Separation and Purification Technology 107 (2013) 158–165

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Separation and Purification Technology

journal homepage: www.elsevier.com/locate/seppur

Filamentous fungi assisted bio-flocculation: A novel alternative technique

for harvesting heterotrophic and autotrophic microalgal cells
Wenguang Zhou, Min Min, Bing Hu, Xiaochen Ma, Yuhuan Liu, Qin Wang, Jian Shi, Paul Chen,
Roger Ruan ⇑
Center for Biorefining, Bioproducts and Biosystems Engineering Department, University of Minnesota, 1390 Eckles Ave., Saint Paul, MN 55108, United States

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

Article history: The energy intensive harvesting of tiny microalgae cells (1–70 lm) from culture broth accounts for at
Received 9 November 2012 least 20–30% of total costs of algal biomass production. The aim of this study was to develop an alterna-
Received in revised form 18 January 2013 tive fungus pelletization assisted bioflocculation method for harvesting microalgae using pellet-forming
Accepted 18 January 2013
fungal strain (Aspergillus oryzae) isolated from municipal wastewater sludge. Facultative heterotrophic
Available online 6 February 2013
microalga Chlorella vulgaris UMN235 was used as a model species. Under heterotrophic growth condition,
the key factors including spore inoculums, organic carbon concentration in medium as well as pH vari-
Keywords:
ation had significantly positive effects on fungus–algae pellet formation. The process parameters of
Aspergillus oryzae
Autotrophic and heterotrophic microalgal
1.2  104 spores/mL, 20 g/L glucose, and pH ranged from 4.0 to 5.0 were found optimal for efficient
cells fungus–algae pellet formation. For autotrophic growth, when pH of culture broth was adjusted to
Cell pelletization 4.0–5.0 with organic carbon addition (10 g/L glucose), almost 100% harvesting efficiency of microalgae
Harvesting was obtained. Moreover, it was observed that diameter and the concentration of fungus–algae pellets
were affected by the shaker rotation. The novel harvesting technology developed in this study might
reduce the microalgae harvesting cost and will have potential to be applied to all types of microalgae spe-
cies as alternative to other traditional harvesting methods.
Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction other commodity materials [6]. Flotation is a mature set of pro-

Microalgae have been recognized as the promising third gener-
ation biofuel feedstock due to their advantages of higher unit area
oil yield than traditional energy crops, not competing with arable
land, and high photosynthetic efficiency and thus high CO2 fixation
capability [1,2]. However, economic viability remains the key
obstacle to the commercialization of algae-based bio-energy pro-
duction. One of the primary concerns is the energy-intensive har-
vesting process which accounts for up to 20–30% of the total cost
of algal biomass production due to small size of algal cells
(typically in the range of 1–70 lm) [3].
Common microalgae harvesting and dewatering operations are
accomplished through centrifugation, flotation, flocculation, filtra-
tion, or combination of above methods [4]. Among these processes,
centrifugation is the most efficient method (over 95% algal biomass
could be obtained) and is widely used for microalgal cells harvest-
ing in lab-scale or pilot-scale microalgae cultivation systems [5].
However, high capital cost, energy input and operational cost im-
pede its further application at a large scale. Currently centrifugation
is only used to harvest microalgae containing high-value bio-prod-
ucts such as highly unsaturated fatty acids, pharmaceuticals and
⇑ Corresponding author. Tel.: +1 612 625 1710; fax: +1 612 624 3005.
E-mail address: ruanx001@umn.edu (R. Ruan).
cesses and is commonly integrated into advanced photo-bioreac-
tors design for simultaneous microalgae removal and cultivation.
The main disadvantage of such an approach is its technical and eco-
nomic viability. Flocculation is carried out with the aid of chemical/
synthetic polymers before harvesting. The use of the polymers re-
duces the value of flocculation technology significantly due to toxic,
expensive, corrosive, and environmental safety issue of these poly-
mers [4]. Filtration method is only used for harvesting microalgae
with properties of long length or formation of large-colony (e.g.,
Spirulina sp. and Micractinium sp.) [5]. Recently, Zhou et al. [7] re-
ported an iron-based self-sedimentation and flocculation of algae
method. Markou et al. [8] found that the change of algal biomass
composition could improve the bio-flocculation of algae for har-
vesting. Both methods provide alternative option for low cost har-
vesting algal biomass for biofuel production in the near future.
Some filamentous fungi such as Rhizopus oryzae, Penicillium
expansum, and Mucor circinelloides, were reported to be capable of
forming large pellets (2–5 mm in diameter) under optimized oper-
ating conditions [9–11], which may provide an alternative cost
effective way to harvest microorganisms. On the other hand, in nat-
ure, it was found that some algae species could be in symbiotic rela-
tionship with other microorganisms such as filamentous fungi
[12,13]. In this symbiosis, algae could fix CO2 through photosynthe-
sis and produce organic carbons and thus promote filamentous

1383-5866/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.seppur.2013.01.030
 W. Zhou et al. / Separation and Purification Technology 107 (2013) 158–165
fungi growth, which could in turn entrap algae by producing fungal
hyphae (a typical example is lichen) [12,13]. Therefore, it is hypoth-
esized that these filamentous fugal strains could entrap microalgal
cells and form fungi–algae pellets, thus allowing efficient algae har-
vesting. However, research in the area especially with regard to
microalgae harvesting using filamentous fungi has not been
reported.
Numerous researches reported that some filamentous fungi
that naturally existed in activated sludge could immobilize or en-
trap the sludge solids by forming bio-flocculation/coagulation
and strengthen the flocculation structure due to their unique fila-
mentous properties [10,14,15]. Therefore in this study, filamentous
fungi were identified and isolated from local municipal wastewater
sludge and used to evaluate their feasibility for bio-flocculation of
a non-flocculating microalgae strain Chlorella vulgaris UMN235.
Self-pelletization under optimized culture conditions was devel-
oped and evaluated as a promising alternative to current harvest-
ing technologies. Furthermore, the lipid content of some fungi
species was reported to be over 30% of total fungal biomass [11],
making high oil fungi a good biodiesel feedstock, which could be
converted into biofuels directly together with oleaginous microal-
gae. Therefore, the lipid content of fungi is also an important selec-
tion criterion.
The objectives of this study are herein (1) to isolate and identify
promising filamentous fungi from local municipal wastewater
sludge which could form pellets under optimized culture condi-
tion; (2) to develop an effective fungal pelletization assisted bio-
flocculation method for effective harvesting autotrophic and
heterotrophic microalgae biomass; (3) to investigate the key fac-
tors affecting the fungi–algae pellet formation and analyze the li-
pid contents and fatty acid profiles of pellets under heterotrophic
and photoautotrophic growth modes. Moreover, the feasibility of
using the pelletized fungi–algae biomass as immobilized cells for
other application was also discussed.
2. Materials and methods
2.1. Isolation and characterization of filamentous fungal strains
Samples collected from local municipal wastewater plant
(MWWP) (Saint Paul, Minnesota, USA) were streaked directly on
potato dextrose agar (PDA) plates with inoculums loop after serial
dilution using methods described previously [16] with minor mod-
ification. The isolated fungal strains were tested for pellets forma-
tion ability by cultivating spores of candidate strains on typical soy
broth and those which could form pellets were further identified
based on mycelia morphological analysis and extracellular com-
pound-secretion ability grown on the PDA agar plates. Subse-
quently, the candidate samples were stained by crystal-violet
and visualized using light microscope [17].
2.2. Algal strain and growth conditions
The algal strain Chlorella vulgaris UMN235 (C. vulgaris UMN235),
stored in our lab [18], was identified as facultative heterotrophic
microalgal strain [18] and was used as the microalgae model for
both heterotrophic and autotrophic growth in this study.
The typical BG-11 autotrophic growth medium was selected to
grow C. vulgaris UMN235 under autotrophic growth and the com-
positions of the medium were described previously [7]. The hetero-
trophic growth medium for C. vulgaris UMN235 heterotrophic
growth was BG-11 medium plus 10 g/L glucose addition.
The enriched seed cultures were inoculated at 10%
(tinoculation/tmedium) on 500 ml liquid medium in 2 L Erlenmeyer
flasks placed on a horizontal shaker (150 rpm). The culture was
159
kept at 25 ± 2 °C under the continuous cool-white fluorescent light
illumination at 120 lmol/(m2 s) for autotrophic growth and under
dark for heterotrophic growth. For filamentous fungi and algae co-
culture, the different initial inoculums (approximate from
1.1  102 to 1.2  106 spores/mL) of fungal spores and different
concentrations of glucose were added to the culture broth. And
pH of culture broth was adjusted accordingly by addition of 1 M
sulfuric acid solution to culture broth.
2.3. Experimental design
In this study, the experiments were carried out in three consec-
utive stages. In the first stage, filamentous fungal strains were iso-
lated from municipal wastewater sludge and preserved on PDA
slant. The spores of candidate fungal strains (approximate
3.5  104/mL as standard spore suspension) were cultivated on
Typical Soy Broth (TSB) for 5 days and pellets-forming fungal
strains were screened and identified based on their morphological
analyses and DNA sequencing. In the second stage, the isolated
promising fungal strain Aspergillus oryzae UMN F07 was studied
for its pellet-forming capability under various culture conditions
including different initial inoculums numbers of fresh spores of
A. oryzae (approximately 1.1  102–1.2  106 mL), pH variation
(ranged from 4.0 to 7.0), and different glucose concentrations (5,
10, 15, 20, and 25 g/L) in the medium cultivated on heterotrophic
growth medium. In the third stage, bioflocculation of Chlorella vul-
garis UMN235 by pellitization of filamentous fungus A. oryzae un-
der autotrophic mode was investigated and optimized parameters
for efficient fungus assisted algae pelletization were determined.
All experiments were carried out in triplicates, and the average val-
ues are reported.
2.4. Spore counting
Fungal spore suspension was obtained by rinsing the PDA slant
with 10 mL distilled water and collected in a sterile tube prior to
use [19]. The number of spores in the suspension was counted by
using an optical microscope (National Optical & Scientific Instru-
ments Inc., San Antonio, TX, USA).
2.5. Microscopic visualization of fungus–algae complex
The pelletized fungus–algae biomass was broke up in order to
perform a microscopic study and were visualized under two differ-
ent kinds of microscopes: Nikon Eclipse E800 (Nikon, Tokyo, Japan)
and Olympus-BH2 with objective lenses of 40 and 100 magnifi-
cation, respectively.
2.6. DNA extraction, polymerase chain reaction (PCR) amplification
and sequencing
The identification of the fungal strain was based on nucleotide
sequence analysis of the internal transcribed space (ITS) region.
The genomic DNA of fungal strain was extracted as described by
Lee et al. [20]. The tested ITS1 region was amplified by PCR with
Primers ITS1: TCCGTAGGTGAACCTGCGG and ITS2: GCTGCG
TTCTTCATCGATGC [21]. The PCR reaction was performed according
to Hinrikson et al. [21]. The purified PCR product was sent to Bio-
medical Genomics Center at University of Minnesota (Saint Paul,
Minnesota, USA) for DNA sequencing and a search of GenBank with
BLAST was used to identify the fungal species with ITS1 gene se-
quences downloaded from the database.
160 W. Zhou et al. / Separation and Purification Technology 107 (2013) 158–165

2.7. Growth and chemical analysis irregular aggregates after 5 days cultivation (data not shown),
implying that pellet-formation for filamentous fungi is strain-spe-
2.7.1. Determination of algal growth cific, which was in agreement with other reports [9–11,25]. The
Optical density (OD) of the inoculated medium at 680 nm was fungal strain was identified as genus A. oryzae based on fungal-
measured daily as the algae density indicator using a spectropho- colony morphology on PDA (Fig. 1) and microscopic images of
tometer (Genesys 5, Spectronic Instruments, UK). A linear relation- the fungal colony and conidia combined with the BLAST result
ship between OD and dry weight (DW, g/L) was determined for from fungi gene library.
algal strain [22]: Dry weight (g/L) = 0.34OD680  0.0068, R2 = 0.997. Diener et al. [26] isolated and identified 38 different fungal
And harvesting ratio of microalgae was calculated according to strains derived from municipal activated sludge. Fakhru’l-Razi
the following formula: et al. [15] also isolated about 70 filamentous fungal strains from
different stages of municipal wastewater treatment plant, sug-
total algal biomass ðgLÞ  suspended biomass ðgLÞ
Harvest ratio ð%Þ ¼  100 gested that activated sludge from municipal wastewater treatment
total algal biomass ðgLÞ
plant could provide a variety of fungi species resources including
filamentous fungi. Moreover, Subramanian et al. [10] reported that
a new pellet-forming fungal strain was isolated from municipal
2.7.2. Glucose analysis wastewater sludge successfully. Therefore, it is reasonable to con-
The concentration of glucose was determined using the dinitro- clude from these reports that municipal wastewater sludge from
salicylic acid assay [23]. local municipal wastewater treatment plant is an ideal resource
for promising filamentous fungal strains collection. This conclusion
2.7.3. Fatty acid profile analysis was also further confirmed by results provided in this study.
Fungus–algae pellets were harvested through simple filtration Many research groups investigated the culture conditions for
with Filter cloth (Wypall X70, Kimberly-Clark Professional) and some pellets-forming filamentous fungi species because fungus
then dried with a freeze dryer (Savant Instruments Inc., USA) be- pellets could help improve mixing, as well as mass and oxygen
fore analysis. Fatty acid content and composition analysis were transfer, and thus enhance the biomass and metabolite productiv-
performed by following two consecutive steps including prepara- ity, reduce energy input and facilitate easy separation compared
tion of fatty acid methyl ester (FAME) and GC–MS analysis. FAME with those of non-pellets fungal strains [9,11]. The fungus pellet
was prepared by a one-step extraction–transesterification method formation is determined by a number of factors including medium
described by Indarti et al. [24], with minor modification. The composition, pH of the medium, agitation rate, and initial spore
detailed procedure was described in our previous report [18]. inoculums and genetic attributes of the specific fungi species, etc
All the experiments were carried out in triplicate and average [9,11,27–29]. In the study, It was found that pH of culture broth
values are reported. Results were analyzed using software JMP dropped dramatically from 7.01 to 4.76 after 5 days cultivation
8.0. ANOVA analysis and Tukey’s post hoc analysis were used to for all isolated fungal strains especially for pellets-forming fungus
determine the significance of difference wherever applicable. strain A. oryzae (Table 1) probably because the fungal growth may
have led to excretion of some unknown acidic metabolites in cul-
3. Results and discussion ture broth [22]. The similar phenomenon was observed by Fujita
et al. [11], suggesting that pH variation may play an important role
3.1. Isolation and identification of fungal strains for pellets-forming in fungal pellet formation. The change in pH of culture broth could
ability derived from municipal wastewater sludge alter the surface properties of fungi, influencing the pellet forma-
tion. The diversity in filamentous fungi which have pellet-forming
Fungi containing samples collected from activated sludge of lo- ability among different fungal strains may be attributed to their
cal MWWP were isolated by serial dilution method grown on PDA different sensitivities to pH [30].
plates and the isolated fungal strains are shown in Table 1 and
Fig. 1. It was observed that some of isolated candidate fungal 3.2. Fungal pelletization assisted bio-flocculation of microalgae
strains showed fungal mycelium on PDA plates after 5-day cultiva-
tion (Fig. 1), suggesting that these fungal strains belong to filamen- Due to relatively small cell size (1–70 lm) and low algal cell
tous fungi. The isolated fungal strains were further investigated for density (typically between 0.5 and 5.0 g/L) of algal biomass, har-
pellets-forming ability by growing fungal spores of these candidate vesting microalgae from culture broth is energy intensive and
fungal strains on TSB broth. It was found that only fungal strain costly. Therefore, development of alternative harvesting technolo-
UMN F07 demonstrated pellet-forming ability (Fig. 2A) (2–4 mm gies for efficient harvesting system is urgently needed. In this
in diameter) while other fungal strains formed loose, fluffy-like study, fungi-assisted bio-flocculation strategy for efficient and
low-cost harvesting relatively diluted algal cells in culture broth
was developed and evaluated.
Table 1
Pellets-forming potential of isolated filamentous fungal strains derived from
3.2.1. Fungal assisted bio-flocculation of heterotrophic microalgal cells
municipal wastewater sludge cultivated on typical soy broth. It was hypothesized that the filamentous fungus A. oryzae could
also assist bio-flocculation of heterotrophic microalgal cells by
Isolated fungal strains Pellet-forming ability Initial pH Final pH
form fungi–algae pellets under certain conditions. Therefore, the
UMN F01  7.02 5.63 main factors affecting formation of fungi–algae pellets such as
UMN F02  7.04 6.34
UMN F03  7.03 5.42
spore inoculums, pH, and glucose concentration were systemati-
UMN F04  7.01 5.23 cally investigated in heterotrophic growth mode.
UMN F05  7.01 5.81 Firstly, the effect of initial inoculums spores and initial pH of
UMN F06  6.98 5.44 medium on fungi–algae pellets formation was studied. Spores
UMN F07 o 7.01 4.76
inoculums of different sizes were added to culture media. It was
UMN F08  7.03 5.81
UMN F09  7.05 5.44 shown that the inoculums sizes ranged from 1.1  102 to
1.2  105 spores/mL could help form fungi–algae pellets under
Note: ‘‘o’’ stands for pellets-forming; ‘‘’’ stands for non-pellets-forming.
heterotrophic growth mode (Table 2a), suggesting the unique pel-
 W. Zhou et al. / Separation and Purification Technology 107 (2013) 158–165
1 2
4 5
7 8
Fig. 1. Morphology of isolated fungal strains grown on potato dextrose agar plates. 1–9 stand for UMN F01, UMN F02, UMN F03, UMN F04, UMN F05, UMN F06, UMN F07,
UMN F08, and UMN F09, respectively.
lets-forming property of the isolated fungi species used in this
study. However, it was found that when initial spores inoculums
was 1.2  106 spores/mL, fungi–algae pellets was not observed
even after 7 days cultivation., which is in agreement with reports
by Foster [31], which demonstrated that fungal cell pelletization
occurred when relatively low spores numbers were inoculated ini-
tially [30]. Since the initial spore concentration of 1.1  104 spores/
mL was found suitable for efficient pellets formation in short
growth period in this study, this inoculums size was used in the
subsequent experiments.
It is worth noting that spore numbers added were positively
correlated with decline of pH in the culture broth, which was prob-
ably due to more acidic chemicals secretion during cultivation (Ta-
ble 2a), suggested that the pH could be controlled by adjusting
fungal spore numbers.
The effect of initial medium pH on fungi–algae pellets forma-
tion was further investigated since pH was considered as another
important factor for pellet formation. The pH of culture broth
was adjusted ranged from 4.5 to 7.0 by 1 M H2SO4. It was observed
that the speed of fungi–algae pellet formation was faster in the
medium with lower initial pH value (Table 2b), which further
confirmed our earlier conjecture. In order to confirm that algae
161
3
6
9
aggregation in this study was not induced or triggered by low
pH, an independent control experiment assessing the effect of low-
ering pH on aggregation/pelletization of algal cells in the absence
of fungus was conducted and the results showed that pH-induced
flocculation of microalgae was not observed when the pH of cul-
ture broth was as low as 4.0 without fungi spores addition (Table
2b).
Liao et al. [9] reported that filamentous fungus strain R. oryzae
grew well and had fast pellet-forming ability when organic carbon
was added in the broth during cultivation, suggesting that organic
carbon have profound impact on fungi growth and cell pelletiza-
tion of filamentous fungi species. Therefore, different initial glu-
cose concentrations ranged from 5 to 25 g/L were studied for fast
fungi–algae pellets-forming ability in heterotrophic growth media.
The results indicated that higher initial glucose concentration pro-
moted formation of fungi–algae pellets in a short cultivation peri-
od, resulting in much lower pH in the culture broth (Table 3).
Similar results were observed when the initial pH of the culture
medium was directly adjusted to 4.0 using H2SO4 (Table 3), further
confirming that pH was the key factor affecting fungi–algae pellet-
ization. Moreover, results also indicated that the pH of culture
broth could be controlled by the initial glucose concentration in
162 W. Zhou et al. / Separation and Purification Technology 107 (2013) 158–165

A B

C D

Fig. 2. Comparison of diameters of fungi pellets/fungi–algae pellets grown on shaker under different rotation speed: (A and C) fungi and fungi-pellets formation with rotation
speed at 150 RPM and (B and D) fungi and fungi-pellets formation with rotation speed at 50 RPM.

did not quantify the effect of key factors including fungal spore
Table 2a
Effect of inoculums size on the fungi–algae pellet formation. inoculums, pH value, agitation and glucose on the fungi–algae pel-
let formation under the heterotrophic and photoautotrophic
Parameter Inoculums size (spores/mL)
growth modes, as well as the optimization the key culture condi-
1.1  102 1.2  103 1.1  104 1.2  105 1.2  106 tions for fungi–algae pellet formation using single-factor experi-
Initial pH 7.01 7.03 7.02 6.98 7.01 mental design in details.
Final pH 5.61 5.32 5.02 4.77 4.41 Besides above mentioned factors, other factors such as metal
Culture time (day) 3.5 4.0 3.0 3.0 7 ions, nutrient profile, and calcium carbonate may also have impor-
Pellet formation o o o o 
tant impact on pellets formation.
Liao et al. [9] found that metal ions of medium, C/N ratio as well
as calcium carbonate had a significant effect on pellet formation of
R. oryzae ATCC 20344. The different results observed above may be
Table 2b
attributed to the properties of different filamentous fungal strains
Effect of pH on the fungi–algae pellet formation.
studied. Typically, for different filamentous fungi species, the
Parameter Inoculums size (spores/mL) structure of mycelia pellets ranged from loose, irregular aggregates
1.1  104 1.1  104 1.1  104 1.1  104 1.1  104 Control to tight spheres caused by different factors mentioned above [34],
Initial pH 7.01 6.01 5.53 5.06 4.00 7.00 which need to be further investigated later.
Final pH 5.02 4.76 4.48 4.39 3.76 4.30 It is worth mentioning that agitation is another key factor
Culture time (day) 3.5 3 2.5 2.5 2.0 5 affecting fungi–algae pellets formation in this study. It was found
Pellet formation o o o o o 
that fungi–algae pellet did not form when no shaking was provided
Note: Control stands for without fungal spore addition in the medium. during the entire experiments (data not shown). Shaker rotation
also affected the size of fungi–algae pellets. As shown in Fig 2A–
D, the diameters (7–9 mm) of fungi pellets/fungi–algae pellets ob-
the medium, probably due to promoting fungi growth in culture
tained from cultures rotated on shaker with 50 rpm rotation are
with high glucose concentration. In summary, the fast and efficient
much larger than those with 150 rpm rotation rate (2–4 mm). Sim-
fungi-based microalgae bioflocculation could be obtained under
ilar phenomena were observed in previous reports [11,28].
the optimized conditions of 1.1  104 spores/mL inoculums size,
20 g/L glucose concentration, and pH ranging from 4.0 to 5.0
(Fig. 2C). Recently, Zhang et al. [32] and Xie et al. [33] also reported 3.2.2. Fungal assisted bio-flocculation of autotrophic microalgal cells
the feasibility of coagulation of tiny microalgae with It was observed that fungus–algae pellets were not formed in
pellet-forming filamentous fungi. However, both of their research the co-culture of microalgal cells and spores (initial inoculums of
 W. Zhou et al. / Separation and Purification Technology 107 (2013) 158–165 163

Table 3
Effect of initial glucose concentration and pH adjustment on fungi–algae pellet A
formation under heterotrophic growth mode.

Initial glucose concentration (g/L) Control

(pH adjustment)
Parameter 5 10 15 20 25 10
pH 6.43 5.57 5.61 5.42 4.51 4.02
Culture time (day) 5 5 4 2.5 2.5 2.5
Pellet formation  o o o o o

Table 4
The fungi–algae pellet-formating ability cultivated under autotrophic growth mode.

Cultivation Pellets-forming ability

(day)
Initial Initial Initial Initial Initial pH
pH pH pH pH (5.04) + glucosea
(7.02) (4.01) (4.51) (5.03)
1      B
2     
3  o o o o
4  – – – –
5  – – – –
6  – – – –

Note: ‘‘a’’ stands for pellets-forming ability after addition organic carbons (10 g/L
glucose) in autotrophic growth medium.
(a) (b) (c)

1.1  104 spores/mL) in flask under autotrophic growth mode

(Table 4). However, when the initial pH in the autotrophic medium
was adjusted to 4.0–5.0, the suspended microalgal cells were
partially entrapped (about 63% of microalgae were harvested) by
filamentous fungus strain A. oryzae. And both formation of fungi-
Fig. 3. Fungi–algae pellets formation grown under autotrophic growth mode:
pellets and the decrease of pH in culture broth were slower com-
(A) fungi–algae pellets cultivated under autotrophic growth mode (pH, 4.43) and
pared with those cultivated in heterotrophic growth mode (Table (B) fungi–algae pellets formation grown on 250 mL flasks: (a) control without any
4). fungal spore addition (pH, 7.0), (b) fungi–algae pellets formation grown under
It is interesting to note that after addition of organic carbon autotrophic growth mode (pH, 4.57), and (c) fungi–algae pellets formation under
(10 g/L) in autotrophic growth medium, and adjustment of the autotrophic growth mode with organic carbon (10 g/L glucose) addition (pH 4.79).

pH of the medium from 4.0 to 5.0, the period of fungi–algae pellets

formation was also shortened to 3 days and almost algal cells were
entrapped in fungi–algae complex with 93% of harvesting
efficiency (Table 4 and Figs. 3A and 4). Moreover, the numbers of
fungi–algae pellets almost doubled compared with those grown
in autotrophic mode (Fig 3B (b and c)).
Filamentous fungi grow fast and show compound performance
when appropriate organic carbons are supplied in the medium [9].
Higher fungi growth is accompanied by greater pH decline (Tables
3 and 4). This could partially explain why much more fungi–algae
pellets formed after addition of extra organic carbon in autotrophic
growth medium.
In addition, other organic carbons might have been generated
and utilized by filamentous fungal strain A. oryzae during the co-
Fig. 4. Microalgae harvesting ratio by filamentous fungus strain Aspergillus oryzae
culture on autotrophic growth medium [35]. Furthermore, the cell under various cultivation conditions. AC: autotrophic culture; HC: heterotrophic
walls of microalgae entrapped inside the pellets may be degraded culture.
by the fungi to produce extra organic carbons [10]. However, the
organic carbons produced through above methods are limited, thus
slower fungi–algae pellets formation than that in heterotrophic created by hydraulic movements may contribute significantly to
growth mode was observed (Fig. 3B). pellet formation. Surface properties of fungi were believed to play
The morphological features of the fungus–algae complex after another major role [36]. It was found that the hydrophobic
breakup were observed using microscopes under 40  and proteins on the mycelial surface of some filamentous might be
100  magnification (Fig. 5A–D). The micrographs showed that al- beneficial to fungus–algae pellet formation because these hydro-
most all microalgal cells were entrapped by filamentous hyphae of phobic proteins could attach to solid surface and assist fungi-
A. oryzae The exact mechanism for fungus–algae pellet formation is solid aggregation/pelletization [36]. The surface charges on
not clearly, and may vary from species to species and strain to microalgae and filamentous fungus might be another reason for
strain [28]. Continuous agitation was considered as one of the fungus–algae pellet formation. Subramanian reported that a new
key reasons for fungus–algae complex formation [9–11,28]. Our isolated pellet-forming fungal strain Penicillium strain BS30 carried
results showed fungus–algae pellets did not form without agitation positive charges [10] while microalgae were typically negatively
(data not shown). Fujia et al [11] reported that shearing stress charged [7], which could cause aggregation of positively charged
164 W. Zhou et al. / Separation and Purification Technology 107 (2013) 158–165

A B

C D

Fig. 5. Microscopic visualization of fungus–algae complex structure: (A and B) breakup of fungus–algae biomass visualized under Nikon-E800 with 40 and
100 magnification, respectively and (C and D) breakup of fungus–algae biomass visualized under Olympus-BH2 with 40 and 100 magnification, respectively.

microalgae and negatively charged fungi. However, surface charge

was considered to vary with pH variation of medium, charges after
biodegradation as well as microbial extracellular-polymeric sub-
stances [37], which might have a reverse effect on pellet formation
and be strain-specific [28]. In summary, the detailed mechanism of
pelletized fungus–algae complex in this study is not very clear up
to now and deserves further investigation.

3.3. Other applications of fungi–algae pellets

Due to the large size (typically 2–4 mm in diameter), the fungi–

algae pellets formed during culture could be harvested directly by
simple filtration method, making such harvesting process more Fig. 6. Fatty acid composition of fungi–algae pellet biomass cultivated on
environmentally friendly and economically viable compared with autotrophic and heterotrophic growth mode.
traditional harvesting technologies. The analyses of the fatty acid
profiles of the harvested fungi–algae pellets grown on autotrophic
ellipsoidal properties. It is well known that microalgae have a high
and heterotrophic growth mode showed that C16–C18 accounted
biosorptive capacity for a variety of metal ions and have been used
for 85.26% and 92.36% of total fatty acid, respectively (Fig. 6), and
for heavy metal removal for decades either by accumulation or
the lipid contents were 17.23% and 36.21% of total algal biomass,
absorption [40]. It was also found that fungi are ideal microorgan-
respectively, which could be converted to high-quality biodiesel
isms for heavy metal remediation due to the surface features of
directly through transesterification process [18].
their cell walls and pigmentation [41]. Therefore, relatively higher
The fungi–algae complex could also be reused as immobiliza-
metals biosorptive capacity might be attained for the immobilized/
tion cells for treatment different types of wastewaters including
pelletized fungus–algae biomass than algae or fungi alone [42].
industrial, municipal, and agricultural wastewaters. The general
Moreover, some heavy metals such as Ni, Si, and Al are good cata-
techniques for immobilization of microorganism (e.g., bacteria,
lysts for thermochemical conversion of harvested fungus–algae
microalgae, yeast, fungi) are to mix these microorganisms with
pellets directly to high quality bio-oil [43]. Nevertheless, the issues
synthetic (e.g., acrylamide, polyurethane, polyvinyl, resins) and/
of scaling-up possibility and economic feasibility of the developed
or natural polymers (e.g., alginate, carraggenan, agar, agarose)
fungi-assisted harvesting system should be further investigated
[38]. However, using these expensive polymers would increase
and evaluated in the near future.
the cost of immobilization process heavily, which limits their
applications in large-scale [39]. Using filamentous fungi as natural
agent for microorganism immobilization could be a good option for 4. Conclusion
economic and efficient wastewater treatment and production of
other high-value byproducts. A filamentous pellets-forming fungal strain (A. oryzae) was iso-
The fungus–algae pellets could be also used as sorbents for bio- lated from municipal wastewater sludge successfully. When co-
sorptive detoxification of heavy/toxic metal-containing industrial cultured with microalgae, fungus–algae pellets were formed with
wastewaters and water recycling due to their unique spherical or continuous agitation provided. The size of initial fungi inoculums
 W. Zhou et al. / Separation and Purification Technology 107 (2013) 158–165
and the pH of the culture media, which changed due to fungi and
algae growth and also was a function of exogenous organic car-
bons, played a key role in the pelletization process. The fungi–algae
pellets have much larger sizes than the algae, allowing harvesting
algae through simple filtration. The developed novel harvesting
technology may provide a solution to problems associated with
current energy-intensive and costly algae harvesting processes.
Moreover, the fungi–algae pellets may be harvested and reused
for treatment of industrial (e.g., heavy metal containing), munici-
pal, and agricultural wastewaters, thus helped recycle and reclaim
water.
Acknowledgments
The work reported here was supported in part by grants from
Minnesota Legislative-Citizen Commission on Minnesota
Resources (LCCMR), China 863 Projects (2012AA021704), NSF of
China (Grant 21177067), the Key Program of Natural Science of
the Jiangsu Province of China (Grant BK2010034) and China Inter-
national Cooperation Projects (2010DFB63750).
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