benefits to microalgal biorefineries, including renewable energy production and sustainable biodiesel production. Dark fermentation has been used for biohydrogen production. Monosaccharides (e.g., glucose and mannose) and polymers, such as cellulose found in microalgal cells, can be used as biohydrogen feedstock. The production of biohydrogen using dark fermentation is explained via two pathways. One pathway produces acetate and the other produces butyrate. Both pathways are shown in Figure 6. Dark fermentation of C. reinhardtii hydrolysate from heat treatment and enzyme hydrolysis using Thermotoga neapolitana had been reportedly shown to be the highest biohydrogen production at 2.5 mol H2/sugar from the enzymatic hydrolysis sample [104]. This suggested that easier digestibility and suitable steps to break the microalgal cell walls are vital to obtain high biohydrogen. Similarly, for the fermentation of Chlorella sorokiniana that was pretreated using the HCl-heat approach, autoclaving, and sonication, the most suitable pretreatment was the HCl-heat method. The fermentation process was able to obtain the maximum biohydrogen amount of 685 dm−3 kg−1 [105]. Biohydrogen production via photofermentation has also been reported previously even though its production was slightly lower than dark fermentation. Hwang et al. [106] concluded that the photofermentation of Chlorella sp. YSL01 and YSL16 was able to produce biohydrogen under cultivation when exposed to continuous illumination. The study mentioned that O2 concentration played a significant role in affecting the biohydrogen production via this approach. The information generated could give new insights into the development of biomimetic photovoltaic cells using microalgae as suggested. Table 5 summarizes the types of pretreatment, fermentation conditions, and biohydrogen yields based on different microalgae strains.

Table 5: The types of pretreatments, fermentation conditions, and biohydrogen yield based on different microalgae strains.

[Click here to view]


7. CHALLENGES AND FUTURE PROSPECTS

Even though there are many studies on the potential of the microalgae biomass as a feedstock for various products, several challenges exist and limitations arise in the aspect of upstream to downstream stages including cultivations for biomass production and subsequent final product processing.

Manipulation of microalgae cultivation at the upstream stage is important when solving the conflict between biomass production and carbohydrate accumulation. Minimal effort is needed when selecting the most suitable condition to obtain maximum biomass and carbohydrate production. Notably, the growth and carbohydrate accumulation of microalgae are species-dependent and require approximately two weeks to obtain maximum biomass concentration. A comprehensive investigation on cultivation for specific indigenous microalgae regarding biomass production and carbohydrate accumulation is important to ensure the feasibility of biofuel and fine chemical production. This is especially true when using a cheap medium within a short period of time. Cultivation using wastewater and CO2 from flue gas has been suggested as a promising approach to overcome cultivation issues. Further exploration on microalgae cultivation either mixotrophic or heterotrophic methods using cheap carbon sources could indirectly secure energy and food supply in the local industry. For example, by-products from agriculture or chemical industries for indigenous strain need to be well established,

Other common challenges in the production of biofuel and chemicals from microalgae carbohydrate are pretreatment and hydrolysis for sugar extraction prior to the fermentation process. Recently, many pretreatment and hydrolysis technologies have been introduced to extract sugar from microalgal biomass. However, most of the technology involves a two-step process. Utilized and substantial amounts of chemicals such as alkaline and acids as a catalyst during the process are required. The innovation on single pretreatment and hydrolysis steps should be explored to improve the process that could significantly reduce reaction time and energy consumption. This will eventually reduce production costs. Technologies such as supercritical liquid extractions, supercritical CO2 extractions, or ionic liquid on different microalgal species are believed to be explored to establish a database for biofuel and chemical production from microalgal carbohydrates. Recent studies have indicated a low sugar production at high initial feedstock results in low hydrolysis efficiency. Thus, improvement of hydrolysis performance is needed in order to obtain a high sugar level for the fermentation process.

Generally, the production of biofuels and fine chemicals from microalgae carbohydrates involves bioconversion using microorganisms as biocatalysts. At this stage, several challenges and limitations associated with these processes have been identified. For instance, low final product production may result due to the presence of bacterial inhibitors which limit growth. Advance fermentation technology known to reduce inhibitors generated during the fermentation process is needed to improve fermentation performance. A single bioreactor with an extraction apparatus would be beneficial in reducing the reaction process and fermentation performance.


8. CONCLUSIONS

Based on the discussion above, it is clear that microalgal carbohydrates have great potential in the chemical platform for the production of various types of chemicals. High carbohydrate content in microalgal biomass could be promising for biohydrogen, liquid fuels, organic acids, and solvent production. But there are several limitations to the development of the economically feasible microalgal biorefinery. Increasing the carbohydrate yield in the microalgae through various strategies such as cultivating the microalgae using manipulation of the cultivation system under optimum abiotic conditions is essential. Further innovation to push the establishment of affordable and environmentally friendly pretreatment methods is particularly needed to make carbohydrate-based products preferable. Therefore, the development of a systematic approach via the integration of biotechnology and chemical conversion approach is required to ensure the feasibility of microalgal carbohydrate biorefinery.


9. ACKNOWLEDGEMENT

The authors would like to acknowledge the School of Industrial Technology, Universiti Sains Malaysia (USM), for their consistent support to write this review paper. The project and manuscript preparation were financially supported by the RUI Grant 1001/PTEKIND/8011043 and USM Fellowship RU(1001/CIPS/AUPE001) from Universiti Sains Malaysia.


10. AUTHOR CONTRIBUTIONS

All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be an author as per the international committee of medical journal editors (ICMJE) requirements/guidelines.


11. FUNDING

The project and manuscript preparation were financially supported by the RUI Grant 1001/ PTEKIND/8011043 and USM Fellowship RU(1001/CIPS/ AUPE001) from Universiti Sains Malaysia.


12. ETHICAL APPROVALS

This study does not involve experiments on animals or human subjects.


13. CONFLITS OF INTEREST

The authors declare that there are no conflicts of interest.


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