Biotechnology Advances 31 (2013) 129–139

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Research review paper

Single cell oil production from low-cost substrates: The possibility and potentialof its industrialization

Chao Huang a,b, Xue-fang Chen b,c, Lian Xiong a,b, Xin-de Chen a,b,⁎, Long-long Ma a,b, Yong Chen a,b

a Key Laboratory of Renewable Energy and Gas Hydrate, Chinese Academy of Sciences, Guangzhou 510640, PR Chinab Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou 510640, PR Chinac Graduate University of Chinese Academy of Sciences, Beijing 100049, PR China

⁎ Corresponding author. Tel./fax: +86 20 37213916.E-mail address: cxd_cxd@hotmail.com (X.D. Chen).

0734-9750/$ – see front matter © 2012 Elsevier Inc. Allhttp://dx.doi.org/10.1016/j.biotechadv.2012.08.010

a b s t r a c t

a r t i c l e i n f o

Article history:Received 18 April 2012Received in revised form 20 August 2012Accepted 25 August 2012Available online 31 August 2012

Keywords:Single cell oilLow-cost substrateLignocellulosic biomassBiodieselFunctional oils

Currently, single cell oils (SCO) attract much attention because of their bi-function as a supplier of functionaloils and feedstock for biodiesel production. However, high fermentation costs prevent their further applica-tion, and the possibility and potential of their industrialization is suspected. Therefore, various low-cost, hy-drophilic and hydrophobic substrates were utilized for SCO production. Of these substrates, lignocellulosicbiomass, which is the most available and renewable source in nature, might be an ideal raw material forSCO production. Although many reviews on SCO have been published, few have focused on SCO productionfrom low-cost substrates or evaluated the possibility and potential of its industrialization. Therefore, this re-view mainly presents information on SCO and its production using low-cost substrates and mostly focuses onlignocellulosic biomass. Finally, the possibility and potential of SCO industrialization is evaluated.

© 2012 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1302. Functions and application of single cell oils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

2.1. Supplier of valuable lipids and medically important polyunsaturated fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . 1302.2. Alternative lipid feedstock for biodiesel production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1312.3. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

3. Low-cost substrates for single cell oil production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1323.1. Single cell oil production on hydrophilic materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

3.1.1. Molasses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1323.1.2. Raw materials from the food industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1323.1.3. Wastewaters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1323.1.4. Glycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1333.1.5. Whey . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

3.2. Single cell oil production on hydrophobic materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1334. SCO production from lignocellulosic biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

4.1. The history of lignocellulosic biomass utilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1334.2. Microbial oil production from lignocellulosic biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

5. The possibility of industrialization of SCO production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1345.1. Fermentation substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1355.2. Lipid fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

5.2.1. Oleaginous microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1355.2.2. Fermentation mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1355.2.3. Simultaneous production of SCO and other valuable by-products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1355.2.4. Other related factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

5.3. Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1366. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

rights reserved.

130 C. Huang et al. / Biotechnology Advances 31 (2013) 129–139

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

Table 1Functions and application of single cell oils.

Functions andapplication

Strains Reference

Substitutes of cocoabutter

Yarrowia lipolytica (Papanikolaou et al., 2001)Apiotrichumcurvatum

(Davies et al., 1990)(Ykema et al., 1989)(Ykema et al., 1990)

Supplier ofpolyunsaturatedfatty acids

γ-Linolenic acid Cunninghamellaechinulata

(Fakas et al., 2007)

Mortierella isabellina (Papanikolaou et al., 2004a,2004b)

Mortierellaramanniana

(Dyal et al., 2005)

Mortierella alpine (Jang et al., 2005)Mucor rouxii (Mamatha et al., 2008)

Docosahexaenoic acid Schizochytrium spS31

(Wu et al., 2005)

Schizochytrium G13/2S

(Ganuza and Izquierdo, 2007)

Schizochytriumlimacinum

(Chi et al., 2007)

Parietochloris incise (Bigogno et al., 2002)Crypthecodiniumcohnii

(de Swaaf et al., 2003a, 2003b;Ratledge et al., 2001)

Arachidonic acid Sirodotia Kylin (Bhosale et al., 2009)Mortierella alpine (Peng et al., 2010)

Eicosapentaenoic acid Candidaguilliermondii

(Guo and Ota, 2000; Guo et al.,1999)

Achlya sp. ma-2801 (Aki et al., 1998)Alternative feedstockfor biodieselproduction

Rhodotorula glutinis (Easterling et al., 2009)Rhorosporidiumtoruloides

(Li et al., 2007)

Trichosporonfermentans

(Zhu et al., 2008)

Lipomyces starkeyi (Angerbauer et al., 2008; Zhao etal., 2008)

Nannochloropsisoculata

(Chiu et al., 2009)

Neochlorisoleoabundans

(Li et al., 2008b)

Cladophora fracta (Demirbas, 2009)Chlorellaprotothecoides

(Demirbas, 2009; Xiong et al.,2008; Xu et al., 2006)

Chlorella vulgaris (Liu et al., 2008)Cunninghamellajaponica

(Sergeeva et al., 2008)

E. coli (Lu et al., 2008)

1. Introduction

Microbial oils, namely, single cell oils (SCO), which are lipids that areproduced by oleaginous microorganisms, have been of potential inter-est tomany researchers in the past decades due to their significant func-tions and specific characteristics. Traditionally, microorganisms, whichinclude bacteria, yeasts, molds and microalgae, that can accumulatelipids to more than 20% of their dry weight are considered oleaginousmicroorganisms (Ratledge, 1991). Research on SCO has a long history.Before the 1980s, uncovering the biochemistry and metabolism oflipid accumulation by oleaginous microorganisms was the focus ofmany scientists (Botham and Ratledge, 1978; Botham and Ratledge,1979; Gill et al., 1977; Ratledge and Hall, 1977). In the subsequent 20years, such biochemical processes and SCO production were of interestto more people because SCO could play critical roles in maintaininghuman health (Sijtsma and Swaaf, 2004) by replacing some expensivematerials such as cocoa butter (Beopoulos et al., 2009). During thoseyears, the process of lipid accumulation was more completely elucidat-ed, and the studies varied. Furthermore, researchers continued to payattention to biochemical mechanisms to explain how a microorganismaccumulates lipids in its body (Alvarez and Steinbuchel, 2002; Evansand Ratledge, 1983a; Holdsworth and Ratledge, 1991; Ratledge, 2004;Tehlivets et al., 2007). The studies in this field involved the key enzymesof those processes and their regulation of lipid accumulation (Morgunovet al., 2004; Polakowski et al., 1999; Ratledge et al., 1997; Savitha et al.,1997; Wheeler et al., 1990; Wynn et al., 1997; Wynn et al., 1999;Zhang et al., 2007) and key intermediates for lipid biosynthesis(Burton et al., 2005; Sheridan et al., 1990). Additionally, screening foroptimal oleaginous microorganisms became a key mission of many sci-entists in the field of SCO production (Evans and Ratledge, 1983b;Granger et al., 1992; Papanikolaou et al., 2004a; Papanikolaou et al.,2007; Ratledge, 1988b; Wu et al., 2005). Other related and interestingaspects such as detective methods for SCO were also explored (Erogluand Melis, 2008; Kimura et al., 2004; Peng and Chen, 2008).

As more evidence on the medical significance of SCO accumulates,especially on critical polyunsaturated fatty acids such as γ-linolenicacid (GLA), arachidonic acid (ARA), docosahexaenoic acid (DHA) andeicosapentaenoic acid (EPA) (Ward and Singh, 2005), the interest inSCO production continuously increases. Concurrently, because the costof lipid feedstock is still high for biodiesel production, more and moregroups attempt to use SCO, whose composition is similar to traditionalvegetable oils, as alternatives for biodiesel production (Li et al.,2008a). Obviously, SCO will play a more important role in today'sworld, especially because of its potential to solve the current energy cri-sis. Because the mechanisms for lipid accumulation by microorganismsare now clarified, more researchers are concentrating on reducing thecost and improving the productivity of SCO production. Because fer-mentation substrates for SCOproduction are costly, it is wise and neces-sary to find low-cost, alternative feedstock for microbial oil production.

It is obvious that SCO will play a more critical role in the future,and low-cost substrates for SCO production will play a key role inthe industrialization of SCO production. Several scientists havereviewed SCO and its production over the past decades (Meng et al.,2009; Ratledge and Cohen, 2008; Ward and Singh, 2005); however,few have focused on substances that are used for SCO production. Inthis review, a brief explanation and introduction of the function andapplication of SCO will be given first. Then, the progress of SCO pro-duction using low-cost substrates will be presented, with a focus onSCO production from lignocellulosic biomass. Finally, the possibilityand potential of SCO industrialization will be evaluated.

2. Functions and application of single cell oils

SCO and its production has been a hot topic over the recent years,and the production and application of these oils was reviewed inother articles (Papanikolaou and Aggelis, 2011a; Papanikolaou andAggelis, 2011b). In this section, the function and value of single celloils will be summarized (Table 1).

2.1. Supplier of valuable lipids and medically important polyunsaturatedfatty acids

People have been attracted to SCO since the 1980s when peopleattempted to produce substitutes of cocoa butter when it was inshort supply (Ward and Singh, 2005). To overcome this shortage,many researchers attempted to find SCO with a similar composition

131C. Huang et al. / Biotechnology Advances 31 (2013) 129–139

to cocoa butter. During the 1990s, many groups attempted to use SCOas substitutes for cocoa butter (Davies and Holdsworth, 1992; Davieset al., 1990; Ykema et al., 1989; Ykema et al., 1990). Later,Papanikolaou and his co-workers proved the capability of Yarrowialipolytica to accumulate lipids that could be used as substitutes forcocoa butter (Papanikolaou et al., 2001) and reduced the fermenta-tion cost of this substance by using agro-industrial residues asmedia (Papanikolaou et al., 2003). Because the price of cocoa butteris continuing to increase due to its short supply, other methodssuch as the conversion of olive or palm oils to cocoa butter equiva-lents by lipase transesterification made us more curious about SCOas cocoa butter substitutes (Ward and Singh, 2005). However, it isworth noting that non-conventional biocatalysis has not yet been ap-plied to large-scale operations of cocoa butter substitutes, but cocoabutter substitutes have been produced with the aid of oleaginous mi-croorganisms in large-scale operations (Papanikolaou and Aggelis,2011b).

Many researchers have focused on the production of polyunsaturat-ed fatty acids, such as the n-6 and n-3 series, by bacteria, fungi or algaerather than simply finding lipids with rare fatty acid compositions thatare similar to cocoa butter (Ratledge, 1993). Among these compounds,omega-3/6 fatty acids, such as γ-linolenic acid (GLA), arachidonic acid(ARA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA),were focused on most in the past decades due to their benefits tohuman health (Berquin et al., 2008; SanGiovanni and Chew, 2005;Youdim et al., 2000).

GLA has selective anticancer properties (Kenny et al., 2000); there-fore, its production by different microorganisms was studied. It wasproven that GLA could accumulate in Cunninghamella echinulata (Fakaset al., 2007), Mortierella isabellina (Papanikolaou et al., 2004a),Mortierella ramanniana (Dyal et al., 2005), Mortierella alpine (Jang et al.,2005) andMucor rouxii (Mamatha et al., 2008). Other important polyun-saturated fatty acids such as DHA, ARA and EPA also attracted attentionfor their importance to humans by contributing to the development ofthe infant brain, the function of the eye, the synthesis of hormones andsignalingmolecules and because of their beneficial effects on the cardio-vascular system (Meyer et al., 2003; Sakuradani and Shimizu, 2003;SanGiovanni and Chew, 2005). Green oleaginous algae were usuallyused to produce DHA. Of these organisms, Schizochytrium sp., which in-clude Schizochytrium sp S31 (Wu et al., 2005), Schizochytrium G13/2S(Ganuza and Izquierdo, 2007), and Schizochytrium limacinum (Chi et al.,2007), were used in many cases. Additionally, other oleaginousmicroalgae such as Parietochloris incise (Bigogno et al., 2002) andCrypthecodinium cohnii (de Swaaf et al., 2003a; De Swaaf et al., 2003b;Ratledge et al., 2001) were used for DHA production. In many situations,microalgae such as the red alga Sirodotia Kylin could produce ARA andDHA (Bhosale et al., 2009), and some Mortierella species such asMortierella alpine were used to produce ARA (Peng et al., 2010). Severalmicroorganisms can accumulate EPA and DHA. For example, the oleagi-nous yeast Candida guilliermondii (Guo and Ota, 2000; Guo et al., 1999)was shown to potentially produce EPA and DHA. Similarly, the fungusAchlya sp. ma-2801 (Aki et al., 1998) was isolated for EPA and DHAproduction.

2.2. Alternative lipid feedstock for biodiesel production

Because of the rising price and decreasing supply of crude oils,more researchers are focusing on clean and renewable alternatives,such as biodiesel, to traditional petroleum diesel fuel (Demirbas,2007; Ma and Hanna, 1999). Two main problems exist in biodieselproduction: its high cost, of which raw materials amount to approxi-mately 75% of the total production cost, and the consumption of largeamounts of vegetable oils for biodiesel production could result in ashortage of edible oils and would increase the price of food(Srinivasan, 2009). Using animal fats, frying oils and waste oilsseems to be a good strategy for reducing the cost of biodiesel.

Unfortunately, the amounts of these resources are too limited tomeet the needs for clean, renewable fuels. New oil sources, such asnon-edible oil plants, may potentially be used in biodiesel production(dos Santos et al., 2008; Gui et al., 2008); however, their low avail-ability prevents their larger application. Hence, microbial oils, whichare more abundant and whose advantages include a short life cycle,less required labor, less affection by venue, season and climate andare easier to scale up (Li et al., 2008a), are now believed to be morepromising candidates for biodiesel production.

Microbial oils, whose compositions are similar to vegetable oils, canbe considered alternatives for biodiesel production, and many oleagi-nous microorganisms, such as bacteria, yeast, fungi and microalgae,have such characteristics. Many articles have described the applicationof these organisms in SCO accumulation for biodiesel production andare described in Table 1. Yeast and microalgae have been used morefrequently than fungi and bacteria for lipid and biodiesel production. Ithas been proven in recent years that the yeasts Rhodotorula glutinis(Easterling et al., 2009), Rhorosporidium toruloides (Li et al., 2007),Trichosporon fermentans (Zhu et al., 2008), and Lipomyces starkeyi(Angerbauer et al., 2008; Zhao et al., 2008) have the potential to producebiodiesel via a transesterification process. Additionallymanymicroalgaestrains such as Nannochloropsis oculata (Chiu et al., 2009), Neochlorisoleoabundans (Li et al., 2008b), Cladophora fracta (Demirbas, 2009),Chlorella protothecoides (Demirbas, 2009; Xiong et al., 2008; Xu et al.,2006), and Chlorella vulgaris (Liu et al., 2008) could supply microbiallipids for biodiesel production.

Although fungi and bacteria have been widely applied in function-al lipid production, these organisms have been used less in biodieselproduction than yeast and microalgae, and only a few fungal species,such as Cunninghamella japonica (Sergeeva et al., 2008), were foundto be potentially utilized in biodiesel production. Few bacteria canbe used for biodiesel production because their lipid composition isnot suitable for biodiesel production. Unfortunately, in spite of advan-tages such as their high growth rate and ease of culturing, it is difficultto use bacteria in biodiesel production. Interestingly, it was reportedthat certain engineered bacteria could directly produce fatty acid es-ters (biodiesel); therefore, metabolic engineering of bacteria couldresult in the identification of new bacterial strains for biodiesel pro-duction (Steen et al., 2011). Recently, overproduction of free fattyacids in E. coli for biodiesel production was successfully accomplished(Lu et al., 2008), which indicates that using bacterial lipids for biodie-sel will be important in the future.

2.3. Discussion

To fulfill the economic value of SCO, it is necessary to know that ithas two functions. The first function is as a source of valuable lipidssuch as DHA, EPA, ARA, GLA, and cocoa butter substitutes. Usually,the market price of these lipids is significantly high; therefore, it isimportant that the fermentation substrate is suitable for an oleagi-nous microorganism to accumulate these valuable lipids.

Secondly, microbial lipids could also be an ideal feedstock for bio-diesel production. When biodiesel feedstock is the object of produc-tion, the quantity of SCO, namely, the lipid yield of the substrates,was of the most importance. To date, the price of petroleum continuesto increase, and in China, the price of gasoline is already higher than8000 RMB/t; thus, it can be expected that biodiesel will be competi-tive in the energy market in the near future. However, it is alsoworth noting that the price of edible oils such as peanut, corn, andsoybean oils is still higher than that of gasoline, diesel, or otherpetrolic fuels, which means that it is still not wise to produce biodie-sel by using lipid feedstock from food sources. Furthermore, whenusing microbial oil for biodiesel production, it is necessary to controlits cost so that it is lower than the price of gasoline and diesel; other-wise, this bioconversion is not competitive for industrialization.Moreover, the cost of biodiesel from SCO includes the fermentation

Table 2Low-cost feedstock for single cell oil production.

Feedstock Strains Reference

Hydrophilic materialsMolasses Trichosporon fermentans (Zhu et al., 2008)

Candida curvata D (Bednarski et al., 1986)Cunninghamella echinulata (Chatzifragkou et al.,

2010)

Raw materials fromfood industry

Starch hydrolysates Mortierella alpine (Zhu et al., 2003)Banana juice Apiotrichum curvatum (Vega et al., 1988)Tomato wastehydrolysate

Cunninghamella echinulata (Fakas et al., 2008)

N-acetylglucosaminehydrolysate

Cryptococcus curvatus (Wu et al., 2011; Zhanget al., 2011)

Sweet sorghumextracts

Mortierella isabellina (Economou et al.,2011a)

WastewatersSewage sludge Lipomyces starkeyi (Angerbauer et al.,

2008)Olive oil millwastewaters

Lipomyces starkeyi (Yousuf et al., 2010)

Monosodiumglutamatewastewater

Rhodotorula glutinis (Xue et al., 2006; Xueet al., 2008; Xue et al.,2010)

Glycerol Yarrowia lipolytica (Papanikolaou et al.,2002; Papanikolaouet al., 2003)

Mortierella isabellina (Papanikolaou et al.,2008)

Schizochytrium limacinum (Chi et al., 2007)(Pyle et al., 2008)(Ethier et al., 2011)

Whey Apiotrichum curvatum (Davies andHoldsworth,1992; Davies et al.,1990;Ykema et al., 1988;Ykemaet al., 1989; Ykemaet al., 1990)

Apiotrichum curvatum,Cryptococcus albidus, Lipomycesstarkeyi, and Rhodosporidiumtoruloides

(Akhtar et al., 1998)

Cryptococcus curvatus, Candidabombicola

(Daniel et al., 1999)

Mortierella isabellina (Vamvakaki et al.,2010)

Hydrophobic materialsVegetable oils Different oleaginous yeasts and

moulds(Aggelis and Sourdis,1997)

Industrial fats Yarrowia lipolytica (Papanikolaou andAggelis, 2003;Papanikolaou et al.,2001)

132 C. Huang et al. / Biotechnology Advances 31 (2013) 129–139

process and subsequent transesterification. Because the cost oftransesterification is relatively stable, the cost of fermentation de-cides the potential for SCO to be industrialized.

Fermentation substrates are critical for the control of productioncost of valuable SCO or biodiesel feedstock that is produced fromSCO. First, the cost of these substrates must be low enough to makethe product competitive in the market. Second, the substrate mustnot influence the quality of the microbial oil, especially its concentra-tion of valuable lipids (i.e., DHA, EPA, ARA, GLA, or cocoa butter sub-stitutes) or the lipid composition of SCO (which is suitable forbiodiesel production).

The fermentation substrate is not the only determinant for indus-trialization of SCO; however, this factor is undoubtedly the base ofSCO production and directly influences the possibility and potentialof its industrialization. Thus, many researchers have focused on find-ing suitable substrates for SCO production, and this finding will befurther reviewed in this work.

3. Low-cost substrates for single cell oil production

As mentioned above, many special functions of SCO impart its sig-nificance on various areas. However, when compared with ethanol,its application is on a smaller scale because of the higher cost of fer-mentation. To reduce the cost of microbial oil production, many ef-forts focused on using low-cost materials as media for SCOproduction. Generally, two types of lipid synthesis exist in oleaginousmicroorganisms: the “de novo” and “ex novo” lipid accumulation pro-cesses (Papanikolaou and Aggelis, 2011b). The former process is usu-ally carried out on hydrophilic materials and usually requiresnitrogen-limited culture conditions. In contrast, “ex novo” lipid pro-duction is the production of SCO through fermentation on hydropho-bic materials. Based on this point, the low-cost substrates that areused for microbial oil production can be divided into hydrophilicand hydrophobic (Table 2). The costs of these substrates are partlygiven (Table 3).

3.1. Single cell oil production on hydrophilic materials

3.1.1. MolassesFor a long time, fermentation was usually carried out on varying

sugars. Thus, low-cost materials that contain various sugars could beused for microbial oil production. Molasses, from sugarcane or beet, isan industrial by-product of sugar manufacturing. Generally, molassescontains fructose, sucrose, and glucose; therefore, this sugar was con-sidered an ideal raw material for cheap medium culture formulationsand was widely used in the fermentation industry for ethanol (Doelleand Doelle, 1990), hydrogen (Tanisho and Ishiwata, 1995), lactic acid(Wee et al., 2004), and levan (Han and Watson, 1992). Sugarcane(Zhu et al., 2008) and beet molasses (Bednarski et al., 1986) havebeen used for SCO production. More recently, Cunninghamellaechinulata showed great potential in the decolorization-detoxificationof waste molasses and in efficiently using molasses for SCO production(Chatzifragkou et al., 2010). Although oleaginous microorganisms cangrow well on molasses medium due to its high sugar content, the highnitrogen content of this sugar prevents its lipid accumulation (Zhu etal., 2008).

3.1.2. Raw materials from the food industryMany starch hydrolysates from different foods contain various fer-

mentable sugars; therefore, these materials could also serve as carbonsources for oleaginous microorganisms. For example, the ARA yield ofMortierella alpine could be as high as 1. 47 g/L on cornstarch hydroly-sate (Zhu et al., 2003), and banana juice (Vega et al., 1988) has beenapplied as a substance for SCO production. Moreover, nitrogen fromtomato waste hydrolysates was used to increase the glucose uptakeand lipid accumulation of Cunninghamella echinulata (Fakas et al.,

2008). More recently, N-acetylglucosamine, which is the major carbo-hydrate of the hydrolysate of shrimp processing waste, was used toproduce microbial oil (Wu et al., 2011; Zhang et al., 2011). Lastly,sweet sorghum extracts were also shown to be potential carbonsources for lipid production by Mortierella isabellina (Economou etal., 2011a).

3.1.3. WastewatersWorld-wide utilization of sewage sludge and wastewaters in

agro-industry has gained attention due to the great abundance ofthis waste and its harmful impact on the environment. The treatmentof such waters and sludge consumes a large amount of energy and re-sults in high costs for treatment (Yang et al., 2005). It is worth notingthat many sugar (polysaccharides)-based materials have been pres-ent in these substances; thus, these materials could be a carbon

Table 3Potential cost of different fermentation substrates.

Substrate Source Cost

Molasses Sugarcane industry $180–250/ta

Starch Corn industry US $310–399/metric tona

Glycerol China US $600–700/metric tona

Whey China US $5–20/kga

Vegetable oils Singapore US $1–2/La

Wastewaters, waste animaloil, and etc.

From various places orindustries

NDb

Lignocellulosic biomassRice straw China 500 RMB/tc

Bagasse China 350 RMB/tc

Corncob China 200–350 RMB/tc

Wheat straw China 350 RMB/tc

a The data were according to information published on www.alibaba.com.b These materials were generated as waste-products of different industries; thus,

their values were difficult to evaluate.c The data were according to information published on price.zz91.com.

133C. Huang et al. / Biotechnology Advances 31 (2013) 129–139

source for SCO production by aerobic microorganisms. Some re-searchers have focused on this application in the recent years. For ex-ample, Angerbauer et al. (2008) used sewage sludge as a substance toproduce SCO. In another study, the bioconversion of olive oil millwastewaters into lipids was performed by Yousuf et al. (2010). Fur-thermore, Xue et al. (2006) first used monosodium glutamate waste-waters as feedstock to synthesize lipids that were to be used as theraw material for the production of biodiesel. Because the biomassand lipid content was too low, this group (Xue et al., 2008) optimizedthe process by analyzing the effect of nitrogen and glucose on lipidaccumulation. Currently, the fermentation process designed by thisgroup was scaled up to a 300-L fermentor (Xue et al., 2010).

3.1.4. GlycerolTraditionally, glycerol was produced by microbial fermentation or

chemical synthesis, but it can also be recovered from soapmanufacturing (Wang et al., 2001). Because soap has been replacedby detergents, the cost of glycerol has increased again. During the bio-diesel production process, oils/fats (i.e., triglycerides) are mixed withmethyl alcohol and alkaline catalysts to produce esters of free fattyacids, and glycerol is the primary by-product. As a result, a largeamount of glycerol would be produced in the biodiesel industry andcould make glycerol an ideal, low-cost feedstock for fermentation.Glycerol's role as an important carbon source for industrial microbiol-ogy has been discussed in other reviews (da Silva et al., 2009), and itcan undoubtedly be used as a low-cost carbon source for lipid fer-mentation. In fact, glycerol has been used as a carbon source for singlecell oil production. Papanikolaou and his co-workers (Papanikolaouet al., 2002; Papanikolaou et al., 2003) performed studies on SCO pro-duction by Yarrowia lipolytica on agro-industrial residues includingtechnical glycerol. More recently, this same group (Papanikolaou etal., 2008) expanded the application of glycerol to be used as feedstockto produce single cell oil, 1,3-propanediol and citric acid byMortierella isabellina. Glycerol was also used as a substance for pro-ducing SCO, especially during DHA production, by some oleaginousalga such as Schizochytrium limacinum (Chi et al., 2007; Ethier et al.,2011; Pyle et al., 2008).

3.1.5. WheyWhey is a by-product of cheese and casein from bovine milk, and it

is an attractive substrate for SCO production due to its great availability,especially in Europe and the United States (Ahn et al., 2000; Koller etal., 2005). For example, Ykema and Davies used cheese whey as a sub-strate for SCO production by Apiotrichum curvatum (Davies andHoldsworth, 1992; Davies et al., 1990; Ykema et al., 1988; Ykema etal., 1989; Ykema et al., 1990). Later, whey was shown to be capable

of being used by other oleaginous microorganisms (Akhtar et al.,1998; Daniel et al., 1999). More recently, Mortierella isabellina had anoutstanding performance in biomass, fat and γ-linolenic acid produc-tion on cheese whey (Vamvakaki et al., 2010).

3.2. Single cell oil production on hydrophobic materials

As mentioned above, hydrophobic substrates are also an impor-tant feedstock for SCO production (Papanikolaou and Aggelis,2011a; Papanikolaou and Aggelis, 2011b), and this bioconversionprocess requires no nitrogen-limited condition. To date, various hy-drophobic substances can be produced during many industry pro-cesses every year, and many of these substances have been used forSCO production in the past decades. For instance, Aggelis andSourdis (1997) focused on the ability of oleaginous microorganismsto use vegetable oils as a substance for growth and lipid accumula-tion. Similarly, Papanikolaou and Aggelis (2003) and Papanikolaouet al. (2001) modeled the lipid degradation and accumulation ofYarrowia lipolytica by cultivating it on media containing industrialfats. Interestingly, when cultured on mixtures of saturated free fattyacids (such as stearin, which is the industrial derivative of animalfat), technical glycerol (the main by-product of biodiesel productionfacilities) and glucose, Yarrowia lipolytica could accumulate somecocoa butter-like lipids (Papanikolaou et al., 2003).

One of the important functions of single cell oils is that it could be apotential substitute for valuable lipids such as cocoa butter. Usually, thisfunction is barely fulfilled by the “de novo” lipid accumulation processbecause, in many cases, the lipid composition of microorganisms doesnot change significantly during the lipid fermentation process (Huanget al., 2009; Zhu et al., 2008). In contrast, during the “ex novo” lipid ac-cumulation process, “new” fatty acid profiles (at extra- and intra-cellular levels) that did not previously exist in the substrate fat couldbe produced (this process is referred to as the “bio-modification” offats and oils by oleaginous microorganisms), and, thus, an “improve-ment” and “up-grade” of the fatty materials that utilized as substratesto produce “tailor-made” lipids of high-added value, such as cocoa but-ter substitutes, can be performed (Papanikolaou and Aggelis, 2011a;Papanikolaou and Aggelis, 2011b). Despite the availability of hydropho-bic substrates (mostly fromwaste oils or the food industry) beingmuchless than that of hydrophilic substrates, the important functions of hy-drophobic substrates will make them a continued focus of futurestudies.

4. SCO production from lignocellulosic biomass

Although the above-mentioned, low-cost substrates could de-crease the cost of SCO production, two disadvantages might occurthat would prevent the industrialization process. First, transportationof the raw materials is usually a problem. Second, the supply of theraw materials cannot be continuous and is, thus, unfavorable for sta-ble SCO production. Based on these issues, lignocellulosic biomass,which is the most available and renewable source for SCO in nature,has been a focus of many researchers in recent years.

4.1. The history of lignocellulosic biomass utilization

For more than half a century, cellulosic biomass was considered agreat potential raw material for producing bio-fuel (Himmel et al.,2007) and other valuable chemicals (Carvalho et al., 2004; Qureshiet al., 2008a) due to its availability and lower cost compared toother substrates (Schubert, 2006). Compared with food crops orgrains, lignocellulosic residues have more advantages, which includehaving fewer competing uses, less interference on the food economyand less strain on environmental resources (Stephanopoulos, 2007).Lignocellulosic biomass consists of three main components: cellulose,

134 C. Huang et al. / Biotechnology Advances 31 (2013) 129–139

hemicellulose, and lignin, and the relative proportion of these compo-nents depend on the material source (Reddy and Yang, 2005).

Generally, the utilization of lignocellulosic biomass includes theprocess of biomass degradation and its subsequent fermentation orbioconversion (Rubin, 2008). Breakdown of lignocellulosic biomassgenerates long-chain polysaccharides, which are then convertedinto their corresponding monosaccharides. Early chemical processesfor cellulose degradation usually used acid treatment (LaForge andHudson, 1918), and, even today, heat and acidic treatment are usedin industrial processes for biomass degradation in spite of disadvan-tages such as it being a slow and inefficient process (Mosier et al.,2005). Additionally, acid hydrolysis would produce inhibitors suchas weak acids, aldehydes, and aromatic compounds, which would de-crease the overall rate of fermentation (Palmqvist and Hahn-Hagerdal, 2000b). Because some companies have reduced theircosts, cellulases offer a potential biological method for sugar genera-tion (Schubert, 2006). However, this method requires pretreatmentto increase its hydrolysis rate, and these treatments were summa-rized in other reviews (Mosier et al., 2005; Wyman et al., 2005). Ad-ditionally, biomass-degraded microbes have served as a new meansfor the breakdown of lignocellulosic residues, and the enzymes thatwere derived from these microorganisms were promising for indus-trial processes (Gilbert, 2007). Novel enzymes and enzyme systemswere also found despite the fact that they mostly present in microbesthat were difficult to culture (Hugenholtz, 2002). Obviously, the mi-crobial strategies for lignocellulosic biomass degradation have greaterpotential than traditional physicochemical methods due to theirmuch higher efficiencies.

Complete hydrolysis of lignocellulosic materials produce a variety ofsoluble, fermentable sugars that consist mainly of pentoses (e.g., xyloseand arabinose) and hexoses (e.g., glucose and mannose), and the ratioof hexoses to pentoses typically ranges from 1.5:1 to 3:1 (Balan et al.,2008; Balan et al., 2009; Lau and Dale, 2009; Rahman et al., 2006;Sassner et al., 2005). Among these sugars, glucose is usually found inthe highest concentration, and this amount is followed by that of xylose(from hardwoods or agricultural residues) or mannose (from soft-woods) and other sugars, which are present at rather lower concentra-tions (Chu and Lee, 2007). As a result, the microorganisms that arepotentially used for bioconversion must efficiently utilize hexoses andpentoses. More concretely, the highly efficient utilization of lignocellu-losic biomass for the production of valuable chemicals depends onobtaining microorganisms that are capable of using pentoses, whichare difficult to metabolize by most microorganisms, as a carbon sourceby screening the natural surroundings or by mutagenesis using tradi-tional or genetic engineering methods. As a result, finding a pentose-fermenting strain is critical for any bioconversion that uses lignocellu-losic biomass as feedstock.

4.2. Microbial oil production from lignocellulosic biomass

Lignocellulosic biomass has been used in many fields, and someproducts such as ethanol have been successfully scaled up to industri-al levels. However, producing microbial oil from lignocellulosic bio-mass is still at its initial stages. Zhao first stated the possibility ofSCO production on lignocellulosic hydrolysates and mentioned theBM2BD (biomass-to-biodiesel) plan, which states that using lignocel-lulosic biomass for biodiesel production includes three steps: first, lig-nocellulosic biomass is converted into fermentable sugars; second, thesugars are converted intomicrobial lipids by oleaginousmicroorganisms;and third, microbial lipids are translated into biodiesel (Zhao, 2005). Thehydrolysis of lignocellulosic biomasswasfirst accomplishedmore than 30years ago, and many of those technologies are relatively mature now(Gallezot, 2007; Hamelinck et al., 2005; Kumar et al., 2008; Wyman,1994). Furthermore, the bioconversion from lipid to biodiesel, whichincludes chemical and biological methods, has also been studied formany years (Adamczak et al., 2009; Demirbas, 2007). Therefore, more

works have focused on screening suitable strains for lipid production onlignocellulosic hydrolysates. To date, the reported oleaginous microor-ganisms that can use low-cost substrates for SCO production mainly in-clude the following: Yarrowia lipolytica (Papanikolaou and Aggelis,2002; Papanikolaou et al., 2003; Papanikolaou et al., 2006), Lipomycesstarkeyi (Angerbauer et al., 2008; Zhao et al., 2008), Rhodotorula glutinis(Easterling et al., 2009; Xue et al., 2008), and Rhodosporidium toruloides(Akhtar et al., 1998; Li et al., 2007); however, few oleaginousmicroorgan-isms were reported to use lignocellulosic hydrolysates, especially for acidlignocellulosic hydrolysates, to accumulate lipids.

Two problems influence SCO production on lignocellulosic hydro-lysates. First, few oleaginous microorganisms can use xylose for SCOproduction because xylose is the second most abundant componentof lignocellulosic hydrolysates (Rubin, 2008). Fortunately, it hasbeen proven that some oleaginous strains such as Lipomyces starkeyi(Zhao et al., 2008), Mortierella isabellina (Fakas et al., 2009), andTrichosporon fermentans (Zhu et al., 2008) can use xylose for SCO pro-duction. Moreover, T. fermentans has been used in microbial oil pro-duction on rice straw (Huang et al., 2009) and bagasse hydrolysates(Huang et al., 2012b). More recently, five oleaginous yeast strains,Cryptococcus curvatus, Rhodotorula glutinis, Rhodosporidium toruloides,Lipomyces starkeyi, and Yarrowia lipolytica, were used for SCO produc-tion on wheat straw hydrolysates, and Cryptococcus curvatus showedthe highest lipid yield (Yu et al., 2011). Additionally, rice hull(Economou et al., 2011b) and sugarcane bagasse hydrolysates(Tsigie et al., 2011) were utilized by Mortierella isabellina andYarrowia lipolytica for SCO production, respectively. Recently, ourwork has shown that another oleaginous yeast, Trichosporon dermatis,could produce a high lipid yield and lipid coefficient on corncob enzy-matic hydrolysates (Huang et al., 2012a). All of these works showedthe great potential of SCO production from lignocellulosic biomass.

Oleaginous microorganisms must also suffer from inhibitors thatare present in lignocellulosic hydrolysates (Almeida et al., 2007). Dif-ferent methods of detoxification could remove some inhibitors fromlignocellulosic hydrolysates (Palmqvist and Hahn-Hagerdal, 2000a),or adaptation (Martin et al., 2007) or modification (Lewis Liu et al.,2009) of the microorganisms could increase their tolerance to inhib-itors. Studies on the effects of inhibitors on growth and the accumu-lation of metabolites could direct the optimization of detoxificationand fermentation processes and result in strain improvement(Palmqvist and Hahn-Hagerdal, 2000b). Currently, a few works havefocused on the effect of inhibitors on the growth and lipid accumula-tion of oleaginous microorganisms (Chen et al., 2009; Hu et al., 2009;Huang et al., 2011a; Huang et al., 2012c). Obviously, it is necessary toperformmore studies to uncover the mechanisms of the inhibitors onoleaginous strains.

In summary, the possibility and potential of SCO production fromlignocellulosic hydrolysates has been proven. The use of lignocellu-losic hydrolysates as substrates could serve as the basis for the indus-trialization of SCO production. However, many problems in thisprocess still exist that must be solved, and this will be discussed inthe next section.

5. The possibility of industrialization of SCO production

Finally, we will evaluate the possibility of industrialization of SCOproduction. First, it is worth noting that the market price of SCO (datamainly from China, Table 4)makes it desirable for these oils to be indus-trialized in the future. Thus, if we can control the cost of SCO productionso that it is lower than itsmarket price, profits could bemade after its in-dustrialization. Generally, from feedstock to product, the industrializa-tion of SCO production basically includes choosing of the fermentationsubstrates, selection of the oleaginous microorganisms, fermentationand its equipment design and building, downstreamprocessing, packag-ing and selling (Fig. 1). During these processes, many factors must beconsidered to fulfill the industrialization of SCO.

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5.1. Fermentation substrates

Throughout this review, we emphasized the importance of fer-mentation substrates. It is difficult to compare the cost of the differ-ent, low-cost feedstock that was mentioned above because theircosts depended on their supplying sources. Undoubtedly, hydropho-bic materials are critical for producing valuable SCO, especiallycocoa butter substitutes. However, it is worth noting that the avail-ability and quantity of lignocellulosic biomass were significant advan-tages when compared to other low-cost raw materials. Thus, the SCOindustry should be built near lignocellulosic biomass sources such asfarms, forests, and grass, and collecting lignocellulosic biomass suchas straw, bagasse, and corncob should be relatively simple. The priceof some lignocellulosic biomasses in China is shown in Table 3,which indicates that these raw materials are cheap enough for the in-dustrialization of SCO production. To date, the lignocellulosic biomassthat is used for SCO production mainly includes rice straw, rice hull,wheat straw, and sugarcane bagasse (Table 5). To find more suitablelignocellulosic biomasses that can be used for SCO production, it isnecessary to utilize more lignocellulosic biomasses and to comparethe lipid yields that occur on different lignocellulosic hydrolysates(Somerville et al., 2010). Meanwhile, if the fermentation factory isnot located near the feedstock, the cost of the supply chain and trans-portation of lignocellulosic biomass should also be considered(Richard, 2010).

Moreover, most studies on lipid production using lignocellulosicbiomass mainly used simple hydrolysis and detoxification methods,and, thus, the lipid yield on lignocellulosic hydrolysates was low(Economou et al., 2011b; Huang et al., 2009; Huang et al., 2012b;Tsigie et al., 2011; Yu et al., 2011). It is possible that the methods forthe pretreatment and hydrolysis of lignocellulosic biomass were com-plex, and, thus, many researchers focused more on the fermentationprocess instead. The sugar concentration of different lignocellulosic hy-drolysates is usually low,which is not beneficial for the industrializationof SCO production (Economou et al., 2011b; Huang et al., 2009; Tsigie etal., 2011; Yu et al., 2011). Furthermore, most works on SCO productionfrom lignocellulosic biomass used acid for hydrolysis, but fewworks fo-cused on using enzymatic lignocellulosic hydrolysates for microbial oilproduction. Recently, our work showed that corncob enzymatic hydro-lysate could be a promising substrate for SCO production (Huang et al.,2012a). Additionally, by using the patent technology (Chen, 2011) ofZHONGKE New Energy Co. Ltd. (Ying-Kou, Liao-Ning, China), thesugar concentration of lignocellulosic acid hydrolysates could be in-creased to greater than 70 g/L, and a low inhibitor concentration can

Table 4Potential market price of different oilsa.

Function Oils Price

Functional oils, beneficialfor human health

γ-Linolenic acid US $50–100/kgDocosahexaenoicacid

US $100–150/kg

Arachidonic acid US $75–83/kgEicosapentaenoicacid (10%)

US $55–75/kg

Making chocolate,biscuits, and bakedgoods, as well as somepharmaceuticals,ointments, andtoiletries.

Cocoa butter(could besubstituted by SCO)

US $3180/metric ton

Daily edible oil for human Peanut oil US $2200–2800/metric tonCorn oil US $1500–1800/metric tonRapeseed oil US $1692–1794/metric tonSoybean oil US $1505–1630/metric tonOlive oil US $4982–5638/metric ton

Energy and fuels Biodiesel (could beproduced from SCO)

Related to the internationalpetroleum price

a The data were according to information published on www.alibaba.com.

be achieved. Thus, using more well-designed hydrolysis reactionscould increase the sugar concentration for lipid production and reducethe generation of inhibitory by-products during hydrolysis. Technolo-gies for pretreating lignocellulosic biomass have been studied formany years, and many of those technologies are mature enough for in-dustrial production (Hendriks and Zeeman, 2009; Kumar et al., 2009;Wyman et al., 2005). Additionally, detoxification methods have been afocus over the past decades (Mussatto and Roberto, 2004; PalmqvistandHahn-Hagerdal, 2000b). Therefore, using propermethods and tech-nologies for pretreatment, hydrolysis, and detoxification could makethe industrial production of SCO possible in the future.

5.2. Lipid fermentation

5.2.1. Oleaginous microorganismsOleaginous microorganisms whose lipid yield is suitable for indus-

trial production are numerous (Papanikolaou and Aggelis, 2011a;Papanikolaou and Aggelis, 2011b; Ratledge, 2004); however, oleagi-nous microorganisms that can utilize lignocellulosic hydrolysates arefew (Table 5). Therefore, it is necessary to discover more oleaginousstrains that have high lipid yield on lignocellulosic biomass hydroly-sates. Recently, many works showed that oleaginous yeasts that belongto Trichosporon, such as Trichosporon fermentans (Huang et al., 2009),Trichosporon dermatis (Huang et al., 2012a), and Trichosporon cutaneum(Huang et al., 2011b), have great potential for SCO production from lig-nocellulosic biomass. Thus, future works should focus more on theserelatively “new” strains for SCO production. Finally, gene modificationof oleaginous microorganisms is also possible, and many works havecurrently begun to do this critical work (Tang et al., 2009; Tang et al.,2010).

5.2.2. Fermentation modeTo date, SCO production from lignocellulosic biomass was usually

carried out through a batch fermentation mode (Economou et al.,2011b; Huang et al., 2009; Huang et al., 2012b; Tsigie et al., 2011;Yu et al., 2011). On a synthetic medium, the sugar concentration usu-ally caused a substrate inhibitory effect on growth and lipid accumu-lation (Li et al., 2007), and fed-batch fermentation may help toovercome this disadvantage. By utilizing fed-batch fermentation, thebiomass and lipid yield of oleaginous microorganisms could be great-er than 100 and 65 g/L, respectively (Lin et al., 2011). However, nowork has used the fed-batch or continuous mode on SCO productionon lignocellulosic hydrolysates; thus, the lipid yield on lignocellulosichydrolysates is currently barely higher than 15 g/L. It is possible thatusing a fed-batch or continuous fermentation mode could fulfill thehigh-cell-density cultivation on lignocellulosic hydrolysates, andthis is undoubtedly beneficial for the industrialization of SCOproduction.

5.2.3. Simultaneous production of SCO and other valuable by-productsThe theoretical yield of lipids on glucose and xylose is merely 33%

and 34%, respectively (Ratledge, 1988a). However, in most cases, alipid yield of 20–22% is considered ideal (Papanikolaou and Aggelis,2011a). In addition to single cell oils, other chemical compoundscould be present in the cell biomass of an oleaginous microorganism.For example, many oleaginous microorganisms were used to supplysingle cell proteins (Taniguchi et al., 1982; Ziino et al., 1999). Addi-tionally, the biomass of oleaginous microorganisms might containother valuable compounds such as other carbohydrates and nucleicacids (Xiong et al., 2010). For example, the combined production oflipids and carotenoids by the oleaginous yeast Rhodotorula glutiniswould be wise (Saenge et al., 2011). Furthermore, the simultaneousproduction of SCO and extra-cellular compounds such as amylase orpoly-galacturonase was also feasible in practice (Papanikolaou et al.,2003; Papanikolaou et al., 2007), and to obtain high lipid yields onsubstrates, many works also focused on the mass and energy balance

Lignocellulosicbiomass

Different methods for hydrolysis

Different methods for detoxification

Lipid fermentation

Various oleaginous microorganisms

Differentfermentation mode

Biomass collectionWastewaters treatment

Lipid extractionBiodiesel production

Lipid refinery

Strains modifications

Packageand selling

Fermentation equipment design and building

Fig. 1. Process of SCO industrialization using lignocellulosic biomass as substrates.

136 C. Huang et al. / Biotechnology Advances 31 (2013) 129–139

of lipid production by oleaginous microorganisms (Pan and Rhee,1986; Zhou et al., 2011). In addition, during the fermentation process,many gases such as CO2, CO, and H2 might also be generated(Sarapatka, 1993), and these bio-gases could be used in many chem-ical processes. In conclusion, to make industrial production of SCOpossible, comprehensive utilization the by-products is also necessarybecause it can increase the profits of SCO production.

5.2.4. Other related factorsOne important issue during lipid fermentation is the degradation

of previously accumulated lipids by oleaginous microorganisms. Inmany cases, oleaginous microorganisms utilize cellular lipids as a car-bon source to maintain their growth when the carbon sources thatare present in the medium are nearly exhausted (Huang et al.,2009; Huang et al., 2012a). Undoubtedly, this process would signifi-cantly reduce the quantity of lipid that is produced. It is worth notingthat this bioprocess could be controlled (Fakas et al., 2007; Fakas etal., 2008; Papanikolaou and Aggelis, 2011a; Papanikolaou et al.,2001; Papanikolaou et al., 2004b). Moreover, using state-of-the-artmodeling approaches to evaluate the lipid accumulation/degradationprocess might also help to understand and control the “lipid turn-over” phenomenon (Economou et al., 2011a; Meeuwse et al., 2011a;Meeuwse et al., 2011b).

To fulfill high-cell-concentration cultivation, adaption might beanother strategy (Ganuza and Izquierdo, 2007) and could help in

Table 5Microbial oil production on lignocellulosic hydrolysates.

Lignocellulosicbiomass

Strains Reference

Rice straw Trichosporon fermentans (Huang et al., 2009)Wheat straw Cryptococcus curvatus, Rhodotorula glutinis,

Rhodosporidium toruloides, Lipomycesstarkeyi, and Yarrowia lipolytica

(Yu et al., 2011)

Rice hull Mortierella isabellina (Economou et al.,2011b)

Corncob Trichosporon dermatis (Huang et al.,2012a)

Sugarcanebagasse

Trichosporon fermentans (Huang et al., 2012b)Yarrowia lipolytica (Tsigie et al., 2011)

overcoming the inhibition that is present in lignocellulosic hydroly-sates (Huang et al., 2011a). Especially, some special technologiesthat are used in bio-ethanol or bio-butanol production from lignocel-lulosic biomass such as simultaneous saccharification and fermenta-tion (Hari Krishna et al., 2001; Qureshi et al., 2008b) could be usedin microbial oil production when the oleaginous strain has the abilityto accumulate lipid on cellobiose (Huang et al., 2009). However, theoleaginous microorganism should also be adapted to high tempera-ture because saccharification is usually carried out at elevated tem-perature (Olofsson et al., 2008).

Methods for microbial oil extraction are also necessary. Tradition-ally, the extraction of lipids required using an organic solvent such asmethanol and chloroform (Bligh and Dyer, 1959), and many studieshave used this method or a modification of it (Xue et al., 2010; Zhaoet al., 2008; Zhu et al., 2008). Some works attempted to simulta-neously fulfill lipid extraction and transesterification (Carrapiso andGarcía, 2000; Fakas et al., 2009), and accomplishing this could savethe amount of solvent that is used and decrease the productiontime. Therefore, this would be a good direction to explore, especiallywhen a microbial lipid is used as the feedstock for biodieselproduction.

5.3. Outlook

As mentioned above, the industrialization of SCO production re-quires solving many related problems. It is optimistic to see thatusing lignocellulosic biomass could be the basis of industrializingSCO production because its cost is much lower than that of other sub-strates (Table 3). Additionally, this type of biomass is highly availablein nature and has renewable characteristics. Recently, ZHONGKE NewEnergy Co. Ltd. (Ying-Kou, Liao-Ning, China) initially calculated thecost of SCO production from lignocellulosic biomass (Table 6). Gener-ally, by using their novel hydrolysis technology, 1 t of lignocellulosicbiomass could generate approximately 0.45 t of fermentable sugars.Therefore, if the lipid yield from the consumption of these ferment-able sugars was approximately 22%, 10 t of fermentable sugarscould produce approximately 1 t of SCO. In China, the price of differ-ent lignocellulosic residues is approximately 300 RMB/t, and 10 t ofthese residues cost approximately 3000 RMB. The other cost of SCOproduction was relatively fixed and required an additional 4500RMB/t (see Table 6). Thus, the overall cost of SCO production from lig-nocellulosic residues was approximately 7500 RMB/t. As shown in

Table 6Potential cost of 1 t of SCO production from lignocellulosic biomassa.

Different units Content and quantity Cost

Lignocellulosic biomass(corn, straw, bagasseand etc.)

Raw materials for hydrolysis,approximately 10 t

3000RMB

Materials forhydrolysis anddetoxification

Acid, lime, activated carbon and etc. 750 RMB

Energy for differenttreatment

Basically includes electricity, steam 1000RMB

Downstreamprocedure

Collection of biomass, extractionof lipid, biodiesel production or lipid refinery

1500RMB

Wastewaterstreatment

Using traditional IC or UASB for treatment 750 RMB

Wage expense Operation and management 500 RMBTotal 7500

RMB

a Data were kindly offered by the ZHONGKE New Energy Co. Ltd. (Ying-Kou,Liao-Ning, China)

137C. Huang et al. / Biotechnology Advances 31 (2013) 129–139

Table 4, the market price of SCO and its related products is usuallyhigher than 10,000 RMB/t. Thus, using lignocellulosic biomass as asubstrate for SCO production could make significant profits if its relat-ed technology could be applied in its industrialization process. Over-all, if we could handle the above-mentioned problems, the possibilityof SCO industrialization is high and could be fulfilled.

6. Conclusion

Undoubtedly, the market for SCO is attractive due to its importantfunction. Like other bio-refineries, SCO production from lignocellulos-ic biomass is a biological and chemical process. Therefore, to fulfill itsindustrialization, researchers should focus on the lipid accumulationprocess, and the chemical methods, especially hydrolysis andpretreatment technologies, should be mastered by specialists ofSCO. Furthermore, the cooperation between biological and chemicalscientists is critical. Microbial oil production from lignocellulosic bio-mass offers a new direction for bio-refinery, and it will have a greatfuture if the above-mentioned problems are handled properly.

Acknowledgements

The authors acknowledge the financial support of the industrializa-tion project of the High-New Technology of the Guangdong province(2009B011200008), the important project of Knowledge InnovationEngineering at the Chinese Academy of Sciences (KSCX2-YW-G-063)and the support plan project of National Science and Technology(2012BAD32B07).

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