Algal biomass conversion to bioethanol - a step-by-step assessment.

Biotechnology Journal

Biotechnol. J. 2014, 9, 73–86

DOI 10.1002/biot.201200353

www.biotechnology-journal.com

Review

Algal biomass conversion to bioethanol – a step-by-step assessment Razif Harun1, 2, Jason W. S. Yip1, Selvakumar Thiruvenkadam2, Wan A. W. A. K. Ghani2, Tamara Cherrington1 and Michael K. Danquah3 1 Department

of Chemical Engineering, Monash University, Victoria, Australia of Chemical and Environmental Engineering, Universiti Putra Malaysia, Serdang, Malaysia 3 Department of Chemical and Petroleum Engineering, Curtin University of Technology, Sarawak, Malaysia 2 Department

The continuous growth in global population and the ongoing development of countries such as China and India have contributed to a rapid increase in worldwide energy demand. Fossil fuels such as oil and gas are finite resources, and their current rate of consumption cannot be sustained. This, coupled with fossil fuels’ role as pollutants and their contribution to global warming, has led to increased interest in alternative sources of energy production. Bioethanol, presently produced from energy crops, is one such promising alternative future energy source and much research is underway in optimizing its production. The economic and temporal constraints that crop feedstocks pose are the main downfalls in terms of the commercial viability of bioethanol production. As an alternative to crop feedstocks, significant research efforts have been put into utilizing algal biomass as a feedstock for bioethanol production. Whilst the overall process can vary, the conversion of biomass to bioethanol usually contains the following steps: (i) pretreatment of feedstock; (ii) hydrolysis; and (iii) fermentation of bioethanol. This paper reviews different technologies utilized in the pretreatment and fermentation steps, and critically assesses their applicability to bioethanol production from algal biomass. Two different established fermentation routes, single-stage fermentation and two-stage gasification/fermentation processes, are discussed. The viability of algal biomass as an alternative feedstock has been assessed adequately, and further research optimisation must be guided toward the development of cost-effective scalable methods to produce high bioethanol yield under optimum economy.

Received 28 FEB 2013 Revised 18 SEP 2013 Accepted 15 OCT 2013

Keywords: Algae · Bioethanol · Fermentation · Gasification · Hydrolysis

1 Introduction Correspondence: Dr. Razif Harun, Department of Chemical and Environmental Engineering, Universiti Putra Malaysia, 43400 Serdang, Malaysia E-mail: [email protected] Additional correspondence: Prof. Michael K. Danquah, Department of Chemical and Petroleum Engineering, Curtin University of Technology, 98009 Sarawak, Malaysia E-mail: [email protected] Abbreviations: A/F, air-to-fuel ratio; DI, deionized water; ELA, extremely low acid pretreatment; FR, floating residue; FT, Fischer-Tropsch synthesis process; S/F, steam-to-fuel ratio; SHF, separate hydrolysis and fermentation; SSF, simultaneous saccharification and fermentation; WIS, water insoluble solids

© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

The increasing human population has resulted in accelerated universal energy consumption [1], and this has elevated prices in the competitive energy market. Table 1 shows the annual increase in worldwide energy consumption from 2002 to 2012. This has triggered the need to find alternative energy sources such as biofuels. Biofuels have gained immense popularity in recent years as they show significant potential to replace the depleting non-renewable fuels. The long-term growth of research and technological development of biofuels has resulted in four generational classifications [2]. First and second generation biofuels relate to the biofuels that are produced from cellulosic biomass, non-food crops, agricultural

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Table 1. Global energy consumption from 2002–2012 [1]

Year Fuel

2002

2003

2004

2005

2006

2007

2008

2009

2010

2011

2012

Oil (Mbbla)/day) Natural gas (×102 bcmb)) Coal (x102 Mtoe) Nuclear energy (x101 Mtoecc)) Hydroelectricity (x101 Mtoe)

75.0 25.2 24.0 61.0 60.0

77.6 26.2 26.0 60.0 60.0

81.0 27.0 28.0 62.4 64.0

82.0 27.8 30.0 63.0 66.2

82.4 28.8 31.0 64.0 69.0

82.2 25.0 32.1 62.1 70.0

83.0 30.5 33.2 62.0 73.0

81.2 30.0 34.0 61.4 74.0

83.2 32.0 35.4 63.0 78.2

84.2 33.0 38.0 60.0 79.4

86.1 34.0 38.4 56.0 83.1

a) Mbbl, one thousand barrels b) bcm, billion cubic meters c) Mtoe, million tons oil equivalent

wastes, and energy crops while the difference between these two generations lies in the technical aspects. Biofuels from algae are classed as third-generation biofuel, commonly known as algae fuel. Fourth-generation biofuels apply the most advanced technologies and genetically engineered microbial systems. Among the liquid biofuels, bioethanol has reached significant production levels in countries such as the USA and Brazil, attaining 69% and 19.6% of global production respectively (as of 2012) [http://ethanolrfa.org/pages/ World-Fuel-Ethanol-Production]. Bioethanol, or perhaps fuel ethanol, is a biomass-derived, biodegradable, and environmentally friendly fuel produced from different feedstocks such as cellulosic biomass, agricultural waste, and wood waste. Bioethanol is produced from biomass by the fermentation of available carbohydrates, usually simple sugars, into bioethanol and carbon dioxide, via the following chemical process: C n H 2 nOn  sugar  

n n C H OH  CO2  Heat 3 2 5 3

(1)

Most carbohydrate molecules ((CH2O) n) have the potential to produce bioethanol; however, the primary sources for current bioethanol production are sugar, starch, cellulose, and hemicellulose [3]. In addition to bioethanol’s easy storage and distribution, the superior characteristics of bioethanol alone and blended with naturally occurring fossil fuels have made it a highly suitable automobile fuel. Unburned hydrocarbon and carbon monoxide emission levels of bioethanol combustion are significantly low when compared with gasoline combustion [4]. Significant research efforts have been made into the development of various pretreatment techniques (physical, chemical, physiochemical, and biological) with the aim of enhancing bioethanol production from agricultural wastes such as sugarcane bagasse, wheat straw, rice straw, and corn straw [5–10]. Despite extensive research into second generation biofuels, the conventional methods of bioethanol production from food and feed crops in the USA and Brazil have led to socioeconomic and environmental concerns [11]. As these crops are also foodstuffs, there is the potential for rising food prices as food and fuel markets compete for scarce arable land in what is termed the “fuel versus

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food” debate. Further issues include the discrepancy between the amount of land used to grow crops and the actual contribution to the liquid fuel demands of the economy. Lignocellulosic biomass is cheap and plentiful; however, the cost of converting the biomass into ethanol is higher than for other feedstocks [12]. Like other feedstocks, algae undergoes a process consisting of pretreatment, hydrolysis and fermentation to produce bioethanol. Each step contains many variables, many distinctive methods exist, and the optimization of these steps is required to maximize the bioethanol yield. Various processes, namely hydrothermal liquefaction, pyrolysis, fermentation, gasification, transesterification, and anaerobic digestion, produce different biofuels from algal biomass [13]. High-carbohydrate content from holocellulose-based cell walls and starch-based cytoplasm has made algal biomass a suitable feedstock for bioethanol production. During microbial hydrolysis these carbohydrate polymers are broken into simple sugars, followed by fermentation to yield bioethanol [14]. Based on size and morphological characteristics, algae are generally divided into two types: microalgae and macroalgae. The photoautotrophic microalgal species are beneficial as they utilize CO2 from the atmosphere for their growth via photosynthesis and also demonstrate better photosynthetic efficiency than land plants [15]. However, the photosynthetic compounds from algal growth eventually self-decompose releasing CO2 back into the atmosphere. In order to avoid this, the algal biomass should be converted to energy-value products [16]. Starch-rich microalgae are extensively studied for the production of bioethanol via fermentation and different pretreatment methods have been evaluated to release the fermentable sugars from algal biomass in order to enhance bioethanol production [16–18]. Microalga genera such as: (i) Chlorella; (ii) Dunaliella; (iii) Chlamydomonas; (iv) Scenedesmus; (v) Nannochloropsis; and (vi) Spirulina are some of the most abundant organisms on earth, existing in salt or fresh water. The advantages of utilizing microalgae for biofuel production are: (i) quick growth rates; (ii) less water intake compared to conventional crop feedstocks; (iii) high-efficiency; (iv) CO2 mitigation; (v) inexpensive farming; (vi) short harvesting



Biotechnol. J. 2014, 9, 73–86


times; and (vii) a minimal amount of nitrous oxide release. On the other hand, the few disadvantages include high investment costs, and low biomass concentration due to poor efficiency of light utilization or poor cultivation medium [2]. Bioethanol production from algal biomass takes place by either the sugar or syngas pathway. Algae are directly fermented to produce bioethanol by the sugar pathway, while when processed via the syngas pathway, hydrocarbons of algal biomass are converted to syngas through gasification followed by fermentation of syngas to produce bioethanol. To date, many research groups have investigated bioethanol production from microalgae, while there have been comparatively few studies examining the use of macroalgae (e.g. seaweed) for bioethanol fermentation due to the presence of sugars such as mannitol and laminarin in macroalgae [19]. With an immense algal accumulation occuring along the Malaysian coastline, there is a strong urge to recycle this biomass into practicable resources instead of composting it. One possible way of recycling this biomass could be by converting it into bioethanol or biodiesel, an alternative fuel, which benefits both the environment and human society. Being one of the world’s largest producers and exporters of palm oil Malaysia currently produces biofuels that meet 16% of the country’s energy demand, comprising palm oil waste (51%), wood waste (27%), and husk and straw residues (2%) [20]. In this review, previous studies on various bioethanol production strategies from algal feedstocks are explored and briefly summarized.

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nickel, molybdenum, selenium, copper, zinc, boron, manganese and chloride) [22, 23]. NaCl present in salt waters mostly affects the sucrose metabolic pathway, and an immediate salt stress causes accumulation of carbohydrates. Also, high salt stress inhibits protein synthesis through deactivation of ATP-synthase [21]. Temperature remains a crucial factor during the cultivation processes as it impacts carbohydrate accumulation, so must remain constant throughout the process. The scarcity of natural algal growth opened the way for industrial reactors. Open ponds are classic, simple, and cost-effective way to achieve mass cultivation of algae. Other commercial reactors include photobioreactors and closed systems. A commonly engaged open pond is the raceway pond imitating a racetrack, where algae, nutrients and water circulate around it with the help of paddlewheels [2]. However, raceway ponds are not commercially favorable due to high risk of microbial contamination and high water loss from evaporation; albeit this system has advantages, such as easy operation and low energy consumption. Closed photobioreactors are the most commercialized reactors with notable improvements being the maintenance of optimal growth conditions and single-strained algal growth [24]. Xu et al. [25] listed the desirable features for an effective culture system as: (i) maximum light supply, (ii) good gas-liquid mass transfer, (iii) easy to control, (iv) low exposure to contamination, (v) minimum capital and production cost, and (vi) demanding minimal area for cultivation. Different cultivation systems for algae growth and the production of various by-products have been reported [2, 24, 26–28].

2 Algae cultivation techniques Phototrophic, heterotrophic, and mixotrophic are general algal classifications based on culturing methods. Phototrophic algae consume light and CO2 as energy and inorganic carbon source whereas heterotrophic algae utilize the organic substrate as both energy and carbon source. Mixotrophic algae can grow using either the phototrophic or heterotrophic pathway, depending upon the availability of organic carbon sources and light intensity. The phototrophic method of cultivating algae has proven to be technically and commercially feasible. CO2 from flue gases emitted out from power plants and heavy industries can be utilized for algal growth, contributing to biological fixation of CO2 [15]. This is an added benefit of phototrophic and mixotrophic culture systems. Nitrogen, carbon and phosphorus are macronutrients that nourish the algae. Rendering CO2 as an inorganic carbon source also controls pH and the level of CO2 localizes intracellular starch accumulation in the algal cells, to either the stroma or pyrenoid [21]. Generally municipal and agro-industrial wastes and wastewaters contain nitrogen, carbon and phosphorus in sufficient quantities for algal growth. Algal cultures also rely on micronutrients such as sulfur, magnesium, calcium, and traces of minerals (iron, cobalt,


3 Pretreatment and saccharification of algal biomass Direct fermentation of algal biomass from complex carbohydrates to bioethanol consists of four steps: (i) pretreatment; (ii) saccharification; (iii) fermentation; and (iv) product recovery. Algae provide an interesting option and unlike other biomass such as wheatgrass and bagasse, contain no lignin, hence the removal of lignin is unnecessary. Lignin removal is actually a rate-limiting step for other feedstocks, hence its absence reduces the costs, time, and difficulty of the conversion process [29]. The fermentative performance for bioethanol production strongly depends on the pretreatment and saccharification conditions of the algal biomass. Under optimized conditions of pretreatment and saccharification, cell wall-bound carbohydrates become accessible for hydrolysis and thereafter are converted into simple soluble fermentable sugars by saccharification. However, non-optimal pretreatment and saccharification conditions can further degrade fermentable sugars to undesirable products such as formic acid, acetic acid, and some furanic compounds [30–32]. Algal carbohydrates comprise a mixture of neutral sugars,

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amino sugars, and uronic acids and the composition differs depending on species and growth conditions [33]. Hence, an effective pretreatment must aim to enhance bioethanol production by improving the carbohydrate accessibility and consequently accelerating the rate of fermentation. Five basic points for evaluating pretreatment techniques are: (i) determination of sugar and carbohydrate content in the liquid- and solid-fractions respectively after filtering pretreated samples; (ii) enzymatic hydrolysis of the pretreated sample; (iii) screening of pretreated samples for inhibitory effects and neutralizing them prior to fermentation; (iv) selecting bioethanol source from either liquid hydrolyzate or WIS (water insoluble solids), after sugar and carbohydrate analyses; (v) assessing pretreated samples for producing other value-added products [31, 34]. Cellulose is the main constituent of algal cell walls and is derived from β-D-glucopyranose units, which condense through β-1,4-glycosidic bonds forming a crystalline structure [35]. The hydrolysis rate of completely disordered crystalline or amorphous cellulose is higher than partially disordered structure, which shows that the initial degree of crystallinity is a crucial element during the pretreatment process. In recent years, many studies have been performed on various pretreatment techniques to improve bioethanol fermentation. Generally, pretreatment is intended to disrupt cells releasing complex carbohydrates, while saccharification is intended to split complex carbohydrates into their monosaccharide components. Saccharification is considered a two-step process involving a pretreatment technique (either physical or chemical or both) followed by enzymatic hydrolysis.

3.1 Chemical pretreatment of algal biomass Extremely low acid (ELA) pretreatment is a widely studied technique, [31] in which the acid pretreatment destroys the algal cell wall by breaking the intra- and inter-molecular hydrogen bonds; further dilution of the acid releases carbohydrates to the liquid hydrolyzate [36]. Maximum sugar recovery during ELA pretreatment has been achieved by combinational optimization of three parameters namely: (i) pretreatment time; (ii) temperature; and (iii) acid concentration [36]. The effect of temperature (ranging from 50 to 180°C) on ELA pretreatment (0.10% H2SO4 for 20 min) was reported by Lee et al. [31] and the maximum glucan content (32%) was obtained at 170°C with brown macroalgae Laminaria japonica. Further studies by Lee et al. [32] with similar ELA pretreatment (0.06% H2SO4 at 170°C for 20 min) on macroalgae Saccharina japonica showed an increase in the glucan content and enzyme digestibility of ELA-treated S. japonica by 4- and 2-fold respectively compared to untreated algae [32]. Zhou et al. [36] noted the decomposition of glu-

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can to formic acid via glucose at elevated temperatures and observed the formation of degraded products. In some cases, degraded products in pretreated hydrolyzate are removed prior to fermentation to avoid process inhibition. Activated charcoal was used to remove hydroxymethyl furfural after subjecting Kappaphycus alvarezii to dilute acid pretreatment [30]. In addition to these degraded products, fermentation inhibitors are produced during pretreatment by the process of neutralization. High concentrations of Na2SO4 generated during sulfuric acid neutralization are observed to inhibit fermentation of Ulva reticulate [37]. To avoid this pretreatment, at low acid concentration could be considered since pretreated samples are hydrolyzed directly without a neutralization step. At high acid concentrations sugar recovery is not affected, and instead remains constant after an optimal acid concentration with an increase in the concentration of degraded products. However, a contrary result was observed with Undaria pinnatifida, which produced only monosaccharide sugars at high temperatures during dilute acid hydrolysis [38]. Alkaline pretreatment for algal biomass was first explored by Harun et al. [10] and showed a maximum glucose yield at optimum conditions of 0.75% NaOH at 120°C for 30 min, showing the effect of NaOH in splitting intermolecular bonds of hemicelluloses with other polymeric components. Alkaline pretreatment of the green alga Ulva lactuca showed contrasting results as gelling during alkaline pretreatment (0.05–0.2 N Ca(OH)2 at 121°C for 15 min) occurred, dismissing this pretreatment option for this algal species [39].

3.2 Mechanical pretreatment of algal biomass Physical pretreatments apply mechanical force to disrupt the cell wall using technologies such as ultrasonication, milling, supercritical CO2 exposure, and extraction. When studying microalgae as a carbohydrate feedstock for yeast fermentation, Zhao et al. [40] discovered the ultrasonic assisted extraction (UAE) method to be more effective for algal biomass as compared with conventional solvent extraction (CSE) and fluidized bed extraction (FBE) methods. When treating microalgae with ultrasonic waves, repetitive compression and rarefaction of the waves cause cavities or microbubbles, which finally collapse to generate a mechanical shear force, thereby disrupting the cell wall and membranes [41]. Ultrasonication pretreatment on Scenedesmus obliquus (YSW15) gave similar findings, the resulting cell damage allowing carbohydrates from the cell interior to diffuse to the cell surface and/or within the periplasmic membrane [42]. Yoon et al. [43] proposed using gamma radiation to weaken the cell wall because high-activity radicals are produced through chain scission reactions during algal biomass irradiation, and the release of soluble organic compounds influences the saccharification step. Furthermore, gamma radiation was shown to improve the saccharification





Table 2. Various hydrolysis treatments methods and their bioethanol yields

Hydrolysis type

Hydrolysis source

Acid

HCl/ MgCl2

SHF

Chlorella sp.

Micro

0.47

[36]

Alkaline

NaOH

SHF

Chlorococcum infusionum

Micro

0.261

[10]

Chemical

H2SO4

SHF

Chlorococcum humicola

Micro

0.48

[9]

Chemicalb)

H2SO4

SHF

Chlorella vulgaris

Micro

0.233

[61]

Chemoenzymaticc)

HCl/ H2SO4 + amyloglucosidase + endocellulase + β-glucosidase

SHF

Dunaliella tertiolecta

Micro

0.14

[46]

Enzymatic

α-amylase + amyloglucosidase

SHF

Chlamydomonas reinhardtii

Micro

0.235

[18]

Enzymatic

endoglucanase + β-glucanase + amyloglucosidase

SSF

Laminaria japonica

Macro

0.196

[38]

Enzymaticb)

cellulase + amylase

SHF

C. vulgaris

Micro

0.178

[61]

Enzymaticd)

cellulase + β-glucosidase

SHF

Gracilaria verrucosa

Macro

0.43

[14]

Enzymatice)

cellulase + β-glucosidase

SSF

Saccharina japonica

Macro

0.111

[31]

Enzymaticb)

cellulase + Amylase

SSF

C. vulgaris

Micro

0.214

[61]

Physicalc)

supercritical CO2

SHF

Chlorococum sp.

Micro

0.383

[45]

a) b) c) d) e)

Fermentation Modea)

Algae species

Algae type

Yield Reference (g ethanol/g algae)

SHF: separate hydrolysis and fermentation; SSF: simultaneous saccharification and fermentation Sonicated algal biomass was utilized Lipid-extracted algal biomass was utilized Agar pulp was extracted after alkali treatment and hydrolyzed Algal biomass received extremely low acid pretreatment.

yield in addition to increasing the concentration of reducing sugars. This pretreatment could be effective for the saccharification of high lignin biomass. Hydrothermal pretreatment fractionates the algal biomass into lipid and sugar phases, where it is further processed for biodiesel and bioethanol production respectively. Response surface methodology (RSM) was designed to obtain optimal hydrothermal conditions. Maximum fractionation was observed at a reaction temperature of 115.5°C, reaction time of 46.7 min and solid loading of 25% (w/w) for the microalgae Schizocytrium sp. [44]. Supercritical fluid exposure is an alternative pretreatment technique. Supercritical CO2 treatment was employed to obtain lipidextracted biomass of Chlorococum sp., with comparative studies showing lipid-extracted biomass producing 60% more bioethanol than the corresponding intact algal biomass [45].

3.3 Enzymatic pretreatment of algal biomass Choi et al. [18] described the liquefaction of starch that occurs during treatment of Chlamydomonas reinhardtii (UTEX90). The commercially available hydrolytic enzymes α-amylase and amyloglucosidase were employed to perform liquefaction and saccharification, respectively. Even though bioethanol yield was low compared with yield obtained using acid pretreatment, α-amylase disrupted the cell wall completely, releasing all carbohydrates with-


out the need for any further pretreatment. Maximum enzyme degradation activity was achieved by optimizing enzymatic hydrolysis reactions at several temperature and pH conditions and for a different residence time period. These physicochemical parameters have been investigated by Lee et al. [38] and they found the optimum enzymatic conditions of 45°C, pH 4.6, and 60 min for extraction of glucose from C. reinhardtii and U. pinnatifida. While documenting ethanol production from K. alvarezii, Hargreaves et al. [30] reported the adverse effect of enzymatic hydrolysis efficiency and the effect of glucose yield over solid concentration. Harun and Danquah [8] studied the kinetics of enzymatic hydrolysis and the effect of hydrolysis conditions on kinetic parameters. Table 2 summarizes various hydrolysis treatments and the bioethanol yields reported in various studies. Similar to the algal bioethanol process, a new conceptual “zero-waste” biorefinery algal system has been developed and this concept proposes the production of bioethanol from remaining algal residual pulp waste after production of high-value products such as biodiesel, agar, and phycocolloids. The conversion of defatted or lipidextracted microalgae biomass to bioethanol could be more economically attractive than direct conversion of microalgae to bioethanol. The residual pulps of Dunaliella tertiolecta and Gracilaria verrucosa, which remain after biodiesel and agar production respectively, were investigated for bioethanol production [14, 46]. Earlier acid

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Biotechnol. J. 2014, 9, 73–86



hydrolysis of agar, a main constituent of red algae, was found to produce high carbohydrate content during acid pretreatment (9.8% HCl and 11.6% H2SO4) of Gelidium amansii [39]. Similarly, marine algae processing waste, such as cellulose-rich floating residue (FR) from the alginate extraction process, produced maximum fermentable sugars from dilute acid pretreatment (0.1% H2SO4 at 121°C for 60 min) [47].

monly operated by two methods, namely SHF and SSF. A complete assessment of the fermentation process is generally based on cell growth, reducing sugar consumption and bioethanol production profiles [46]. Environmental and operational factors such as (i) nutrient levels; (ii) alkalinity; (iii) concentration of toxic substances; (iv) temperature; and (v) pH optimum of the fermenting microorganism greatly influence bioethanol generation from algal biomass [50].

4 Single-stage fermentation process

4.1 Microbial fermentation of algal biomass

Various fermentation paths commonly utilized to convert algal biomass into bioethanol are shown in Figure 1, based on Taherzadeh and Karimi [48]. The compositional analysis of the feedstock determines which process is the best option; however in general the following steps occur: (i) biomass pretreatment to rupture the cell walls; (ii) enzymatic hydrolysis of the cellulosic components to form simple sugars, and finally (iii) alcoholic fermentation of simple sugars to produce bioethanol. Depending on which combination of steps occurs, the processes are denoted as follows [48, 49]: (i) separate hydrolysis and fermentation (SHF), in which the hydrolysis of pretreated biomass and fermentation happen in two different units; (ii) separate hydrolysis and co-fermentation (SHCF), in which both the hemicellulose sugars (pentose) and cellulose sugars (hexose) are fermented simultaneously after a separate hydrolysis; (iii) simultaneous saccharification and fermentation (SSF), in which cellulosic hydrolysis and fermentation happen together in a single reactor; (iv) simultaneous saccharification and co-fermentation (SSCF), in which both hemicelluloses and cellulose are hydrolyzed and fermented simultaneously; and (v) consolidated bioprocessing (CBP), in which enzyme production, hydrolysis, and fermentation of all sugars occurs in a single unit. Singlestage or direct fermentation of algal feedstock is com-

One of the biggest hurdles for effective fermentation is the inability of commonly used microorganisms to convert hemicellulosic sugars (xylose and arabinose) into bioethanol. To achieve economically feasible bioethanol production, all potential substrates (i.e. glucose and mannose from cellulose, and xylose and arabinose from hemicellulose) must be utilized. Naturally occurring microorganisms that convert xylose (the primary pentose from hemicellulose) into bioethanol do exist and include species of bacteria, fungi, and yeasts. However, each microorganism has its own disadvantages, which make it unsuitable for industrial use, as outlined in Table 3. In order to achieve high levels of bioethanol production it is essential to carry out fermentation with suitable bioethanol producers (fermenting organisms) since each producer has a very narrow substrate range. Many organisms exhibit different degrees of fermentation but their poor tolerance to ethanol accumulation prevents their application to largescale production systems. These limitations resulted in the development of recombinant strains tolerant to high ethanol concentrations and capable of effectively producing bioethanol by metabolizing various sugars. Glucose fermentation to bioethanol is generally performed by Saccharomyces cerevisiae, a species commonly utilized in industrial fermentation. S. cerevisiae is also capable of

Figure 1. Various routes for algae biomass – bioethanol processing.

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Table 3. Advantages and disadvantages of various natural microorganisms regarding industrial ethanol production. Adapted from [98] with permission.

Organism

Anaerobic bacteria Escherichia coli Zymomonas mobilis Saccharomyces cerevisiae Pichia stipitis Filamentous fungi a) b) c) d)

Natural sugar utilization pathwaysa)

Major productsb)

Glu

Man

Gal

Xyl

Ara

EtOH

Other

+ + + + + +

+ + – + + +

+ + – + + +

+ + – – + +

+ + – – + +

+ – + + + +

+ + – – – –

Tolerancec)

O2 neededd) pH

Alcohols Acids Hydrolysate – – + ++ – ++

– – – ++ – ++

– – – ++ – ++

– – – – + –

Neutral Neutral Neutral Acidic Acidic Acidic

+: Fermentation possible; –: Fermentation not possible +: Major product(s); –: Minor product(s) ++: High tolerance; +: Moderate tolerance; –: Poor tolerance +: O2 needed; –: O2 not needed

fermenting galactose [51]. Scholz et al. [52] observed the changes in S. cerevisiae physiology over the fermentation period and noted the nonlinear relationship between algal biomass and turbidity. This nonlinearity of growth after glucose consumption was due to changes in cell physiology and/or accumulation of secondary substrates [52]. During G. amansii fermentation, Brettanomyces custersii (KCCM11490) is preferred as the bioethanol producer over S. cerevisiae [53]. B. custersii (KCCM11490) produces high bioethanol yields from galactose-rich hydrolyzate of G. amansii, since it ferments galactose more rapidly than other sugars present in the hydrolyzate [53]. Bioethanol production from galactose involves three biochemical pathways: (i) the D-galactose-6-phosphate pathway; (ii) the Leloir pathway; and (iii) the Entner-Deudoroff pathway [51]. When compared with anaerobic fermentations of Enterobacter sp. (JMP3), the aerobic process showed a higher microbial population and Enterobacter sp. (JMP3) consumed more glucose than mannitol [54]. Nevertheless, the highest bioethanol yield was observed during anaerobic conditions in which mannitol was used as a sole carbon source. Mannitol fermentation is effectively accomplished by Escherichia coli (KO11) [39]. In specific cases researchers have utilized two different microorganisms to ferment various saccharified products to produce bioethanol. S. cerevisiae and E. coli (KO11) were used sequentially to ferment a L. japonica hydrolyzate [39]. The sequential culturing strategy proved to be highly beneficial in terms of bioethanol yield, but there was no difference observed in the bioethanol production rate of E. coli (KO11) fermented alone and S. cerevisiae/E. coli (KO11) co-fermentation. Another group of researchers investigated the impact of culturing four different yeasts (Pichia angophorae [KCTC 17574], Pichia stipitis [KCTC 7228], S. cerevisiae [KCCM 1129] and Pachysolen tannophilus [KCTC 7937]) with Bacillus sp. (JS1) for ethanol production from SSF of S. japonica [55]. In this SSF, Bacillus licheniformis acts as a saccharifying agent while the yeasts performed the fermentation. P. angophorae yielded the highest ethanol concentration of the yeasts. After concentrating FR, a by-product of algi-


nate extraction process, by rotary evaporator, Ge et al. [47] tested the difference between fermentations of FR hydrolyzate and concentrated FR hydrolyzate. The results showed no significant difference in ethanol yield. The glycolytic pathway, a pathway common to virtually all organisms, describes the oxidation of glucose to form pyruvate along with NADH and ATP. In the absence of oxygen, pyruvate is directed towards two-step alcoholic fermentation to yield ethanol and CO2. During fermentation pyruvate is catalyzed by pyruvate decarboxylase to form acetaldehyde, then acetaldehyde is reduced by alcohol dehydrogenase to yield ethanol [56]. This conversion of pyruvate to ethanol can also be affected by intracellular NADH/NAD+ ratio [54]. E. coli, a well-characterized microorganism for various genetic manipulations, was engineered by introducing Zymomonas mobilis pdc (pyruvate decarboxylase) and adhB (alcohol dehydrogenase II) genes to form an ethanologenic strain, E. coli (KO11) [57]. Ethanol production was studied in an engineered mutant C. reinhardtii strain (cw15) and the maximum ethanol production rate, μmax 14 ± 1 mL/g h was derived by following Michaelis–Menten kinetics [52]. With advancements in recombinant DNA technology, genetic manipulation of marine algal species can be achieved by various strategies namely: (i) trans-conjugation; (ii) natural transformation; (iii) induced transformation; (iv) electroporation; (v) biolistic transformation; (vi) glass beads; (vii) silicon carbon whiskers method; (viii) microinjection; (ix) artificial transposon method; (x) recombinant eukaryotic algal viruses; and (xi) Agrobacterium tumefaciens mediated genetic transformation [58]. The study of the recovery of bioethanol, produced by SSF using pretreated S. japonica, over different fermentation suspensions such as: (i) sodium citrate buffer; (ii) deionized water (DI); and (iii) liquid hydrolyzate showed no significant differences [32]. Furthermore, S. japonica suspended in DI is found to be an economical and effective way to recover bioethanol from the treated biomass. The primary microorganism, S. cerevisiae (DK410362), consumes the glucan in DI-suspended

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Table 4. Bioethanol production from SSF and SHF tested on various algal strains

Fermentation Algal feedstock Type

Hydrolysis

Separate Hydrolysis and Fermentation (SHF)

Simultaneous Saccharification and Fermentation (SSF)

Source

Bioethanol Yield

Reference

Treatment conditions

Source

Process conditions

Chlamydomonas Glutase fasciata

40°C for 30 min

Saccharomyces cerevisiae

100 rpm and 40°C for 30 h

0.194 g ethanol/g algae

[99]

Chlorella vulgaris Cellulase + Amylase

200 rpm and 45°C

Zymomonas mobilis

30°C in desktop fermentation


[61]

Schizocytrium sp. Amylase

37°C at 150 rpm for 24 h

Escherichia coli

150 rpm and 37°C


[44]

Laminaria japonica

Sulfuric acid

121°C for 15 min

E. coli

150 rpm and 37°C

0.4 g ethanol/g carbohydrate

[39]

Saccharina japonica

Bacillus licheniformis

200 rpm and 30°C for 7.5 days

Pichia angophorae


7.7 g ethanol/ L algae hydrolysate

[55]

C. vulgaris

Cellulase + Amylase

200 rpm and 45°C Z. mobilis



[61]

C. vulgaris

Sulfuric acid

121°C for 20 min.

Z. mobilis



[61]

Dunaliella tertiolecta

HCl/H2SO4 + cellulase + amyloglucosidase

121°C for 15 min

S. cerevisiae



[46]

Gelidium amansii

Sulfuric acid

150°C and 3.0– 3.5 bar pressure

Brettanomyces custersii

150 rpm and 30°C

27.6 g ethanol/ L algae hydrolysate

[53]

Scenedesmus abundans

Cellulase

37°C for 30 min

S. cerevisiae


0.103 g ethanol/ g algae

[60]

L. japonica

Cellulase + Cellubiose


S. cerevisiae

30°C for 36 h

0.143 L ethanol/ kg algae

[47]

S. japonica to form bioethanol and thereafter the remaining DI-suspended algal biomass is available for the production of other bioproducts from the left over carbohydrates, such as mannitol and alginate. Various microorganisms namely Clostridium sp. [32], Enterobacter sp. (JMP3) [54], E. coli (KO11) [39] are able to ferment mannitol to bioethanol while Vibrio sp. produce bioethanol from alginate.

4.2 Microbial fermentation conditions of algal biomass Temperature affects microbial activity and can repress bioethanol production. In the case of S. japonica, thermal stress at higher temperatures (>46°C) result in metabolically inactive yeast cells [32]. WIS-loading level also affects bioethanol production. Bioethanol production doubled in pretreated S. japonica with an increase in WIS loading from 3% to 6% but further increase in WIS loading above 6% resulted in a decrease in bioethanol production. possibly due to inefficient mixing and enzyme deactiva-

80

Fermentation

tion. The option of increasing WIS loading is limited in algal species with high hygroscopic properties. Concerning cultivation strategies, continuous reactors have been reported to show better performance, in terms of bioethanol production, than batch reactors. Fermentation of water hyacinth by S. cerevisiae demonstrated that bioethanol production during continuous mode is 1.5 times higher than in batch reactors [59]. Bioethanol produced from hydrolyzate of G. amansii in continuous mode was increased by more than 50 times compared to the production found in batch reactors [53]. This huge increase is attributed to the accumulation of inhibitory compounds that occurs during the course of the fermentation process in the batch system, with ten times greater levels of inhibitory compounds reported during the batch mode compared to the continuous mode. Although microbes in batch reactors metabolize organic acids when fermentable sugars are exhausted completely this phenomenon is absent in continuous reactors because of the continuous delivery of reducing sugars. This phenomenon was inferred from the steep decrease in con-



Biotechnol. J. 2014, 9, 73–86


centration of formic and levulinic acid after 32 h cultivation in batch reactors of G. amansii hydrolysate. Different methods of hydrolysis pretreatments influence the overall bioethanol production from an algal species as inferred from the studies of three different pretreatments methods (H2SO4, H2SO4 + amylases,, H2SO4 + cellulases) on microalga Mychonastes afer (PKUAC 9) and Scenedesmus abundans (PKUAC 12) [60]. The pH value is another influential fermentation parameter. Prior to fermentation with Z. mobilis, the hydrolyzate pH of Chlorella vulgaris was adjusted from 5.0 to 6.0 by adding CaCO3 [61]. KOH can also be used to maintain pH 6.0 [50]. The predominant sugars in the hydrolyzate after saccharification determine which fermenting microorganisms are optimal for culturing during fermentation. Comparative studies on bioethanol yield from yeast Yarrowia lipolytica displayed higher yields from lipid-extracted biomass compared to lipid-rich biomass, indicating the formation of reducing sugars is inhibited by algal lipids [50]. This is in contrast to findings made on Schizocytrium sp. where the presence of lipids had no effect on bioethanol fermentation using E. coli (KO11) [44]. The addition of lactose can also be a viable solution to enhance bioethanol production. On fermentation studies with Spirogyra sp. biomass saccharified with the mycelial mat of Aspergillus niger, cellulose activity was triggered by the addition of lactose, resulting in enhanced bioethanol production [52]. Even ethanol concentrations as low as 30 g/L have been found to restrict microbial growth. Restricted microbial growth can be activated by employing ethanol recovery through adsorption by adding activated carbon, or adding solid polymeric material for microbial encapsulation. Jones et al. [57] have demonstrated the application of Ca-Alginate beads to encapsulate microorganisms upholding ethanol production post adsorption. A major problem during the course of adsorption is declining pH in the flasks and this can be controlled by adding NaOH or KOH. Another disadvantage is the prolonged fermentation time required due to the buffering of carboxyl groups present in the encapsulating alginate polymer matrix. Previous studies on SSF and SHF tested on various algal strains are listed in Table 4.

5 Two stage gasification/ fermentation processes 5.1 Gasification of algal biomass Gasification is a chemical process where biomass is subjected to a high temperature range (700–1000°C) to produce a combustible gas mixture. The product gas mixture is generally referred as syngas, or synthetic gas, which usually consist of carbon monoxide, hydrogen, carbon dioxide, minor amounts of methane, trace gases. This



process uses a gasifying agent such as: air, hydrogen, oxygen, carbon dioxide, steam, or their mixtures. Production of high-quality syngas is mostly engineered by utilizing proper gasifying agent(s) [62]. Gasification remains as a most promising technique for recovering energy from various waste materials. Gasifiers are classified under two categories: fixed bed and fluidized bed gasifiers. A compatible gasifier is selected based on feedstock type, air flow in fuel column, and the kind of combustion bed [63]. Researchers have widely reported the gasification process of various materials, namely waste rubber [64], oil palm wastes [65–67], oil refinery sludge [68], wastewater sludge [69], co-gasification of polyethylene, and woodchips [70]. Aside from the above mentioned waste materials, some additional progress has been made in the gasification of algae such as direct gasification, catalytic gasification, hydrothermal gasification, and steam gasification of algae to gaseous products [71–73]. Unlike terrestrial biomass; the absence of lignin in algae makes them less resistant, which is beneficial for the gasification process [74]. Gasification reaction rates mainly rely on biomass type and the operating conditions, namely particle size, char porosity, and mineral content, and the temperature and pressure of gasifying agents [75]. Sanchez–Silva et al. [13] reported the effect of: (i) temperature; (ii) initial sample weight; (iii) particle size; (iv) steam concentration; and (v) sweep gas flow on gasification of Nannochloropsis gaditana microalgae in a bubbler system setup. Several algal feedstocks processed for SSF gasification and wet gasification with the combined-cycle gas turbine (CCGT) proved algae can be an efficient feedstock with high conversion rates [76]. Erkiaga et al. [77] investigated the use of in situ catalyst (olivine and γ-Al2O3) for syngas production with reduced tar formation (

Evolutionary engineering of Saccharomyces cerevisiae for efficient conversion of red algal biosugars to bioethanol.

An overview of key pretreatment processes for biological conversion of lignocellulosic biomass to bioethanol.

Biotechnological Strategies to Improve Plant Biomass Quality for Bioethanol Production.

Miscanthus as cellulosic biomass for bioethanol production.

Algal biomass analysis by laser-based analytical techniques--a review.

Hydrogen from algal biomass: A review of production process.

Bioethanol from lignocellulosic biomass: current findings determine research priorities.

Algal biomass production and wastewater treatment in high rate algal ponds receiving disinfected effluent.

Conversion of lignocellulosic biomass to nanocellulose: structure and chemical process.

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Efficient conversion of solar energy to biomass and electricity.

Ultrasound pretreatment of filamentous algal biomass for enhanced biogas production.

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Thermochemical conversion of microalgal biomass into biofuels: a review.

Comparing new and conventional methods to estimate benthic algal biomass and composition in freshwaters.

Erratum to: A novel solid state fermentation coupled with gas stripping enhancing the sweet sorghum stalk conversion performance for bioethanol.

Investigation of a novel acid-catalyzed ionic liquid pretreatment method to improve biomass enzymatic hydrolysis conversion.

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Study of algal biomass harvesting through cationic cassia gum, a natural plant based biopolymer.

The influence of light intensity and photoperiod on duckweed biomass and starch accumulation for bioethanol production.

Particulate size of microalgal biomass affects hydrolysate properties and bioethanol concentration.