Bioresource Technology 232 (2017) 398–407

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Review

Recent advances and industrial viewpoint for biological treatment

of wastewaters by oleaginous microorganisms
Chao Huang a,b,c, Mu-Tan Luo b,d, Xue-Fang Chen a,b,c, Lian Xiong a,b,c, Xiao-Mei Li a,b,c, Xin-De Chen a,b,c,⇑
a
CAS Key Laboratory of Renewable Energy, Guangzhou 510640, PR China
b
Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou 510640, PR China
c
Guangdong Provincial Key Laboratory of New and Renewable Energy Research and Development, Guangzhou 510640, PR China
d
University of Chinese Academy of Sciences, Beijing 100049, PR China

h i g h l i g h t s

 Biological treatment of wastewaters by oleaginous microorganisms is reviewed.

 The potential of industrialization of this technology is discussed.
 The issues for the industrial application of this technology are presented.
 Critical outlook for development of this technology is given.

a r t i c l e i n f o a b s t r a c t

Article history: Recently, technology of using oleaginous microorganisms for biological treatment of wastewaters has
Received 29 December 2016 become one hot topic in biochemical and environmental engineering for its advantages such as easy
Received in revised form 13 February 2017 for operation in basic bioreactor, having potential to produce valuable bio-products, efficient wastewa-
Accepted 15 February 2017
ters treatment in short period, etc. To promote its industrialization, this article provides some compre-
Available online 20 February 2017
hensive analysis of this technology such as its advances, issues, and outlook especially from industrial
viewpoint. In detail, the types of wastewaters can be treated and the kinds of oleaginous microorganisms
Keywords:
used for biological treatment are introduced, the potential of industrial application and issues (relatively
Biological wastewaters treatment
Oleaginous microorganisms
low COD removal, low lipid yield, cost of operation, and lack of scale up application) of this technology
Industrial application are presented, and some critical outlook mainly on co-culture method, combination with other treat-
Co-culture ments, process controlling and adjusting are discussed systematically. By this article, some important
Combination with other treatments information to develop this technology can be obtained.
Process controlling and adjusting Ó 2017 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
2. Wastewaters treated by oleaginous microorganisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
2.1. Treatment of wastewaters from food industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
2.2. Treatment of municipal wastewater or sludge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
2.3. Treatment of fermentation wastewaters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
3. Oleaginous microorganisms used for biological treatment of wastewaters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401
3.1. Oleaginous yeasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401
3.2. Oleaginous microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401
3.3. Oleaginous fungi and bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402
4. Potential for industrial application of wastewaters treatment by oleaginous microorganisms and the main issues of this technology . . . . . . . 402
4.1. Potential for industrial application of wastewaters treatment by oleaginous microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402
4.2. Issues of wastewaters treatment by oleaginous microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402

⇑ Corresponding author at: No. 2 Nengyuan Road, Tianhe District, Guangzhou 510640, PR China.
E-mail address: cxd_cxd@hotmail.com (X.-D. Chen).

http://dx.doi.org/10.1016/j.biortech.2017.02.055
0960-8524/Ó 2017 Elsevier Ltd. All rights reserved.
 C. Huang et al. / Bioresource Technology 232 (2017) 398–407 399

4.2.1. Low COD removal after biological treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402

4.2.2. Low lipid yield of oleaginous microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402
4.2.3. Cost of operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403
4.2.4. Lack of large scale application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403
5. Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403
5.1. Co-culture of microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403
5.1.1. Co-culture of microorganisms in different fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403
5.1.2. Co-culture of oleaginous microorganisms for biological treatment of wastewaters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403
5.1.3. Co-culture of oleaginous microorganisms and other microorganisms for biological treatment of wastewaters . . . . . . . . . . . . . 404
5.2. Combination with chemical or physical treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404
5.3. Controlling and adjusting the fermentation process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404
6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405

1. Introduction
In modern society, wastewaters are always one critical problem
in city organization and industrial manufacture. Biological treat-
ment of wastewaters is usually applied for many urban or indus-
trial wastewaters in the field of food, fermentation, papermaking,
and so on. For biological treatment of wastewaters, aerobic diges-
tion and anaerobic digestion are two main technologies that both
have a long history (Chan et al., 2009). Usually, these technologies
require some special equipment such as UASB, EASB, etc. (Colussi
et al., 2009; Wei et al., 2011), and thus the upfront investment is
usually high. Recently, wastewaters treatment by the oleaginous
microorganisms attracts much attention for its many advantages:
one the hand, this technology can be carried out in basic bioreactor
and fermenter (the bioreactor and fermenter which can be used for
common fermentation) and thus requires little upfront invest-
ment; on the other hand, this technology can not only treat the
wastewaters efficiently within short period but also can generate
some valuable bio-products (Fig. 1). From the past decades, various
research articles reported the technology of using oleaginous
microorganisms for biological treatment of wastewaters, but few
articles gave some comprehensive analysis of this technology such
as its advances, issues, and outlook especially from industrial view-
point. In this review article, the advances of wastewaters treat-
ment by oleaginous microorganisms are introduced firstly
including the types of wastewaters can be treated and the kinds
of oleaginous microorganisms used for biological treatment, then
the potential of industrial application and the issues of this tech-
nology such as relatively low COD removal, low lipid yield, cost
of operation, and lack of scale up application are presented. Finally,
some critical outlooks of this technology including the application
of co-culture, combination of chemical or physical methods, and
controlling and adjusting the fermentation process are discussed
systematically. Overall, this review article can offer important
information to develop the technology of using oleaginous
microorganisms for biological treatment of wastewaters.
2. Wastewaters treated by oleaginous microorganisms
In recent research articles, food industry wastewaters, munici-
pal wastewater (or sludge), and fermentation wastewaters are
the three main types of wastewaters which can be treated by
oleaginous microorganisms (Table 1). In this part, the details for
these bio-processes are introduced systematically.
2.1. Treatment of wastewaters from food industry
In modern society, food industry is important. Many food pro-
cessing such as olive oil-producing industry (Paraskeva and

Fig. 1. Technology of using oleaginous microorganisms for biological treatment of wastewaters and its advantages.
400 C. Huang et al. / Bioresource Technology 232 (2017) 398–407

Table 1
Biological treatment of some wastewaters by oleaginous microorganisms.

Wastewater Microorganism COD removal (%) Reference

Wastewaters from food industry
Monosodium glutamate wastewater Rhodotorula glutinis 85.51 Xue et al. (2006)
Olive oil mill wastewater Lipomyces starkeyi NAa Yousuf et al. (2010)
Potato processing wastewater Aspergillus oryzae 74.2 Muniraj et al. (2013)
Soybean processing wastewater Chlorella pyrenoidosa 77.8 Hongyang et al. (2011)
Starch wastewater Rhodotorula glutinis 80 Xue et al. (2010)
Municipal wastewater or sludge
Sewage sludge Lipomyces starkeyi NA Angerbauer et al. (2008)
Paper mill sludge Cryptococcus vishniaccii NA Deeba et al. (2016)
Municipal wastewater Rhodotorula glutinis, Rhorosporidium toruloides, and Cryptococcus curvatus NA Pirozzi et al. (2013)
Municipal wastewater Co-culture of oleaginous yeast and algae 88 Chi et al. (2011)
Municipal wastewater Oleaginous consortium (yeast and bacteria) 81 Hall et al. (2011)
Fermentation wastewaters
Brewery effluents Rhodotorula glutinis NA Schneider et al. (2013)
Bioethanol wastewater Rhodosporidium toruloides 72.3 Zhou et al. (2013)
Distillery wastewater Cryptococcus curvatus 80 Gonzalez-Garcia et al. (2013)
Distillery wastewater Rhodotorula glutinis 84 Gonzalez-Garcia et al. (2013)
ABE fermentation wastewater Trichosporon coremiiforme 68.0 Chen et al. (2012)
ABE fermentation wastewater Trichosporon dermatis 68.2 Peng et al. (2013)
ABE fermentation wastewater Trichosporon cutaneum 68.0 Xiong et al. (2015)
Fermentation effluent in Riboflavin (B2) Chlorella pyrenoidosa 89.2 Sun et al. (2013)
a
NA means not available.

Diamadopoulos, 2006), starch industry (Field et al., 1987), monoso-
dium glutamate industry (Liu et al., 2007), etc. will generate great
number of wastewaters. Usually, wastewaters from food industry
has high BOD5/COD (high fermentability) and thus can be treated
by traditional anaerobic digestion (Hassan and Nelson, 2012).
Recently, many researches showed that oleaginous microorgan-
isms can also be used for the treatment of wastewaters from food
industry. Firstly, oleaginous yeast Rhodotorula glutinis was used for
microbial oil production in monosodium glutamate wastewater
that the COD removal could reach 85.51%, but the lipid yield was
not high (less than 0.25 g/L) (Xue et al., 2006). However, the lipid
yield could increase greatly by adding glucose to monosodium glu-
tamate wastewater with the strategy of glucose feedback addition
(Xue et al., 2008). For another, oleaginous yeast Lipomyces starkeyi
was used to treat olive oil mill wastewater and generate lipids suit-
able for biodiesel production (Yousuf et al., 2010). In addition,
oleaginous filamentous fungus Aspergillus oryzae was used to treat
potato processing wastewater that the removal of COD was 74.2%
(Muniraj et al., 2013). Moreover, it is found that soybean process-
ing wastewater could be used for the cultivation of oleaginous
microalga Chlorella pyrenoidosa and the COD removal was close
to 80% (Hongyang et al., 2011). Additionally, starch wastewater
was used as raw material for microbial lipid production by Rhodo-
torula glutinis in pilot-scale and this initially showed the potential
of industrial application of using oleaginous microorganisms for
wastewaters treatment (Xue et al., 2010).
2.2. Treatment of municipal wastewater or sludge
As the increase of human population and development of indus-
try, the treatment of municipal wastewater attracts more and more
attention. Traditionally, anaerobic–aerobic treatment is the basic
method for the treatment of municipal wastewater (Chan et al.,
2009). At the same time, sewage sludge, the residue produced dur-
ing the process of wastewater treatment, its treatment becomes
another challenge in wastewater management (Fytili and
Zabaniotou, 2008). In the recent years, oleaginous microorganisms
were proven to be possible to treat the municipal wastewater or
sludge. For example, oleaginous yeast Lipomyces starkeyi was used
to treat sewage sludge and accumulate microbial lipid that the
lipid content could be as high as 68% (Angerbauer et al., 2008).
Besides sewage sludge, it is also found that paper mill sludge can
be converted into neutral lipids by oleaginous yeast Cryptococcus
vishniaccii for biodiesel production (Deeba et al., 2016). On the
other hand, oleaginous yeasts such as Rhodotorula glutinis,
Rhorosporidium toruloides, and Cryptococcus curvatus, could be used
for municipal wastewater treatment (Pirozzi et al., 2013). For the
treatment of municipal wastewater, co-culture of various microor-
ganisms such as oleaginous yeast and algae could be one efficient
strategy (Chi et al., 2011). Also, nine oleaginous yeasts and one
oleaginous bacterium were used for municipal wastewater treat-
ment and the COD removal was approximately 81% (Hall et al.,
2011).
2.3. Treatment of fermentation wastewaters
Fermentation is one important biological technology that can
synthesize various valuable compounds to satisfy the requirement
of chemical industry, energy, food, etc. However, various wastew-
aters are usually generated after fermentation. Generally, the bio-
logical treatment of fermentation wastewaters usually uses
anaerobic–aerobic method by activated sludge with some special
equipment (Lu et al., 1999; Pant and Adholeya, 2007). In the past
years, it is also found that oleaginous microorganisms can treat
the wastewaters from different fermentation industries. Ethanol
fermentation will generate large amount of wastewater especially
after recovery of the products from fermentation broth by distilla-
tion. This wastewater was shown as one promising substrate for
oleaginous microorganisms. For instance, brewery effluent was
used as fermentation substrate to accumulate microbial oil and
carotenoid simultaneously by oleaginous yeast Rhodotorula glutinis
(Schneider et al., 2013). For another, oleaginous yeast Rhodosporid-
ium toruloides was used to treat bioethanol wastewater, and the
COD removal, yeast biomass, lipid content could reach 72.3%,
3.8 g/L, and 34.9%, respectively (Zhou et al., 2013). In addition,
oleaginous yeasts Rhodotorula glutinis and Cryptococcus curvatus
were used to treat distillery wastewater, and the COD removal
could reach 80% (Gonzalez-Garcia et al., 2013). Similar to ethanol
fermentation, ABE (acetone-butanol-ethanol) fermentation also
generates great amount of wastewater after recovery of the
 C. Huang et al. / Bioresource Technology 232 (2017) 398–407 401

Table 2
Oleaginous microorganisms used for wastewaters treatment.

Microorganism Substrate Biomass (g/ Lipid content Reference

L) (%)
Oleaginous yeasts
Rhodotorula glutinis Brewery effluents 5.22 <15 Schneider et al. (2013)
Rhodotorula Olive mill wastewater 9.61 NAa Jarboui et al. (2013)
mucilaginosa
Trichosporon cutaneum ABE fermentation wastewater 4.9 14.7 Xiong et al. (2015)
Trichosporon dermatis ABE fermentation wastewater 7.4 13.5 Peng et al. (2013)
Trichosporon ABE fermentation wastewater 5.8 19.1 Chen et al. (2012)
coremiiforme
Cryptococcus curvatus Distillery wastewater 5.19 25.2 Gonzalez-Garcia et al.
(2013)
Cryptococcus albidus Pulp and paper mill effluent NA NA Singhal and Thakur (2012)
Cryptococcus laurentii Winery wastewater NA NA Santos et al. (2014)
Cryptococcus podzolicus Pulp mill wastewater NA NA Fernandes et al. (2014)
Rhodosporidium Distillery wastewater 8.12 43.65 Ling et al. (2013)
toruloides
Lipomyces starkeyi Potato starch wastewater 2.59 8.88 Liu et al. (2013)
Yarrowia lipolytica Olive mill wastewater-based media 7.0 17.1 Sarris et al. (2011)
Oleaginous microalgae
Chlorella vulgaris Artificial wastewater 0.69 42 Feng et al. (2011)
Chlorella protothecoides Thiocyanate wastewater 1.3 30.6 Ryu et al. (2014)
Chlorella pyrenoidosa Piggery wastewater Close to 0.3 Close to 15 Wang et al. (2012)
Chlorella ellipsoidea Domestic secondary effluent 0.425 43 Yang et al. (2011)
Scenedesmus acutus Municipal wastewater 1.18 28.3 de Alva et al. (2013)
Scenedesmus obliquus Fish pond (FP) discharges, and municipal secondary settling tank (MSST) 2.0 52.5 Mandal and Mallick (2011)
discharges
Scenedesmus bijuga Food wastewater effluent 1.49 35.06 Shin et al. (2015)
Neochloris Anaerobically digested dairy manure Close to 1.0 14.8 Levine et al. (2011)
oleoabundans
Botryococcus braunii Nitrate-rich wastewater supplemented with CO2 2.26 30.3 Yeesang and Cheirsilp
(2014)
Arthrospira platensis Olive-oil mill wastewater 1.70 16.91 Markou et al. (2012)
Oleaginous fungi and bacteria
Aspergillus oryzae Potato processing wastewater NA (Highest lipid yield was Muniraj et al. (2013)
3.5 g/L)
Mucor circinelloides Wastewaters from equalization tank 0.60 22.11 Bhanja et al. (2014)
Trichoderma reesei Wastewaters from equalization tank 0.68 9.82 Bhanja et al. (2014)
Rhodococcus opacus Primary effluent wastewater NA NA Hall et al. (2011)
Rhodococcus sp. RHA1 Chemithermomechanical pulping effluent NA NA Du et al. (2011)
a
NA means not available.

products from fermentation broth by distillation. It is also shown
that oleaginous microorganisms can treat the ABE fermentation
wastewater that the COD removal was close to 70% (Chen et al.,
2012; Peng et al., 2013; Xiong et al., 2015). Other wastewaters
from fermentation can also be treated by oleaginous microorgan-
isms. For instance, oleaginous microalga Chlorella pyrenoidosa
was used to treat fermentation effluent in Riboflavin (B2) and the
COD removal was close to 90% (Sun et al., 2013).
3. Oleaginous microorganisms used for biological treatment of
wastewaters
Generally, various microorganisms including yeasts, algae,
fungi, and bacteria could accumulate lipid to 20% of their dry
weight, and so called oleaginous microorganisms (Ratledge,
1991). To date, all kinds of these microorganisms have been uti-
lized for biological treatment of different wastewaters (Table 2)
and the details are introduced in the following parts.
3.1. Oleaginous yeasts
Recently, many oleaginous yeasts have been applied for micro-
bial oil production due to their excellent capacity for substrate con-
sumption, lipid production, fast growth rate, easy for culture,
stable yield, etc. (Sitepu et al., 2014). For the biological treatment
of wastewaters, the oleaginous yeasts used mainly includes the
yeasts from genus of Rhodotorula such as Rhodotorula glutinis
(Cheirsilp et al., 2011; Chi et al., 2011; Schneider et al., 2013;
Xue et al., 2008) and Rhodotorula mucilaginosa (Jarboui et al.,
2012, 2013), genus of Trichosporon such as Trichosporon cutaneum
(Dragičević et al., 2010; Xiong et al., 2015), Trichosporon dermatis
(Peng et al., 2013), and Trichosporon coremiiforme (Chen et al.,
2012), genus of Cryptococcus such as Cryptococcus curvatus (Chi
et al., 2011; Gonzalez-Garcia et al., 2013), Cryptococcus albidus
(Singhal and Thakur, 2012), Cryptococcus laurentii (Santos et al.,
2014), and Cryptococcus podzolicus (Fernandes et al., 2014), and
some other kinds of oleaginous yeasts such as Rhodosporidium
toruloides (Ling et al., 2013; Zhou et al., 2013), Lipomyces starkeyi
(Angerbauer et al., 2008; Huang et al., 2011; Liu et al., 2013), Yar-
rowia lipolytica (Lan et al., 2009; Lanciotti et al., 2005; Sarris et al.,
2011) and so on.
3.2. Oleaginous microalgae
For a long time, oleaginous microalga is considered as one
important oleaginous microorganism for bio-fuel production
(Lam and Lee, 2012). In spite that oleaginous microalga seldom
uses some special substrates such as lignocellulosic hydrolysates
for fermentation (Huang et al., 2013), but it has many advantages
which make it has great potential for wastewaters treatment. Com-
pared with yeast, microalga can survive in the wastewaters with
low nutrient concentration due to its autotrophic character (Cai
402 C. Huang et al. / Bioresource Technology 232 (2017) 398–407
et al., 2013). More specially, it requires little aseptic environment
and thus can be operated in the ‘‘open-door” environment
(Ratledge and Cohen, 2008). In addition, many by-products such
as b-carotene and algal polysaccharide with high value can also
be synthesized by different microalgae (Raja et al., 2007; Zhu
et al., 2014). Although the growth period of microalga is relatively
longer when compared with other microorganisms and its collec-
tion is also one industrial issue (Barros et al., 2015), it still has great
potential for industrialization for considering its above excellent
capacity. For the biological treatment of wastewaters, the oleagi-
nous microalgae used mainly includes the microalgae from genus
of Chlorella such as Chlorella vulgaris (Feng et al., 2011; Ji et al.,
2013), Chlorella protothecoides (Ryu et al., 2014), Chlorella pyrenoi-
dosa (Wang et al., 2012), and Chlorella ellipsoidea (Yang et al.,
2011), genus of Scenedesmus such as Scenedesmus acutus (de Alva
et al., 2013), Scenedesmus obliquus (Mandal and Mallick, 2011),
and Scenedesmus bijuga (Shin et al., 2015), and some other kinds
of oleaginous microalgae such as Neochloris oleoabundans (Levine
et al., 2011), Botryococcus braunii (Yeesang and Cheirsilp, 2014),
Arthrospira platensis (Markou et al., 2012) and so on.
3.3. Oleaginous fungi and bacteria
Compared with microalgae and yeasts, fungi and bacteria were
applied relatively less in biological treatment of wastewaters.
However, these microorganisms could generate many special
products which yeasts and microalgae seldom accumulate
(Sancholle et al., 2004; Wältermann et al., 2005) and thus they
have special application in industry. To date, the oleaginous fungi
and bacteria applied for biological treatment of wastewaters
mainly include Aspergillus oryzae (Muniraj et al., 2013), Mucor
circinelloides and Trichoderma reesei (Bhanja et al., 2014), Rhodococ-
cus opacus (Hall et al., 2011), Rhodococcus sp. (Du et al., 2011) and
so on.
4. Potential for industrial application of wastewaters treatment
by oleaginous microorganisms and the main issues of this
technology
4.1. Potential for industrial application of wastewaters treatment by
oleaginous microorganisms
Basically, the industrialization of biochemical technology
requires many factors (Saxena et al., 2009). The substrate is the
most critical one that a suitable substrate for industrial biochemi-
cal technology should be low-cost and available (Huang et al.,
2013). Wastewater is usually wastes from industry, agriculture or
city, and thus its cost is extremely low. In addition, various
wastewaters exist from different place, thus it has great availabil-
ity. Therefore, wastewater can be considered as one promising sub-
strate for industrial biochemical technology. Besides substrate, the
cost of bioconversion is also important. The biological treatment of
wastewaters by oleaginous microorganisms merely requires a
short fermentation period (usually several days), and moderate
temperature (usually room temperature is suitable), and thus the
energy consumption of this technology is small. The basic fermen-
tation equipment could be used for this bioconversion and there-
fore it requires no extra and expensive building of new
equipment. In addition, the floor area of this technology is less than
that of traditional biological treatment. Overall, the cost of this bio-
conversion is suitable for industrial application.
Usually, the downstream technology plays an important role in
biochemical engineering. For the biological treatment of wastewa-
ters by oleaginous microorganisms, the downstream technology
mainly includes two parts, namely the biomass harvesting and
collection, and microbial lipid recovery. For biomass harvesting
and collection, both centrifugation and flocculation can be applied
(Christenson and Sims, 2011). For lipid recovery, many methods
for cell disruption (ultra-sonication, high-pressure homogeniza-
tion, French pressing, bead milling, microwave, chemical lysis,
osmotic shock) and lipid extraction (organic solvent extraction,
supercritical fluid extraction, ultrasound-assisted extraction,
microwave-assisted extraction) are used widely (Halim et al.,
2012). It is worth noting that all these technologies should avoid
high temperature and pressure to reduce cost in industrial
application.
Considering the cost from upstream to downstream technology,
the biological treatment of wastewaters by oleaginous microor-
ganisms has great potential for industrialization. To date, some
pilot scale researches have been carried out for the biological treat-
ment of wastewaters by oleaginous microorganisms (Fernandes
et al., 2014; Xue et al., 2010; Zhu et al., 2013) and the information
is critical for the industrial application because it proves the possi-
bility of using this technology in large scale. In future, more appli-
cation should be carried out in various industries.
4.2. Issues of wastewaters treatment by oleaginous microorganisms
4.2.1. Low COD removal after biological treatment
As one novel technology for biological treatment of wastewa-
ters, although it has a relatively short period for treatment, the
COD degradation of biological treatment by oleaginous microor-
ganisms is still usually lower than that of traditional technologies
of both aerobic and anaerobic ones. In fact, by the traditional
anaerobic–aerobic biological treatment, the total COD removal of
industrial and municipal wastewaters can be higher than 80%
and even higher than 90% (Chan et al., 2009). And in many situa-
tions, the biological treatment of wastewaters by oleaginous
microorganisms had a relatively low COD removal and even lower
than 80% (Table 1). Undoubtedly, relatively low COD removal of
this technology makes it less competitive for industrial application.
Therefore, to fulfill the industrialization of wastewaters treatment
by oleaginous microorganisms, the most important mission is to
increase the COD removal of this technology to satisfy the require-
ment of water standard.
4.2.2. Low lipid yield of oleaginous microorganisms
Compared with traditional lipid fermentation on different
media containing high concentration of fermentation substrates
(Huang et al., 2013; Sitepu et al., 2014), the biomass and lipid con-
tent of oleaginous microorganisms in different wastewaters are
small (even less than 2.0 g/L and 20%, respectively, see Table 2)
and thus the lipid yield of oleaginous microorganisms is low. If
the microbial oil is the final products, the economic value of this
bioconversion is undoubtedly less competitive. The unsuitable C/
N ratio of wastewaters for lipid synthesis might explain this phe-
nomenon (Papanikolaou and Aggelis, 2011). To increase the lipid
yield of oleaginous microorganisms in wastewaters, more
exogenous carbon sources could be added into different media
(Huang et al., 2011; Xue et al., 2008), but this will also increase
the cost of substrate. Thus, unless the method to increase the lipid
yield of oleaginous microorganisms could be found from either
genetic or engineering viewpoint, the better idea is still explore
the value of by-products of lipid fermentation. In fact, besides
microbial lipid, many other valuable products such as chlorophyll
(Farooq et al., 2013; Li et al., 2008), carotenoid (Saenge et al.,
2011; Schneider et al., 2013), polysaccharides (Xiong et al., 2016;
Zhu et al., 2014), citric acid (Lanciotti et al., 2005; Sarris et al.,
2011), and even the biomass of oleaginous microorganisms
(Zheng et al., 2005) might also be generated during the biological
treatment of wastewaters by oleaginous microorganisms. Thus, it
 C. Huang et al. / Bioresource Technology 232 (2017) 398–407
Table 3
Potential market price of different products generated from fermentation of
oleaginous microorganisms.a
Product Function and application
Chlorophyll Cancer prevention, liver protection,
healing of stomach ulcers and intestine
ulcers, antibacterial, removal of odors;
Mainly applied as pharmaceutical stuff,
food pigment, and daily use
Carotenoid Promoting the healthy of eye and skin,
eliminating free-radicals, improving
immunity, preventing cardiopathy and
cancer, resisting arteriosclerosis, etc.;
Mainly applied in food field,
pharmaceutical field, and cosmetics field
Polysaccharides Reducing blood pressure, controlling blood
sugar levels, using as natural flavor
enhancer, stimulating immune organ
development, enhancing anti-virus
function and reducing the infection of
pathogens, promoting animal growth, etc.;
Mainly applied in food field,
pharmaceutical field, and cosmetic field
Citric acid Mainly used as flavoring agent,
preservative and antistaling agent in food
and beverage industry, used as
antioxidant, plasticizer, detergent in
chemical, cosmetics and detergent
industries
Microbial Mainly used as feed for animals (cattle,
biomass poultry, pigs, fish, etc.)
a
The data were according to information published on www.alibaba.com.
is wise and necessary to explore the collection, separation, and
purification of these products to increase the profits of the whole
technology (Table 3).
4.2.3. Cost of operation
As mentioned above, the cost of biological wastewaters treat-
ment by oleaginous microorganisms is suitable for industrial appli-
cation, but for the actual industrialization, more factors on the
practical cost should be considered more. First of all, in spite that
this bioconversion has great potential for industrialization due to
the almost ‘‘zero” cost of wastewaters, it still needs to consider
the location of the fermentation plant. In fact, most wastewaters
used for biological treatment by oleaginous microorganisms come
from different industrial plants (see references in Table 1), and
therefore the cost of transportation should be added into the total
cost of this technology. In practical application, the bioreactor for
oleaginous microorganisms should be built near the sources of
wastewaters and thus the cost of transportation can be saved. As
mentioned above, basic bioreactor can be applied for the wastew-
aters treatment by oleaginous microorganisms, but for some
industries such as olive oil producing industry (Paraskeva and
Diamadopoulos, 2006), soybean processing industry (Hongyang
et al., 2011), and paper mill processing industry (Deeba et al.,
2016), basic bioreactor must also be built for the wastewaters
treatment. Considering the equipment building cost, the biological
treatment by oleaginous microorganisms is most suitable for fer-
mentation industry because the fermentation equipment could
also be used for the wastewaters treatment by oleaginous microor-
ganisms (see references in Table 1). In some situations, pretreat-
ment of wastewaters (Deeba et al., 2016; Ji et al., 2013) will also
increase the total cost of this technology and this should be consid-
ered in the industrial application. Last but not least, downstream
technology of lipid fermentation including biomass collection
(Christenson and Sims, 2011) and lipid recovery (Halim et al.,
2012) will also increase the total cost of this technology. Overall,
403
the cost of operation should be considered for all aspects and not
merely for the ‘‘wastewater” itself.
Market price
4.2.4. Lack of large scale application
US $20–200/ Although the possibility and potential of using oleaginous
Kilogram
microorganisms for wastewaters treatment have been proven,
and some scale up of this technology have been carried out as well
(Fernandes et al., 2014; Xue et al., 2010; Zhu et al., 2013), most
US $20–500/ researches introduced above are still in laboratory scale. Thus,
Kilogram more researches should be focused on the scale up of this technol-
ogy and only when this technology applied in pilot or industrial
scale long term experiments with many examples can make it
more possible for industrialization.
US $20–300/
Kilogram
5. Outlook
5.1. Co-culture of microorganisms
5.1.1. Co-culture of microorganisms in different fields
US $700– Traditionally, the core of anaerobic-aerobic treatment of
1000/Metric organic wastewaters is activated sludge. It is worth noting that
Ton
activated sludge is not pure microorganism and it is one complex
microbial population containing bacteria, fungi, protozoa, meta-
zoan, etc. (Eckenfelder, 1998; Seviour and Blackall, 2012). The co-
US $1–1.5/ culture of various microorganisms makes the COD of organic
Kilogram
wastewaters decrease efficiently. In fact, co-culture is one
advanced technology which has been applied widely in the fields
of chemical engineering, food, environment etc. For chemical engi-
neering, co-culture has been applied in the fermentation system of
organic acids production before 1990s (Tang et al., 1988). Nowa-
days, co-culture is usually used in ethanol production and the
related review reported that more than 25 co-culture systems in
100 references, showing that the utilization of glucose and xylose
were more efficiently in co-culture fermentation system (Chen,
2011). Specially, co-culture is usually used in bioethanol produc-
tion by simultaneous saccharification and fermentation on ligno-
cellulosic biomass (Golias et al., 2002). Other alcohol compounds
fermentation such as ABE fermentation can also use co-culture to
improve products yield (Tran et al., 2010). Also, simultaneous sac-
charification and fermentation by co-culture can be used for
hydrogen production (Wang et al., 2008). For food industry, early
co-culture was usually applied for increasing the protein content
of food (Bhalla and Joshi, 1994). Also, co-culture can offer an addi-
tional safety provision in Cheddar cheese manufacture (Moreno
et al., 2003). Besides, co-culture system was also applied in Wine
Sauvignon Blanc production (Sadoudi et al., 2012), malolactic fer-
mentation (Nehme et al., 2010), and cheese processing industry
(Buriti et al., 2007). For environmental engineering, co-culture is
one common mode: as mentioned above, activated sludge is one
natural microorganism population (Eckenfelder, 1998; Seviour
and Blackall, 2012). Besides, the bio-degradation of organic mate-
rials such as benzene, toluene, ethylbenzene and xylene can also
be fulfilled by co-culture method (Shim et al., 2002).
5.1.2. Co-culture of oleaginous microorganisms for biological
treatment of wastewaters
Although co-culture has been widely used in many areas, the
study on biological treatment of wastewaters by co-culture of
oleaginous microorganisms is just at its beginning. Co-culture of
oleaginous microorganisms belong to similar kinds has been
applied successfully for biological treatment of wastewaters. For
instance, co-culture of different oleaginous yeasts from the genus
of Trichosporon was used to treat ABE fermentation wastewater
(Xiong et al., 2016). Besides co-culture of oleaginous yeasts, het-
erotrophic cultivation of mixed microalgae was also proven to be
404 C. Huang et al. / Bioresource Technology 232 (2017) 398–407
one suitable mode for lipid accumulation and wastewater treat-
ment during sequential growth and starvation phases (Devi et al.,
2012).
Compared with co-culture of oleaginous microorganisms
belong to similar kinds, co-culture of oleaginous microorganisms
belong to different kinds has more potential for biological treat-
ment of wastewaters because wastewaters treatment by co-
culture is based on the different capacity of substrate metabolism
among various microorganisms and the capacity of oleaginous
microorganisms belong to different kinds varies more greatly than
that belongs to similar kinds. Biological treatment of wastewaters
by co-culture of oleaginous microorganisms belong to different
kinds also has some successful examples. For the first one, a con-
sortium consisted of nine known oleaginous yeast (Rhodotorula
glutinis, Cryptococcus curvatus, Cryptococcus albidus, Candida valida,
Candida utilis, Codermyces poitrasii, Rhodosporidium toruloides,
Lipomyces starkeyi, and Pichia angusta) and one oleaginous bacterial
species (Rhodococcus opacus) was used to treat primary effluent
wastewater (Hall et al., 2011). For another, a two-step biological
process of oleaginous yeasts (Cryptococcus curvatus, Yarrowia
lipolytica, and Rhodotorula glutinis) and phototrophic oleaginous
algae (Chlorella sorokiniana) was applied for food waste and munic-
ipal wastewater treatment (Chi et al., 2011). Moreover, a mixed
culture of oleaginous yeast Rhodotorula glutinis and oleaginous
microalga Chlorella vulgaris was carried out to enhance lipid pro-
duction from the effluent from steamed fish process (Cheirsilp
et al., 2011).
5.1.3. Co-culture of oleaginous microorganisms and other
microorganisms for biological treatment of wastewaters
In many situations, merely co-culture of oleaginous microor-
ganisms might not be enough for the biological treatment of
wastewaters. Thus, it is necessary to co-culture of oleaginous
microorganisms and other microorganisms to obtain more COD
or other nutrients removal. For co-culture of oleaginous microor-
ganisms and other microorganisms, microalgae–bacteria-based
system was applied commonly to treat wastewaters and produce
biodiesel and chemical products, and this technology has been
reviewed in some articles (Olguín, 2012). More recently, a cooper-
ative algal-bacterial system that efficiently degrades thiocyanate
(SCN ) was used to prevent the high inhibition on lipid production
and final treat the thiocyanate-containing wastewater efficiently
and obtain a high lipid yield (Ryu et al., 2014). For another, a
two-stage fermentation with bacterium Ethanoligenens harbinense
B49 and oleaginous microalga Scenedesmus sp. R-16 was applied
to increase the energy conversion efficiency and COD removal effi-
ciency (Ren et al., 2014). Besides co-culture of oleaginous microal-
gae and other microorganisms, co-culture of oleaginous yeast and
other microorganism can be also used for biological treatment of
wastewaters. For instance, oleaginous yeast Trichosporon cutaneum
under aerobic conditions and Geotrichum candidum under faculta-
tive anaerobic conditions in a two-step biodegradation process
was successfully used to remove COD, phenolic compounds, and
dark color of olive mill wastewater (Dragičević et al., 2010). For
another, oleaginous yeast Rhodotorula mucilaginosa and fungi
Aspergillus niger were used for olive mill wastewater (OMW) bio-
logical treatment in single pure and mixed cultures (Jarboui
et al., 2013). In addition, co-culture of oleaginous yeast Rhodotorula
glutinis and halotolerant yeasts Candida halophila was applied for
the biological treatment of glutamate fermentation wastewater
(Zheng et al., 2005). More specially, co-culture of oleaginous
microorganisms and activated sludge can be also used for biologi-
cal treatment of wastewaters. For example, two-stage biological
treatment by anaerobic digestion of dairy manure and later fer-
mentation by oleaginous green alga Neochloris oleoabundans was
applied to assimilate 90–95% of the initial nitrate and ammonium
and offer lipid feedstock for biodiesel production (Levine et al.,
2011).
5.2. Combination with chemical or physical treatment
It is possible that some wastewaters contain materials that bio-
logical methods cannot remove completely even using ‘‘co-culture”
mode, and the COD degradation is still lower than that of tradi-
tional aerobic and anaerobic technologies. In this situation, it is
necessary to accompany with some other chemical or physical
methods to fulfill the efficient wastewaters treatment. Undoubt-
edly, this will increase the total cost of wastewaters treatment.
Thus, when in industrial application, it is wise to has one compre-
hensive evaluation on the technologies and equipment used to
make correct choice.
Fenton process is one classical chemical method for wastewa-
ters treatment (Babuponnusami and Muthukumar, 2014), and the
combination of Fenton method and biological method has great
potential to obtain high COD removal in actual wastewaters treat-
ment. For instance, the biological treatment by oleaginous yeast
Cryptococcus laurentii and chemical treatment by Fenton’s reagent
were combined to treat winery wastewater, and a total COD
removal of 98% can be obtained (Santos et al., 2014). Similarly, bio-
logical treatment by oleaginous yeast Cryptococcus podzolicus and
solar photo-Fenton treatment were used together to treat pulp mill
wastewater, and the result showed that the technology could offer
an efficient and practical improvement for current company
wastewater treatment plant (Fernandes et al., 2014).
For physical treatment of wastewaters, adsorption is usually
used for removal of many materials with different absorbents
(Gupta et al., 2009), and using this method can help removing
the materials which are difficult to be removed by merely biolog-
ical treatment with oleaginous microorganisms. For example,
micro/mesoporous hyper-cross-linked polymeric adsorbent can
be used for ABE fermentation wastewater treatment (Lin et al.,
2015), and therefore can be one supplement for biological treat-
ment by oleaginous microorganisms (Chen et al., 2012; Peng
et al., 2013; Xiong et al., 2015). For another, activated carbon and
polymer resin were proven to be suitable absorbent for the treat-
ment of effluents of a Kraft pulp mill (Zhang and Chuang, 2001),
and thus can be supplement for biological treatment by oleaginous
microorganisms (Fernandes et al., 2014; Singhal and Thakur,
2012).
5.3. Controlling and adjusting the fermentation process
Besides above methods, controlling and adjusting the fermenta-
tion process can also be applied to increase the COD and nutrients
removal, and products accumulation of biological wastewaters
treatment by oleaginous microorganisms. For one fermentation
process, medium composition (such as carbon sources, nitrogen
sources, trace elements, etc.) and fermentation condition (such as
inoculum concentration, pH value, temperature, etc.) are two main
factors affecting the products yield and COD removal. For instance,
the optimization of medium composition and fermentation
condition could be used to increase both COD removal and lipid
production (Cheirsilp et al., 2011). For another, oleaginous yeast
Rhodosporidium toruloides was used to treat distillery wastewater
and the lipid production, and COD, TN, and TP removal can be
increased simply by increasing cell density (Ling et al., 2013).
Usually, high initial COD concentration might prevent efficient
COD removal possibly due to the ‘‘substrate inhibition”. To over-
come the influence of high initial COD concentration, dilution of
the wastewater effluent can increase the nutrient removal and
lipid production for biological treatment by oleaginous microor-
ganisms (Ji et al., 2013). On the other hand, it is possible that the
 C. Huang et al. / Bioresource Technology 232 (2017) 398–407
Fig. 2. Technological development of using oleaginous microorganisms for biological treatment of wastewaters.
nutrients in the wastewaters were not enough for lipid fermenta-
tion, and thus adding extra nutrients can increase the biomass
and lipid content of oleaginous microorganisms (Xue et al.,
2008). Recently, advanced fermentation mode was proven to be
beneficial for biological treatment of wastewaters by oleaginous
microorganisms. For example, for the biological wastewaters treat-
ment by oleaginous microalgae, cultivation system in the two-
stage photoautotrophic–photoheterotrophic/mixotrophic mode
was useful to increase the lipid productivity (Farooq et al., 2013).
Besides medium composition and fermentation condition,
microorganism is also one important factor for the biological
wastewaters treatment by oleaginous microorganisms. For
instance, immobilization of yeast cells was found to be beneficial
for the COD and oil removal of oil-containing wastewater by the
treatment of oleaginous yeast Yarrowia lipolytica (Lan et al., 2009).
In summary, development for the technology of using oleagi-
nous microorganisms for biological treatment of wastewaters is
not one simple mission and it requires technology integration of
the outlook mentioned above (Fig. 2), and using other methods
of biological, chemical, and physical ones comprehensively to sat-
isfy different requirements of various industries.
6. Conclusion
From the past decades, technology of using oleaginous microor-
ganisms for biological treatment of wastewaters has become one
hot topic in biochemical and environmental engineering, and it
has great potential for industrial application for its advantages of
both removal of the COD or nutrients in wastewaters and genera-
tion of valuable products. Future researches should focus more on
the development of this technology by the methods of co-culture,
combination with chemical or physical treatment, controlling and
adjusting the fermentation process, etc. accompany with the effort
of exploring the by-products of lipid fermentation by the long term
large scale experiments.
Acknowledgements
The authors acknowledge the financial support of National Nat-
ural Science Foundation of China (51378486, 21606229,
51508547), project of Guangzhou Science and Technology-Pearl
River S&T Nova Program of Guangzhou (201610010014), Youth
Innovation Promotion Association CAS (2015290), project of
Huai-An Science and Technology (HAS2015035), the Science and
Technology Project of Guangdong Province (2016A010105016,
405
2016A010104009), and the Key Research and Development Plan
of Jiangsu Province, China (BE2016706).
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