Accepted Manuscript

Current advances on fermentative biobutanol production using

third generation feedstock

Yue Wang, Shih-Hsin Ho, Hong-Wei Yen, Dillirani Nagarajan,

Nan-Qi Ren, Shuangfei Li, Zhangli Hu, Duu-Jong Lee, Akihiko
Kondo, Jo-Shu Chang

PII: S0734-9750(17)30060-5
DOI: doi: 10.1016/j.biotechadv.2017.06.001
Reference: JBA 7129
To appear in: Biotechnology Advances
Received date: 1 March 2017
Revised date: 8 May 2017
Accepted date: 1 June 2017

Please cite this article as: Yue Wang, Shih-Hsin Ho, Hong-Wei Yen, Dillirani Nagarajan,
Nan-Qi Ren, Shuangfei Li, Zhangli Hu, Duu-Jong Lee, Akihiko Kondo, Jo-Shu Chang ,
Current advances on fermentative biobutanol production using third generation feedstock,
Biotechnology Advances (2017), doi: 10.1016/j.biotechadv.2017.06.001

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 ACCEPTED MANUSCRIPT

A revised manuscript (JBA-D-17-00076R1) submitted to Biotechnology Advance

Current advances on fermentative biobutanol production using third

generation feedstock

Yue Wang1,2,3, Shih-Hsin Ho3, Hong-Wei Yen4, Dillirani Nagarajan5,6, Nan-Qi Ren3,

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Shuangfei Li1,2, Zhangli Hu1,2, Duu-Jong Lee3,6, Akihiko Kondo7,8,9, Jo-Shu Chang3,5,10*

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1
College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, China
2
Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and

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Guangdong Province, College of Optoelectronic Engineering, Shenzhen University,
Shenzhen, China
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3
State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of
Technology, Harbin, China
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4
Department of Chemical and Material Engineering, Tunghai University, Taichung, Taiwan
5
Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan
6
Department of Chemical Engineering, National Taiwan University, Taipei, Taiwan
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7
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe
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University, 1-1 Rokkodai, Nada-ku, Kobe 657-8501, Japan

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8
Core Research for Evolutional Science and Technology, Japan Science and Technology
Agency, 3-5 Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan
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9
Biomass Engineering Program, RIKEN, 1-7-22 Suehiro, Tsurumi-ku, Yokohama, Japan
10
Research Center for Energy Technology and Strategy, National Cheng Kung University,
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Tainan 701, Taiwan

*
Corresponding author: Professor Jo-Shu Chang; e-mail: changjs@mail.ncku.edu.tw

JBA-D-17-00076R1 (Revised manuscript)-clear.doc 6/1/2017

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Abstract

Biobutanol is gaining more attention as a potential alternative to ethanol, and the

demand for fermentative biobutanol production has renewed interest. The main challenge

faced in biobutanol production is the availability of feedstock. Using conventional

agricultural biomass as feedstock is controversial and less efficient, while microalgae, the

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third generation feedstock, are considered promising feedstock for biobutanol production

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due to their high growth rate and high carbohydrates content. This review is primarily

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focused on biobutanol production by using carbohydrate-rich microalgal feedstock. Key

technologies and challenges involved in producing butanol from microalgae are discussed
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in detail and future directions are also presented.
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Keywords: Microalgae, Biobutanol, ABE fermentation, Biomass pretreatment,

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Immobilized cells, microalgae-based carbohydrates

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1. Introduction

Currently, the renewable biofuels market is valued at $26 billion, and is dominated by

bioethanol. Biodiesel and bioethanol are extensively used as fuel additives by the transport

sector in the U.S and Europe, and China (European Biofuels Technology Platform, 2011).

Among the potential liquid biofuels, biobutanol is especially promising due to its superior

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physical and chemical properties. Biobutanol is touted as the only “drop-in” biofuel that can

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be readily blended with gasoline at a content of up to 85%. As estimated by Nexant and

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Chemical strategies, the global butanol market was US$ 5.9 billion in 2011 and is expected

to reach US$ 9.9 billion by 2020 (Fig. 1). Asia accounts for about 47% of the global butanol
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market, with China being the major consumer (34%). The European Union and North
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America account for about 25% and 24% of the market, respectively. Butanol is used as an

alternative energy source in the US, Western Europe, and Japan, accounting for 33%, 19%,
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and 8% of their total energy supplement. Biobutanol blends up to 16% are currently allowed
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in the US and 15% is allowed in Europe. Butanol has low volatility and a lower heat of
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vaporization than ethanol, as well as a higher viscosity, and can potentially replace ethanol
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as a gasoline additive (Qureshi et al., 2001). Butanol fits perfectly into the existing oil
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infrastructure for inbound and outbound transport, because of its non-corrosive and less

hygroscopic nature. Its high octane content makes it energy dense (with a level about 82%

of that of gasoline).

Biological acetone-biobutanol-ethanol (ABE) fermentation has been widely

investigated for its potential to replace chemical synthesis for butanol production (Kumar

and Gayen, 2011; Qureshi et al., 2001; Xue et al., 2013; Xue et al., 2017b). The commercial

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production of renewable biobutanol from cellulosic and non-cellulosic feedstock has been

investigated over the past decade. Currently, there are four leading biobutanol producers in

the world, including Butamax, Gevo, US technology Corporation, and Green Biologics. As

of July 2015, Gevo started retailing butanol blended gasoline in North America, and is

aiming to capture the marine fuel market (Gevo press release, 2015). Moreover, a joint

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venture of DuPont and Bio Architecture Lab, Inc. invested US$8.8 million for research and

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development of seaweed biomass based butanol production. The cost for butanol production

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using fossil fuel is proximately 1.35 dollar/L, and is associated with the price of crude oil.

The price of butanol produced from crops by ABE fermentation is 0.317 dollar/L, which is
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competitive with the price of 0.338 dollar/L for bioethanol (Pfromm et al., 2010).
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Although biobutanol production via the ABE route has many attractive properties,

commercialization of ABE fermentation also faces severe problems (Jang et al., 2012;
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Tashiro et al., 2013). The first challenge of using biobutanol would be the relatively high
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costs of feedstock used for biobutanol production. For example, consumption of corn for
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biobutanol production will increase the cost of corn as it is edible biomass, ultimately
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resulting in the increase of butanol price. Use of lignocellulosic biomass (agricultural waste
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or plant biomass like switch grass) or microalgal biomass cultivated in wastewater as a

fermentation feedstock may have the potential to reduce the price of butanol. The second

major problem associated with the commercial application of biobutanol would be the low

butanol production efficiency of the Clostridial species. This is mainly due to the low

butanol concentration in the fermentation medium (<20 g/L) caused by product inhibition

on the butanol-producing bacteria. This would eventually lead to a low biobutanol

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productivity (<0.3 g/L/h) due to low cell concentration and a low ABE yield (0.28-0.33 g/g)

due to the hetero-fermentation pathway. Finally, energy intensive biobutanol recovery

process is also a major obstacle hindering the use of biobutanol as a liquid fuel.

Typically, biofuels can be produced by using crops or microalgae as the feedstock

(Bellido et al., 2014; Milledge and Heaven, 2014). The first generation feedstock for biofuel

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production were oil seeds and crops rich in starch, like corn, maize and sugarcane. However,

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this proved to be expensive, since it competes with food crops for arable land and the related

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resources. The second generation feedstock, based on forest- and waste-based cellulosic and

lignocellulosic biomass, is now extensively used in ethanol production. These approaches

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were much more successful commercially, but again suffered from high-cost and poor
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sustainability (Chen et al., 2013b). Microalgal biomass emerged as a promising third

generation feedstock for biofuel production. Microalgae, like terrestrial crops, can be used
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for direct energy production by lipid extraction, or as a feedstock for other biofuel
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production via the fermentation process (Lakaniemi et al., 2013). Microalgae have higher
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photosynthetic efficiency and can grow much faster compared to terrestrial plants.
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Microalgae can be cultivated in seawater and wastewater, so they do not compete for
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terrestrial resources with conventional agriculture. Microalgae consist of a wide range of

autotrophic organisms, which grow through photosynthesis, just like land-based plants

(Harun et al., 2010). Moreover, microalgae can mitigate CO2 emissions, and especially

provide valuable microalgal components (such as carbohydrates and glycerol) for

fermentative bacteria. Although microalgae-based biobutanol has great potential to replace

petroleum, there are no commercial microalgae-to-butanol technologies that can overcome

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the related technical and economic barriers and thus achieve sustainability (Zhou et al.,

2014). The main obstacle for commercialization of biobutanol production from microalgal

biomass is the high capital and operating costs for microalgae cultivation. If the microalgae

for butanol fermentation was collected from polluted river/sea, it will be affected by

seasonal variations and steady supply of the feedstock is questionable. However, the cost

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for microalgal biomass production is 400€/1000 kg biomass or 100€/100 kg microalgal

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sugar as mentioned by Wijffels’s group (Norsker et al., 2011).

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Even though the advantages and challenges of microalgae-based bioenergy recovery

have been widely reported, most related studies focused on biodiesel production from
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microalgal lipids (Chen et al., 2011), with few dealing with biobutanol production using
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microalgal biomass (Ellis et al., 2012; van der Wal et al., 2013). To date, there are only

several lab-scale studies that have tried to use different algal biomasses for ABE
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fermentation. However, since Green Biologics (UK), Cobalt Technologies (US) and Russian
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Biotechnologies are now working on lignocellulosic feedstock-based biobutanol through

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ABE fermentation by Clostridia, those successful industrial-level biobutanol fermentation

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can provide guidelines to help produce biobutanol from microalgal feedstock on a large
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scale. Thus, this review is aimed to provide comprehensive information on biobutanol

production using carbohydrate-rich microalgae as feedstock. Microalgae can be divided into

carbohydrate-rich microalgae and lipid-rich microalgae, based on their ability to accumulate

carbohydrates or lipids as energy reserves. The carbohydrates present in starch

accumulating microalgae (higher than 40%) are considered as an appropriate feedstock for

microbial growth and biobutanol production. Therefore, the processes tailored for the

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conversion of microalgae-based biomass into biobutanol are described in this review and

this specific topic apparently deserves further investigations.

2. Carbohydrate-rich microalgae as a renewable feedstock for butanol production

Microalgae are the simplest and oldest photosynthetic plants on earth and very diverse

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both physiologically and genetically. They can be unicellular or simple multicellular

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organisms, and can be autotrophic or heterotrophic or mixotrophic (i.e., combination of both

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autotrophic and heterotrophic). The main constituent components of algae that are of

commercial importance are carbohydrates, lipids, and proteins (Zhu, 2015). Microalgae
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(such as Spirulina sp. and Chlorella sp.) have been consumed by humans as nutritional
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supplement because of their high protein content. The protein content is strain dependent,

and for some microalgae used as nutritional supplements, the protein content can be as high
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as 60%~70%. (Kay, 1991). In contrast, microalgal lipids are used as renewable feedstock
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for biodiesel production due to potentially high lipid productivity with lipid-rich microalgae
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(Chen et al., 2011). On the other hand, microalgal carbohydrates can also be used for biofuel
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production by serving as feedstock for fermentative production of bioethanol or biobutanol

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(Ho et al., 2013a). The percentage of carbohydrates, lipids and proteins varies with

microalgal species as well as with the cultivation conditions (Vitova et al., 2015). Since

proteins content is high normally during log phase, the productivity of proteins can be

improved by optimizing cell growth. In contrast, cellular accumulation of lipids and

carbohydrates in microalgae is known to be induced under stress conditions. The most

commonly used strategy to stimulate lipids or starch accumulation is the employment of

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nutrient depletion (usually nitrogen starvation) conditions (Breuer et al., 2012; Ho et al.,

2012; Li et al., 2008). The energy-rich compounds (carbohydrates or lipids) can be further

converted into biodiesel (e.g., triacylglycerol) and fermentation products (e.g., starch) (Ho

et al., 2014). On-going research indicates that microalgae (with a biomass concentration of

180 g/L) can be directly used as feedstock for biohydrogen fermentation, producing butyric

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acid (5.8 g/L) and hydrogen (4478 mL/L) as the main products. Although lipids are high in

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energy content and easy to store, carbohydrate metabolism is the central energy yielding

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pathway in all organisms. The energy yield from the complete oxidation of fatty acids is

about 9 kcal/g (38 kJ/g), in contrast to about 4 kcal/g (17 kJ/g) for carbohydrates. Algae,
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such as Chloroccum sp., Spirulina platensis, Chlamydomonas reinhardtii, Nitzchia
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ciosterium, Phaeodactylum tnicornutum and Dunaliella tertiolecta, have relatively high

photo conversion efficiency, and are able to accumulate high carbohydrate content
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(potentially higher than 50% of their dry weight) (Markou et al., 2012b). As for sugar-based
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fermentation processes, microalgae containing high carbohydrate content should be a

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perfect substrate alternative for biobutanol production.

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The carbohydrate contents of microalgae are divided into two important functional
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components: (1) as energy reserves, mainly polyglucans in the chloroplasts (e.g., starch or

glycogen), or (2) as a part of the structural component of the cell wall, such as cellulose.

The storage and cell wall polysaccharide composition is species specific, and is not affected

by changes in growth conditions, whereas the composition and structure of the storage lipids

vary with species and with cultivation conditions (Table 1). It is important to note that algal

biomass lacks hemicelluloses and lignin, and some algae lack cell walls, which can reduce

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pretreatment costs (Markou et al., 2012a). Pretreatment of the lignocellulosic materials

releases certain furan aldehydes (such as furfuran, 5 hydroxymethy furfural) that interfere

with the efficiency of the fermentation process, while this problem may be alleviated using

microalgae-based carbohydrates that lack lignin and thus requires only a mild pre-treatment

process. In our previous study, it was observed that the acid hydrolysate (3% H2SO4) of

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microalgal biomass contained only a trace amount of furfural and 5-hydroxymethyl furfural

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(0.5 g/L) (Wang et al., 2016). Butanol fermentation strain C. acetobutylicum can tolerance

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at least 1 g/L furfural and 2 g/L 5-hydroxymethy furfural. The concentration present in

microalgal hydrolysate is too low to generate any negative effect on butanol production
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(Wang, Y. et al., 2015). Therefore, the carbohydrates from microalgae have been the focus
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of several reviews (Chen et al., 2013b; Markou et al., 2012a), but the use of microalgal

sugars as a feedstock for biobutanol production has not been discussed elsewhere.
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In using microalgae-based carbohydrates as feedstock for fermentation, the main task

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is the selection of microalgal strains that has high biomass productivity and carbohydrate
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content. Microalgae that contain glucose-based carbohydrates (starch/cellulose) are the

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most feasible feedstock for fermentation, because of the preferred utilization of simple
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sugars in the feed (Chen et al., 2013b). Many studies show that variations in culture

conditions can enhance starch accumulation in microalgae (Table 2). The effects of light

intensity, CO2 concentration, nitrogen starvation, sulfur starvation and osmotic stress have

all been reported. It is evident from the results of these studies that optimum light intensity

and CO2 concentration, coupled with specific macronutrient starvation, seems to be the best

method to improve the carbohydrate content of microalgae. Nutrient starvation also has

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some adverse effects, as it affects the total biomass and the carbohydrate productivity of the

culture. Sulfur starvation is suggested as the best strategy for the accumulation of starch

(Markou et al., 2012a; Vitova et al., 2015). Recent research showed that under sulfur

deprivation, the carbohydrate concentration increased almost 10-fold and the starch content

was maintained for a long period. Starch accumulation starts immediately after nutrient

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starvation, thereby allowing high production of carbohydrates in microalgae (Vitova et al.,

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2015).

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A two-stage cultivation method has been applied to overcome the reduction in biomass

associated with nutrient starvation conditions (Ho et al., 2013a; Wang, X. et al., 2015). The
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microalgal biomass is first cultivated in nutrient rich medium to promote maximal cell
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growth, and then switched to a nutrient deficient conditions to trigger carbohydrate

accumulation. Ho et al. (Ho et al., 2013a) used nitrogen starvation as a trigger, and
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Scenedesmus obliquus cells accumulated 51% carbohydrates by dry weight (DW), about 80%
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of which was glucose, as released by acid hydrolysis. Wang et al. (Wang, X. et al., 2015)
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developed a two-stage fed batch based photoautotrophic cultivation of C. reinhardtii and

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achieved a carbohydrate content of 71% (maximum yield reported till date) and a 14-fold
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improvement in biomass concentration. The enhanced carbohydrate accumulation was

based on the high affinity CCM (CO2 concentrating mechanism), which occurs with

ambient CO2 concentration (about 0.04%), concentrating the dissolved inorganic carbon

inside the cells (Wang, X. et al., 2015).

The growth of algal culture can also be improved by co-culturing with plant growth

promoting bacteria (PGPB). PGPB are rhizosphere-dwelling bacteria that promote plant

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growth by aiding in nitrogen fixation and supplementing plant growth hormones (Glick,

1995). Chlorella vulgaris has been co-cultured with the well-studied PGPB Azospirillum

brazilense, in an attempt to understand the plant-bacteria interactions. The growth and

pigment production of C. vulgaris was enhanced in the presence of the bacteria (Gonzalez

and Bashan, 2000). A similar study focused on carbohydrate and starch accumulation in two

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species of chlorella in the presence of Azospirillum brazilense. In the presence of PGPB,

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starch accumulation reached maximum after 72 hours of co-immobilization and co-culture.

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Chlorella accumulated up to 94 mg/g dry weight of carbohydrate, and 78% of the

accumulated carbohydrate was starch (Kim et al., 2014). This indicates that it is a promising
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way to improve the carbohydrate content of microalgae, and one that needs to be explored
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further.

Currently, there are only a few reports regarding biobutanol production using
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microalgal feedstock cultivated from wastewater (Anthony et al., 2013; Efremenko et al.,
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2012; Ellis et al., 2012; Jernigan et al., 2013; Potts et al., 2012; van der Wal et al., 2013).
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The reported microalgae were mainly collected from wastewater treatment lagoons or
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natural bays, pretreated to release the component simple sugars, and used as the substrate
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for ABE fermentation (Ellis et al., 2012; Jernigan et al., 2013; Potts et al., 2012; van der

Wal et al., 2013). Efremenko et al. (2012) tried to perform biobutanol fermentation with

immobilized Clostridium acetobutylicum cells using several microalgae species containing

total carbohydrate content of 24.3 to 69.7% per dried weight (DW). However, the soluble

carbohydrate content released after appropriate pretreatment was only 9.7 to 27.9% DW,

indicating the need for advanced pretreatment technology to efficiently release fermentable

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sugars (Table 2). The growth of microalgal biomass in naturally polluted water bodies

seems to have less process costs, thus being economically beneficial to the subsequent

fermentation process. However, when under suitable cultivation, monoculture of microalgae

may give a higher cellular carbohydrate content, and thus the pretreatment process could be

more cost effective (Table 2). In addition, the quality of monocultural microalgae is usually

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more stable, making it more suitable for the purpose of industrial applications.

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3. Genetic engineering of microalgae to improve carbohydrate accumulation

Carbohydrates and their intermediates play a central role in carbon fixation and
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energy storage in microalgae. Energy reserves are preferably stored as many forms of
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polysaccharides in various groups of algae, when the cells are subjected to stress like

nutrient depletion as mentioned in Table 1. Storage glucans are the first to be metabolized
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during dark phase and are important for dark survival periods (Raven and Beardall, 2003).
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In summary, starch is a storage product that is preferably synthesized and mobilized,

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whereas lipid or oil is noted as a long term storage product. This is understandable as energy
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input is lower for starch synthesis compared to fatty acid synthesis, with 66.7% energy
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returns (Johnson and Alric, 2013).

Two main strategies can be applied to increase starch accumulation: increasing starch

synthesis and decreasing starch degradation. Most of the related studies have been done on

food crops to increase the nutritional value or on model plants to understand the metabolism.

The reaction catalyzed by the enzyme ADP-glucose pyrophosphorylase (AGPase) is the rate

limiting step in starch synthesis. Genetic engineering strategies to improve heterologous

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expression of this enzyme can increase starch synthesis. Conversely, starchless mutants of

Chlamydomonas reinhardtii has been used to accumulate oil reserves for conversion to

biodiesel (Li et al., 2011). However, when complemented, these mutants preferably

accumulate starch with minimal oil accumulation (Work et al., 2010). The growth rate of

these starch mutants are impaired and this must be considered when biofuel production from

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these strains are the major goal.

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Increase in carbohydrate accumulation in microalgae has been successfully achieved

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by process engineering strategies, rather than genetic engineering as the carbon partitioning

between lipid and starch synthesis is not completely understood. However, few studies have
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been done, mainly in cyanobacteria because of the ease of genetic engineering in a haploid
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genome. Nobles et al. reported the expression of bacterial cellulose synthesis genes in

Synechococcus leopoliensis strain UTCC 100 (Nobles and Brown, 2008). The recombinant
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cyanobacterium was capable of producing non-crystalline cellulose at a concentration of

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0.22 g/L as extracellular matrix components, which are easy to separate and could be used
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as feedstock for biofuel production. Similarly, Su et al introduced cellulose synthesis genes

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form Acetobacter xylinum in the cyanobacterium Anabaena sp. PCC 7120 (Su et al., 2011).
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The mutant was capable of overcoming wasteful respiration, with efficient flux of

photosynthate carbon, obtaining a total extractable glucose content of 0.53-0.66 mg ml-1

OD750-1. Random mutagenesis of Spirulina platensis with plasma increased growth rate and

carbohydrate productivity of the strain. The mutant has a carbohydrate content of 0.261 and

0.331 g carbohydrate/g biomass and the mutation also expressed high genetic stability (Fang

et al., 2013). Another study utilized similar random mutagenesis on Synechocystis PCC

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6803 using ethyl methane sulfonate. The generated mutants had high biomass production

and cellular carbohydrate content (Patel et al., 2016). The mutant gave 3.6 times higher

biomass concentration and the carbohydrate content was 255 mg/L, indicating the feasibility

of using this strain as biofuel feedstock.

Enhancing photosynthetic efficiency is another way to enhance carbohydrate

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accumulation in microalgae. Evidence suggests that AGPase, the rate limiting enzyme in

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starch synthesis, is allosterically activated by 3-phosphoglycerate (the initial product of CO2

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fixation), linking starch synthesis to photosynthesis (Zabawinski et al., 2001). The carbon

dioxide fixing enzyme RuBisCO (Ribulose-1,5-bisphosphate carboxylase/oxygenase) is

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often dubbed as a slow and confused enzyme as it can utilize both carbon dioxide and
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oxygen as substrate. Utilization of atmospheric levels of CO2 in the presence of abundant

oxygen needs large amounts of enzymes, indicating the significance of accumulation of this
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enzyme in photosynthetic center of various photosynthetic organisms. RuBisCO has been

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targeted by many plant physiologists to enhance photosynthetic efficiency of higher plants

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and increase food production (Whitney et al., 2011). In C. reinhardtii, the small subunit of
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RuBisCO was replaced by the subunits from Arabidopsis and Sunflower plants and were
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evaluated for their efficiency (Genkov et al., 2010). The hybrid enzymes showed 3–11%

increase in CO2/O2 specificity and retain near normal Vmax values. In another study, the large

subunit of RuBisCO from Chlamydomonas was subjected to directed evolution by gene

shuffling (Zhu et al., 2010). Several mutated Rubisco enzymes with increased carboxylase

activity or CO2/O2 specificity were identified. On the other hand, the bicarbonate transporter

gene, ictB, and carbonic anhydrase gene, ecaA, of S. elongatus PCC7942 were

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overexpressed in the same strain from a plasmid, enhancing photosynthetic efficiency

(Chow et al, 2015). This also led to an increased biomass and carbohydrate productivity of

1239 mg/L/d and 564 mg/L/d, respectively. The information provided in this section may

be used as a guideline to genetically engineer microalgae with improved photosynthetic

efficiency and carbohydrate productivities aiding in sustainable biofuels production.

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4. Biobutanol production using microalgal biomass as feedstock

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4.1 Fermentative bacteria for butanol production

The use of Clostridial species on sugar-based feedstock via ABE fermentation is the
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most commonly used process for biobutanol production (Lee et al., 2008). ABE
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fermentation denotes the anaerobic fermentation of sugars in the medium to Acetone,

Butanol and Ethanol in the ratio of 3:6:1. Four main strains of Clostridia are used in
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industrial butanol production, namely, C. acetobutylicum, C. beijerincki, C.

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saccharoperbutylacetonicum and C. saccharobutylicum. Among these, C. acetobutylicum

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is the most studied strain and is best suited for a starch-based medium. C.
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saccharoperbutylacetonicum is versatile with regard to its substrate preference, and can use
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both starch- and sugar-based substrates, with moderate but consistent butanol production

(Ranjan and Moholkar, 2012). C. beijerincki has been reported to have the best tolerance

for the presence of inhibitory compounds in the fermentation medium (furfural and 5-

hydroxymethy furfural mainly), while its butanol productivity may not be the highest among

the four strains mentioned above

Sugars derived from biomass are the most common substrates used for biofuel

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production, as in the first and second generation biofuels. However, the energy yield of n-

butanol from corn or switch grass is very low compared to that obtained for corn- or switch

grass-based ethanol. This is important, because the feedstock price is always reflected in the

fuel price (Pfromm et al., 2010). There is thus a continued search for the most suitable

feedstock for biobutanol production. Microalgae are rich in carbohydrates, which can be

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converted to simple monosaccharides and used for fuel production. Pretreated microalgal

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biomass and algal biomass residues after biodiesel production have already been used for

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bioethanol fermentation (Harun et al., 2009). The underlying process is the same for both

ethanol and butanol fermentation: the algal biomass is harvested, pre-treated to release the
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monosaccharides, and these are fed into the fermentation process. One important thing to
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note is that yeast, the traditionally used organism for ethanol production, cannot metabolize

pentoses and other hexoses, and they prefer glucose-based substrates. However, most
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solventogenic Clostridia can utilize a large variety of substrates from monosaccharides

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including many pentoses and hexoses to polysaccharide (Jones DT, 1986). They are
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inherently saccharolytic and are armed with a repertoire of carbohydrate active enzymes,
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like amylases, cellulases and other endoglucanases. Cheng et al. (Cheng et al., 2015)
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observed that when using microalgae-based feedstock for butanol production, C.

acetobutylicum is capable of converting residual solid matter present in the medium to

butanol. They also showed that the butanol yield was higher (21.96 mg/g-residues) when

using unfiltered hydrolysate as a substrate compared to a lower butanol yield (10.03 mg/g-

residues) with filtered hydrolysate. The duration of batch fermentation with the fermentation

bacteria is between 2-6 days, depending on the conditions and the type of substrate

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employed. The final total concentration of solvents produced ranges from 0.009 to 26.3 g/L

in batch fermentation, and the components can be further purified from the fermentation

broth by distillation (Jang et al., 2012; Tashiro et al., 2013).

The genome of the important industrial clostridial species C. acetobutylicum ATCC

824 is available and a search for sugar transport genes revealed the presence of similar

PT
systems in solventogenic clostridia (Nolling et al., 2001). In C. beijerinckii, sugar transport

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and phosphorylation are accomplished by PTS and non-PTS based passive transport

SC
methods (Mitchell, 1996); however there is a swift decline in PTS activity during

fermentation. As a result, as fermentation proceeds towards solventogenesis, glucose

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transport and phosphorylation are taken up by a non-PTS transport mechanism and a
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cytoplasmic glucokinase (Lee et al., 2005). The hexoses are metabolized by the glycolytic

pathway to pyruvate, and the pentoses enter the pentose phosphate pathway and feed into
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glycolysis or enter anabolic pathways. Both C. beijerinckii and C. acetobutylicum possess

E

genes encoding proteins that facilitate transport and metabolism of xylose. The distribution
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of these xylose utilization genes varies with species. The xylose utilization system is present
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as a large gene cluster in C. beijerinckii, whereas they are distributed in various locations of
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the genome in C. acetobutylicum (Gu et al., 2010). A well-defined arabinose utilization

system, present as a gene cluster, is also documented in many Clostridial species (Zhang et

al., 2012). In C. acetobutylicum, it is present along with the genes for pentose phosphate

pathway in one large gene cluster named CAC1339-CAC1349, and for efficient

transcription it has been divided into seven transcriptional units (Paredes et al., 2004).

Previous studies indicate that in Clostridium acetobutylicum ATCC824, arabinose can be

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metabolized either by the pentose phosphate pathway or the phosphoketolase pathway.

However, the increased production of acetate while growing in arabinose versus in xylose

reveals that phosphoketolase pathway is the preferred way of metabolism (Servinsky et al.,

2012). Also, it is shown that a bi-functional xylulose-5-P/fructose-6-P phosphoketolase,

encoded by the gene CAC1343 was induced in the presence of arabinose, showing the

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possibility of arabinose metabolism by phosphoketolase pathway (Servinsky et al., 2012).

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4.2 Pretreatment of microalgal biomass

Pretreatment is needed for microalgal cell disruption when using microalgal biomass
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as the feedstock, and this can provide microalgal sugars for fermentative bacteria.
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Hydrolysis of the microalgal biomass releases the constituent sugars. Analyses of the

hydrolysates have revealed the composition of the carbohydrate content. The most common
D

among the hexose sugars are glucose and mannose, with others including galactose,
E

rhamnose, fructose, and fucose. Xylose, arabinose, and ribose are the common pentoses.
PT

Galacturonic acid and glycerol are also found, as galacturonic acid is a cell wall component
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and glycerol is accumulated in the cytosol as a response to osmotic stress (Doan et al., 2012).
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All these sugars can be efficiently utilized by Clostridia, as explained in Section 3.1. After

the carbon sources are converted to the central metabolite, pyruvate, they are directed into

ABE fermentation. At present four pretreatment technologies for microalgal biomass are

widely used, including thermal, mechanical, chemical and biological (enzymatic) methods

(Passos et al., 2014). Of these, thermal and mechanical pretreatments are considered

efficient for cell disruption in microalgae. Thermal pretreatments are the most employed

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method in continuous reactors (Passos and Ferrer, 2015). For example, thermal pretreatment

enhanced methane yield from C. vulgaris biomass by 62% (Mahdy et al., 2015). In addition,

chemical pretreatment is usually conducted at high temperatures. For butanol production

using microalgae biomass, acid hydrolysis pretreatment of wastewater microalgae was

conducted using 1.0 M sulfuric acid at 80–90 °C for 120 min (Castro et al., 2015). Moreover,

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the energy-intensive concentration and drying processes of the harvested microalgal

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solution can be eliminated with thermal pretreatment (Kita et al., 2010). The energy profit

SC
ratio (EPR) of the wet process is approximately twice than that of the dry process when

using the thermal pretreatment of microalgae (Saga et al., 2015). Mechanical pretreatment
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is independent of the microalgal species and the associated difference in cell wall structure,
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but it is more energy intensive compared to the other methods mentioned above (Lee et al.,

2012). Chemical pretreatment works better if combined with another method, and a
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combination of thermal and chemical methods is often successful (Mendez et al., 2013).
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The major disadvantage of using chemical method is the inhibitory effect of its leftover
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chemicals on the fermentative ability of the bacterial strain used in ABE fermentation.
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Dilute acid or alkali hydrolysis combined with thermal treatment is most commonly
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employed for biomass hydrolysis in microalgae and the method is also simple and efficient.

The presence of leftover alkali in the hydrolysate might help in delayed acidification of the

fermentation medium in the acidogenesis step. However, it must be noted that these

chemicals could also lead to the production of fermentative inhibitors that might interfere

with the fermentation performance of the Clostridia. It was reported that for algae from

wastewater, the optimal conditions for hydrolysis were found to be 10% dried algae, treated

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with 1 M sulfuric acid for 120 mins at 80-90 °C with a sugar yield of 166.1 g per kg of dry

microalgae. When used in ABE fermentation, 5.23 g/L ABE and 3.74 g/L butanol were

obtained, at a production cost of USD $12.54 per kg of butanol (Castro et al., 2015).

Enzymatic pretreatment seems to be a promising method for microalgae hydrolysis, but it

is not cost effective when commercialized production is considered (Ehimen et al., 2013).

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The microalgal cell wall is predominantly composed of cellulose and other polysaccharides,

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which are species dependent. These components are hydrolyzed to their respective

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monomeric forms, which can then be utilized by Clostridial strains for butanol production.

The enzymes and their hydrolytic activities on microalgal cell walls are highly substrate-
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specific. Thus, the major obstacles to be overcome before applying enzymatic hydrolysis on
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an industrial scale are the development of the specific enzyme cocktail and the increased

price associated with the procurement of enzymes. In the enzymatic hydrolysis trials of
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Chlamydomonas reinhardtii and Chlorella vulgaris, the addition of carbohydrolase

E

(Viscozyme) and protease (Alcalase) resulted in high carbohydrate and protein

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solubilization of both biomasses (86–96%) (Mahdy et al., 2014). In addition, the co-
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culturing of enzymes-secreting bacteria and microalgae has also been performed. When
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Chlorella vulgaris cells were exposed to the growing Flammeovirga yaeyamensis cells,

microalgal cell walls were degraded, as confirmed by enzyme assays (amylase, cellulase

and xylanase), electron microscopy, and the presence of reducing sugars in the medium

(Chen et al., 2013a).

Chemical, thermal and biological pretreatment methods have been reported in studies

of biobutanol fermentation using microalgal biomass as the feedstock (Table 3). The

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thermal microalgal biomass decomposition at 108°C for ABE fermentation had been

examined in several species of microalgae (Mahdy et al., 2014). In general, thermal and

mechanical pretreatments lead to cell disruption, but cannot obtain a high yield of

monosaccharides. Chemical pretreatment can breakdown cell walls and hydrolyze

microalgal carbohydrates into fermentable sugars, but some inhibitory compounds (such as

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furfural, 5-hydroxymethy furfural, acetic acid, oxalic acid, formic acid) are generated during

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the chemical pretreatment process, affecting cell growth and butanol production during

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fermentation. Enzymatic pretreatment can be used as an additional method to increase

soluble sugar recovery from the total carbohydrate content of the microalgal biomass
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without generating inhibitors to the fermentation process (van der Wal et al., 2013).
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However, since the microalgal carbohydrates are mainly starch, instead of cellulose, simple

chemical hydrolysis (e.g., dilute acid hydrolysis) alone can already achieve high sugar yield.
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As a result, additional enzymatic hydrolysis may not be necessary from an economic

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viewpoint when considering the high cost of the hydrolytic enzymes. For large-scale
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microalgal biomass pretreatment, the size reduction of microalgae may be required to yield
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suitably small sized particles to improve the hydrolysis of carbohydrates (Potts et al., 2012).
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Our on-going studies on butanol fermentation from pretreated C. vulgaris biomass

show that chemical pretreatment exhibited better hydrolysis performance when compared

with other pretreatment methods when using a high microalgal biomass concentration of up

to 120 g/L. An innovative sequential alkali-acid hydrolysis method was shown to effectively

produce fermentable sugars for butanol fermentation, as the alkali pretreatment could

remove proteinaceous inhibitors originating from a high concentration of microalgal

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biomass. The release of fermentable sugars from microalgae reached almost 100% after

being pretreated with 1% NaOH, and the butanol production was 13.10 g/L using 60 g/L

sugar obtained from hydrolysis of NaOH pretreated microalgae (Wang et al., 2016).

4.3 Current advances in biobutanol fermentation using microalgae-based sugars

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Typical yields for ABE fermentation range from 0.15 to 0.25 g butanol/g sugar with

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productivities of 0.5 g/h/L (Ezeji, T. et al., 2007; Qureshi and Ezeji, 2008; Qureshi et al.,

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2001). The main limitation of ABE fermentation for biobutanol production is the toxicity of

butanol with regard to Clostridial strains. Clostridia can tolerate only up to 30 g/L solvents
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in the media, which in turn limits the initial sugar concentration in the fermentation
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medium(Xue et al., 2012). Butanol is particularly toxic, as it is lipophilic and can disrupt

the phospholipid bilayer of the cell membrane (Jin et al., 2011). Methods to improve butanol
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fermentation, including improved fermentation technologies, improved product recovery

E

and improved tolerance to solvent toxicity, have been extensively reviewed (Green, 2011;
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Jin et al., 2011). As for butanol fermentation using microalgal biomass as the feedstock,
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the butanol yield and production titer was within the range of 0.02 to 0.29 g butanol/g sugar
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and 0.8 to 13.2 g/L, respectively (Table 3). The performance of ABE fermentation using

pretreated sugarcane bagasse, barley straw, commercial starch and starch based food wastes

are also compared in Table 3. It should be noted that the lignocellulose based feedstock

underwent rigorous pretreatments, acid or alkali followed by enzymatic hydrolysis, due to

its recalcitrant nature, as discussed previously. The fermentative inhibitors generated were

not discussed in these reports and hence the actual effect of these pretreatment methods on

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fermentation performance and yield cannot be evaluated. It would be prudent to say that

microalgal biomass grown under appropriate conditions with high carbohydrate content and

simple pretreatment can serve as an efficient feedstock for ABE fermentation and the

butanol titer obtained is comparable to liquefied starch (Wang et al 2015, Ezeji et al 2007).

The low butanol concentration obtained from microalgae-based feedstock was mainly due

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to the low initial microalgal sugar concentrations and the type of monosaccharides present

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in microalgal hydrolysate. The initial microalgal sugar concentration used for ABE

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fermentation was quite low (ranging from 8 to 23 g/L) in the presence of mixed

monosaccharides. The low microalgal sugar concentration was because of the high cost of
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concentrating microalgal biomass, poor pretreatment efficiency resulting from high biomass
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loading, or the low intracellular carbohydrate contents of the microalgal cell used. However,

it is evident that the addition of an easily metabolized substrate in the fermentation medium,
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such as glucose and butyrate, enhanced butanol production, though it is not considered to
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be economically feasible. For instance, supplementing 1% glucose into a culture medium

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containing the algae-based sugars could raise the butanol production to 10.7 g/L (van der
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Wal et al., 2013). Therefore, to obtain a relatively high initial sugar concentration for ABE
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fermentation, it is inevitable that extra glucose should be added to the medium, which

originally contains a lower concentration of sugars from microalgae. However, this may not

be an efficient strategy for microalgae-based biobutanol production in terms of energy input

and from an economic perspective. Therefore, further research to design an integrated

energy efficient method, from biomass cultivation to final biobutanol fermentation, are

necessary.

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On the other hand, two methods have been much explored to improve biobutanol

production, namely immobilization of Clostridial cells (Jiang et al., 2009; Lee, S.-M. et al.,

2008) and in situ product removal (Chen et al., 2014). The cell immobilization technique is

a promising method with the following advantages over the conventional free-cell

fermentation process: high available cell densities, increased stability of cells, lower

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microbial cell loss, reduction in the negative impact of metabolites accumulated in the

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process of ABE fermentation, and strong tolerance to accumulated solvents (Jiang et al.,

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2009; Lee, S.-M. et al., 2008). Polyvinyl alcohol (Dolejs et al., 2014), fibrous materials

(Jiang et al., 2009) and cyrogel beads (Tripathi et al., 2010) have been used for
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immobilization of Clostridial cells. For instance, Efremenko et al. used PVA cryogel
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immobilized C. acetobutylicum cells for biobutanol fermentation, resulting in a 68%

increase in the butanol yield when compared to conventional free-cell systems (Efremenko
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et al., 2012).
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Conventional fed-batch and continuous fermentation processes do not seem to be

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economically feasible for biobutanol production, due to product inhibition and the biphasic
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nature of ABE fermentation. To overcome this problem, fed-batch culture for ABE
AC

fermentation is usually coupled with in situ recovery process (Ezeji et al., 2004; Xue et al.,

2016a). Coupling of biobutanol fermentation with an in situ removal process was found to

effectively reduce product inhibition, thereby improving the titer and productivity of

biobutanol (Ezeji, T.C. et al., 2007). The technologies applied for in situ removal of butanol

from fermentation broth include adsorption (Wiehn et al., 2014), membrane pervaporation

(Sharif Rohani et al., 2015) liquid–liquid extraction or perstraction (Weilnhammer and Blass,

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1994; Xue et al., 2017a; Xue et al., 2016b), vacuum separation(Wiehn et al., 2014), and gas

stripping(Chen et al., 2014). Shin et al. (Shin et al., 2015) used a polystyrene-b-

polydimethylsiloxane-b-polystyrene triblock copolymer membrane for selective removal of

butanol from aqueous solutions by pervaporation, and were able to alleviate product

inhibition and enhance butanol productivity. In situ removal in a pilot large-scale ABE

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fermentation has also been studied (Potts et al., 2012). The biobutanol concentration in the

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fermentation broth was maintained at about 4 g/L by a gas stripping method to avoid the

SC
effects of butanol toxicity, and this achieved better butanol producing performance than that

reported in the literature (Potts et al., 2012). This value was however lower than a previous
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report, where 153.5 g/L ABE was obtained from corn stover hydrolysate by employing
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vapor stripping-vapor permeation process (Xue et al., 2016b).

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5. Bottlenecks in butanol production from algae and their potential solutions.

E

The use of microalgae-based carbohydrates as a feedstock for ABE fermentation is

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highly advantageous over second generation feedstock, as it answers the most controversial
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land usage problems concerned with conventional agriculture. The use of algae as a
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feedstock fits into the existing ABE fermentation technology with little or no modification

of the technology. Algal biomass is free of lignin and contains a lower amount of

hemicelluloses. The energy intensive pretreatment and hydrolysis treatments, which are

required for the saccharification of lignocellulosic materials, may thus not be necessary for

microalgae feedstock. In contrast, simple acid/alkali hydrolysis would suffice and the

inhibitory compounds (e.g., furans) derived from hydrolysis of lignocellulosic materials

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might also be absent. The monosaccharides (no matter whether 5 C or 6 C sugars) present

in the microalgae hydrolysate can be efficiently utilized by Clostridial strains for ABE

fermentation. Moreover, some Clostridial strains are naturally saccharolytic and can

solubilize sugars from starch and cellulose, and this is helpful from an economic point of

view. However, it should be mentioned that the large amount of residues generated after

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hydrolysis must be disposed properly.

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Despite the fact that carbohydrate-rich microalgae can be used for biobutanol

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production, it is worth noting that the technology is still developing. Moreover, a limited

amount of research has been done on this issue, as most researchers are interested in
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producing biofuels from microalgal lipids and or developing other high value products (e.g.,
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pigments, DHA) from microalgae, rather than carbohydrates. Most of the studies on

carbohydrate accumulation were done to understand the basic carbohydrate metabolism in

D

microalgae. With the turn to the use of algae-based carbohydrates, these techniques are
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exploited for application-oriented studies. However, many bottlenecks, as summarized in

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Table 4, should be addressed for successful use of microalgae based carbohydrates as a

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feedstock for ABE fermentation. As discussed in Section 4.3, the presence of very low
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initial sugar concentrations in the fermentation medium is the main limiting factor for low

solvent titers. It has already been reported that pure glucose at a concentration of 60 g/L is

the appropriate sugar concentration for butanol production by Clostridial strains (Wang et

al., 2014). Such high concentrations of algae-based sugars, particularly glucose, are difficult

to achieve, and other monosaccharides, including galactose, rhamnose and xylose, are

present in the algal biomass hydrolysate. To overcome this problem, strains that accumulate

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high levels of carbohydrates (greater than 50% per dry weight) should be selected as the

fermentation feedstock. With the use of current pre-treatment methods, efficient recovery

of soluble sugars can be achieved if the carbohydrate content of the biomass is high, thereby

minimizing algal biomass loading in the pre-treatment. Various strategies have been

employed to increase carbohydrate accumulation in microalgae, as discussed in Section 2.

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The lack of special media designed for specific metabolite synthesis leads to alterations in

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the basal medium components on a trial and error basis, which is not very effective. Design

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of specific media, keeping in mind the end product (carbohydrates or lipids), must be carried

out. However, such a strategy has not yet been employed and more work is needed in this
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regard.
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Effective pre-treatment can improve the recovery of the sugars from microalgal

carbohydrates (starch, cellulose or glycogen, depending on the algal species). Currently,

D

mild acid hydrolysis followed by thermal pre-treatment is sufficient, and has been employed
E

on a large scale (Castro et al., 2015). Cellulase- or amylase-assisted digestion can also be
PT

used for complete recovery of the sugars, but it is not economically viable. The presence of
CE

residual solid matter in the hydrolysate might also affect the available initial sugar
AC

concentrations. It is imperative that a Clostridial strain that has more substrate versatility

and unaffected butanol productivity is chosen for fermentation. C.

Saccharoperbutylacetonicum and C. beijerincki are more robust when it comes to substrate

specificity as previously mentioned. Specific methods to improve ABE fermentation based

on strain improvement and fermentation technology have also been reviewed recently

(Zheng et al., 2015).

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Another commonly overlooked feature is the presence of inhibitory substances in the

algal biomass hydrolysate after pre-treatment. Results from our experiments indicate that

the use of high concentrations of algal biomass hydrolysate might introduce contaminating

proteins into the fermentation medium. Further analysis revealed that increased

concentration of proteins in the fermentation medium inhibited butanol production (Wang

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et al., 2015b). In such cases, it would be beneficial to choose an algal strain that has a very

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high carbohydrate content, and thus the protein level of the biomass would be low. In recent

SC
studies, the biochemical composition of various algal strains was analyzed, and it is

interesting to note that algal strains with high carbohydrate concentrations (>50% DW) have
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very low protein contents (5-10% DW) (Efremenko et al., 2012; Gonzalez and Bashan,
MA

2000). The low levels of protein in the biomass hydrolysate mean very low concentrations

of possible inhibitory proteins, and thus the effects on butanol production will be negligible.
D

Actively growing microalgal cells in the exponential phase have a very high protein content,
E

most of which is the machinery used by the cells for cell division. Therefore, if the
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microalgae are harvested at the end of exponential phase or at the beginning of stationary
CE

phase or at nitrogen starvation stage, the maximal carbohydrate content can be achieved (Ho
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et al., 2013a). Inhibition of cell division by phosphorus or sulphur starvation and

synchronous culturing of microalgae can accumulate more carbohydrates, based on the

same principle (Vitova et al., 2015). Laurens et al. (Laurens et al., 2015) employed a similar

strategy for algal harvest: early harvest for protein rich algae, mid harvest culture for

carbohydrate rich algae, and late harvest culture for lipid rich algae. They also proposed a

sequential acid hydrolysis and solvent extraction method for algal biomass pre-treatment

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where the carbohydrate, lipid and protein content were separated. The efficiency of this

method with regard to ethanol fermentation was also tested, and 51% glucose to ethanol

conversion was achieved. A more efficient way of utilizing the protein content of microalgae

is to use it as a carbon source for biofuel production. Huo et al. (2012) discussed the use of

protein-rich algae as a feedstock for biofuel production, and hence the closure of the

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atmospheric nitrogen cycle. An E. coli strain that has the capacity to utilize amino acids as

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a carbon source and produce higher alcohols by the ketoacid pathway was engineered, and

SC
the production of C4 and C5 alcohols was demonstrated. Production of up to 3 g/L of higher

alcohol from pre-treated algal and bacterial biomass was achieved, along with 56% of the
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theoretical yield (Huo et al., 2012). Another source of inhibitory compounds in the media
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could be the salts formed as a result of the alkali-based neutralization of the acid-treated

algal biomass hydrolysate for use in the fermentation medium (Markou et al., 2013).
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Maximal levels of salts were produced when HCl and HNO3 were used, and lower salt levels
E

were observed when H2SO4 was used, as it is a diprotic acid. The effect of the salts present
PT

was shown to be inhibitory to the ethanol producer Saccharomyces, but similar studies were
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not done for Clostridial species. This could be one factor to be taken into account when
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assessing the efficiency of pre-treatment methods of algal biomass.

Obtaining more in-depth knowledge about the metabolic pathways in microalgae

leading to nutrient accumulation in the form of carbohydrates and lipids is crucial for the

development of green and sustainable microalgae-based biofuels. Even though it has been

shown that nutritional stress induces starch or lipid accumulation, the exact mechanism

behind such responses is not clear. Genome sequencing of the model organisms and detailed

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gene annotation is the most sophisticated way to overcome this particular hurdle. With the

wealth of genetic data that is now available, the transcriptomes and proteomes can also be

analyzed and the results provide useful information in this regard. About 18 nuclear

genomes and 39 transcriptomes are available for microalgae (Cadoret et al., 2012).

Furthermore, the US Aquatic Species Program and the US Department of Energy Algal

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Feedstock Technology Project 2015 raised interest in this area, and this may help remove

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the information block between algal genomics and industrial applications of algae.

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6. Future prospects: Fourth generation algal biobutanol.
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The production of biofuels from agricultural crop-based feedstock has always been
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limited by the insufficient photosynthetic efficiency of terrestrial plants. To overcome this

problem, microalgae with high photosynthetic efficiency have been used as feedstock for
D

biofuel production. To efficiently tap the photosynthetic efficiency and utilize it to directly
E

convert CO2 to biofuels forms the basis of fourth generation biofuels (Klinthong, 2015). In
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general, the engineered algae can by-pass anabolic pathways for growth, and instead use the
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products of carbon fixation (ATP, NADPH) to convert the fixed CO2 to butanol. A designer
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gene or a designer pathway is usually expressed heterogeneously in the native algae, which

acts upon one of the CO2 fixation pathway metabolites and converts it to butanol. Designer

pathways that can convert the metabolites glyceraldehydes-3-phosphate, 3-

phosphoglycerate and fructose 1,6-diphosphate to butanol have been reported (US Patent

8735651). Another example is based on the concept of photanol, which is related to the

construction of a metabolic chimera between phototrophic and fermentative metabolism in

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a single organism, alternately known as photo-fermentation (Hellingwerf and Teixeira de

Mattos, 2009). The feasibility of such conversion has already been proven in Synechocystis

sp., where the fermentative enzyme ketoacid decarboxylase from Lactobacillus lactis was

expressed heterogeneously, although with the major drawback of the oxygen sensitivity of

the fermentative enzymes (Daroch et al., 2013). Direct conversion of CO2 into biofuels

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(such as iso-butanol) at the expense of solar energy is an ideal fuel generation scenario, and

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recent advances in synthetic biology have made it technologically feasible when using

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engineered phototrophic organisms (e.g., cyanobacteria) as the cell factory (Klinthong, 2015;

Lan and Liao, 2011). However, there is still a large gap between such technological
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developments and the commercial viability of fourth generation biobutanol.
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7. Conclusions
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As a third generation feedstock, microalgae have significant advantages over the

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starch- and lignocellulose-based feedstock conventionally used for biobutanol production.

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Microalgal cultivation can also be used as a bioremediation measure for wastewater

CE

treatment, and even the residual biomass after lipid extraction from algae can be used as a
AC

feedstock. Microalgae that can accumulate very high levels of carbohydrates are best suited

for this, as the butanol production and yield depend on the initial sugar concentration in the

fermentation medium. A better understanding of the algal carbohydrate metabolism at the

genetic level is essential in developing high carbohydrate accumulating strains, either by

strain improvement techniques or by altering cultivation conditions. The storage

polysaccharides starch/glycogen and cellulose in cell walls can be released and hydrolyzed

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by simple and mild treatments, as microalgal cells lack the lignin that makes crop-based

feedstocks difficult to use. Nevertheless, an efficient and cost-effective microalgal biomass

pretreatment is still critical for biobutanol production. Chemical, thermal and biological

methods have already been reported to have relatively high sugar yields. However, the

feasibility of any pretreatment technology should be examined in terms of the generation of

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inhibitors, energy consumption, operating cost, and sugar yield. The reported biobutanol

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yields from microalgal biomass range from 0.02 to 0.29 g biobutanol/g sugar, while the

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biobutanol concentration ranges from 0.8 to 10.9 g/L. The strategies of cell immobilization

and in situ removal of inhibitive products have been applied to obtain higher biobutanol
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yields. Fourth generation biofuels, where CO2 is directly converted into butanol by
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engineered phototrophic microorganisms, seem to be an ideal route for the generation of

fuels, but more advances in the technology are required before reaching the
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commercialization stage.
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Acknowledgments
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This work was supported by Open Project of State Key Laboratory of Urban Water
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Resource and Environment, Harbin Institute of Technology (HCK201607) and China

Postdoctoral Science Foundation. The authors also gratefully acknowledge the support

received from Taiwan’s Ministry of Science and Technology under grant numbers MOST

106-3113-E-006-011, 106-3113-E-006-004-CC2, 104-2221-E-006-227-MY3, and 103-

2221-E-006-190-MY3.

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surfactants in enzymatic hydrolysis for acetone-butanol-ethanol fermentation. Bioresour.
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Zabawinski, C., Van Den Koornhuyse, N., D'Hulst, C., Schlichting, R., Giersch, C.,
Delrue, B., Lacroix, J.M., Preiss, J., Ball, S., 2001. Starchless mutants of
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Chlamydomonas reinhardtii lack the small subunit of a heterotetrameric ADP-glucose

pyrophosphorylase. J Bacteriol 183(3), 1069-1077.
D

Zhang, L., Leyn, S.A., Gu, Y., Jiang, W., Rodionov, D.A., Yang, C., 2012. Ribulokinase
E

and transcriptional regulation of arabinose metabolism in Clostridium acetobutylicum.

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J Bacteriol 194(5), 1055-1064.

Zheng, J., Tashiro, Y., Wang, Q., Sonomoto, K., 2015. Recent advances to improve
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fermentative butanol production: genetic engineering and fermentation technology. J

Biosci Bioeng 119(1), 1-9.
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Zhou, W., Chen, P., Min, M., Ma, X., Wang, J., Griffith, R., Hussain, F., Peng, P., Xie, Q.,
Li, Y., Shi, J., Meng, J., Ruan, R., 2014. Environment-enhancing algal biofuel
production using wastewaters. Renewable and Sustainable Energy Reviews 36, 256-
269.
Zhu, L., 2015. Biorefinery as a promising approach to promote microalgae industry: An
innovative framework. Renewable and Sustainable Energy Reviews 41, 1376-1384.
Zhu, X.G., Long, S.P., Ort, D.R., 2010. Improving photosynthetic efficiency for greater
yield. Annu Rev Plant Biol 61, 235-261.
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Table 1 Composition of microalgal cell wall and storage products (Chen et al., 2013b).

Division Cell wall Storage products

Cyanophyta Lipopolysaccharides, peptidoglycan Cyanophycean starch

Chlorophyta Cellulose, hemicellulose Starch/lipid

Dinophyta Absence or contain few cellulose Starch

Cryptophyta Periplast Starch

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Euglenophyta Absent Paramylum/lipid

Heterokontophyta Naked or covered by scales or with large Leucosin/lipid

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quantities of silica

SC
Rhodophyta Agar, carrageenan, cellulose, calcium Floridean starch

carbonate
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PT
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Table 2 Carbohydrate or starch content of microalgal species

Total carbohydrates
Microalgae species Reference
(% per dry weight)

Arthrospira platensis 40.8 (Efremenko et al., 2012)

Nannochloropsis sp. 56.8 (Efremenko et al., 2012)

Dunaliella tertiolecta 50.6 (Efremenko et al., 2012)

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Dunaliella salina Teod 69.7 (Efremenko et al., 2012)

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Galdieria partita Sentz 50.1 (Efremenko et al., 2012)

SC
Cosmarium sp. 58.4 (Efremenko et al., 2012)

Nostoc sp. 52.3 (Efremenko et al., 2012)

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Chlorella vulgaris IAMC-534 37.0 (starch) (Hirano et al., 1997)

C. vulgaris CCAP 211/11B 55.0 (Illman et al., 2000)

MA

C. vulgaris P12 41.0(starch) (Dragone et al., 2011)

C. vulgaris FSP-E 55.0 (starch) (Ho et al., 2013b)

Chlamydomonas reinhardtii UTEX 90 60.0 (Choi et al., 2010)

D

C. reinhardtii IAM C-238 55.0 (starch) (Kim et al., 2006)

E
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Chlorococum humicola 32.5 (Ike et al., 1997)

Chlorococcum sp. TISTR 8583 26.0 (starch) (Rodjaroen S, 2007)

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S. acutiformis TISTR 8495 16.4 (starch) (Rodjaroen S, 2007)

S. obliquus CNW-N 51.8 (Ho et al., 2012)

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Tetraselmis sp. CS-362 26.0 (Brown et al., 1998)

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Table 3 Comparison of the feedstock pretreatment conditions and biobutanol production performance using the first (starch), second (lignocellulosic
materials) and third (microalgal biomass) generation feedstock.
Butanol
ABE fermentation bacteria Feedstock used Biomass pretreatment
T
Butanol yield (g/g)

P
production (g/L)
Reference

C. beijerinckii NCIMB 8052 Ulva lactuca Sulfuric acid and enzymes pretreatment
I
0.35 ABE g/g sugar

R
3.0
(van der Wal et
al., 2013)

C. acetobutylicum ATCC 824 Ulva lactuca Sulfuric acid and enzymes pretreatment

S C 0.08 ABE g/g sugar 0.8

(van der Wal et
al., 2013)
(van der Wal et
C. acetobutylicum ATCC 824 Ulva lactuca

N U
Sodium hydroxide and enzymes pretreatment nd nd al., 2013)
(Ellis et al.,

A
C. saccharoperbutylacetonicum N1–4 Wastewater algae Sulfuric acid pretreatment 0.201 2.26
2012)

C. saccharoperbutylacetonicum N1–4 Wastewater algae

M
Sulfuric acid and enzymes pretreatment 0.249 7.79
(Ellis et al.,
2012)
C. saccharoperbutylacetonicum ATCC
27021
Wastewater algae
E D
Thermal pretreatment with steam (121°C,
15 psig, 1h)
0.13 g/g algae nd
(Jernigan et al.,
2013)

PT
(Castro et al.,
C. saccharoperbutylacetonicum N1-4 Wastewater algae Sulfuric acid pretreatment nd 3.74
2015)

C. beijerinckii ATCC55025

C E
Ulva lactuca Sulfuric acid pretreatment 0.29 g/g sugar 4.0
(Potts et al.,
2012)

C. saccharoperbutylicum ATCC27021

C. acetobutylicum B1787 A C Ulva lactuca

Arthrospira platensi a
Sulfuric acid pretreatment

Sulfuric acid pretreatment

nd

0.16
2.75 (ABE)

5.26
(Potts et al.,
2012)
(Efremenko et
al., 2012)
(Efremenko et
C. acetobutylicum B1787 Arthrospira platensi b Sulfuric acid pretreatment 0.29 9.13
al., 2012)
C. acetobutylicum B1787 Nannochloropsis sp. Sulfuric acid pretreatment 0.18 10.9 (Efremenko et

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al., 2012)
(Efremenko et
C. acetobutylicum B1787 Nannochloropsis sp. c Sulfuric acid pretreatment 0.21 13.2
al., 2012)
De-oiled biomass of C. (Cheng et al.,
C. acetobutylicum ATCC 824
sorokiniana CY1
Sulfuric acid (2%)+NaOH(2%)

P T
0.09 g/g carbohydrate 3.86
2015)

C. acetobutylicum ATCC 824

Chlorella vulgaris
JSC-6
NaOH (1%) followed by H2SO4 (3%)

R I
0.58 mol/mol sugar
(or 0.24 g/g sugar)
13.1
(Wang et al.,
2016)

Clostridium beijerinckii BA101

Liquefied corn starch
(Commercial)
Starch - based packing
N/A

S C 0.41 g ABE/g sugar 13.4

Ezeji et al,
2007
Jesse et al,
Clostridium beijerinckii BA101
peanuts

N U
Blending, Autoclaving at 121°C for 15 min, 0.37 g ABE/g sugar 21.7 (ABE)
2002
Huang et al,
Clostridium. beijerinckii P260

C. acetobutylicum GX01
Restaurant food waste

Sugarcane bagasse
M A
Blending, Autoclaving at 121°C for 15 min

Alkali pretreatment and enzymatic hydrolysis

0.36–0.38 g ABE/g sugar

0.22 g/g sugar

18.8–20.9 (ABE)

14.17
2015
Pang et al,
2016

Clostridium acetobutylicum CH02 Sugarcane bagasse

E D
Diluted acid (DA) and oxidate ammonolysis
(OA) pretreatment followed by enzymatic 0.25 g/g sugars 7.68 Li et al, 2017

Clostridium acetobutylicum DSM

P T hydrolysis
Dilute acid pretreatment followed by
135.0 g/kg pretreated Yang et al,
1731
Barley straw

C E enzymatic hydrolysis in the presence of

surfactants
straw
7.9
2015

C
a: Bubbling of air enriched with carbon dioxide

A
b: Cultivation under physiological stress conditions (in absence of nitrogen and phosphorus sources) and maximum illumination
c: Addition of 50g/L NaCl and bubbling of air
nd: Data not shown

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Table 4 Bottlenecks in butanol production from microalgae and their potential solutions

Bottlenecks in Butanol production from algae Solutions

Lower Initial sugar concentrations in fermentation 

T
Strategies to increase accumulation of starch/glycogen in microalgae
P
medium 


I
Efficient tailoring of growth medium to enhance productivity (Malcata,
2011)
R

C
Efficient pre-treatment methods to improve soluble sugar

S
concentration(Laurens et al., 2015; Markou et al., 2013)
Strain improvement techniques to improve starch utilization by Clostridial

 N U
strains (Zheng et al., 2015)

Presence of inhibitory protein components after pre-

treatment  A
Use of algae that accumulate very high amounts of carbohydrates
Removal of inhibitory substances by alkali pre-treatment (Wang et al.,
M
2015b)

E D 


Usage of late harvest algae with limited protein content (Laurens et al.,
2015)

P T Methods to utilize protein as carbon source for growth (Huo et al., 2011;
Huo et al., 2012)

viable microalgae
C E
Limited genetic knowledge about commercially 

Widen genome analysis of microalgae (Cadoret et al., 2012)
Develop genetic transformation and selectable marker systems for

A C 
microalgae
Elucidation of metabolic pathways that facilitate accumulation of
carbohydrates.

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10
9.2 9.9
1 .9%
f ~1 7.86
8 R o
C AG
Market size (billion USD)

PT
6 5.9

RI
4

2
SC
NU
0
MA
13
08
09
06
07

12
05

10
11

15

20
14
20
20
20
20
20

20
20

20
20

20

20
20

Year
CAGR: Compound Annual Growth Rate
E D
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Figure 1 Market size of butanol in recent years (Integrated information reported by

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General Electric Company, Nexant, Grand View Research, and PR newswire.).

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Highlights
 Microalgae-based carbohydrates is a potential feedstock for ABE fermentation
 Updated information on microalgae biomass based biobutanol production is
presented
 Biomass pre-treatment methods for efficient butanol production are described
 Enhanced butanol production with immobilized cells and product removal is
discussed

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 Bottlenecks in microalgae-based biobutanol production is critically reviewed

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SC
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MA
E D
PT
CE
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