ORIGINAL PAPER

Growth and biochemical composition of filamentous microalgaeTribonema sp. as potential biofuel feedstock

Hui Wang • Bei Ji • Junfeng Wang •

Fajin Guo • Wenjun Zhou • Lili Gao •

Tian Zhong Liu

Received: 21 March 2014 / Accepted: 9 June 2014

� Springer-Verlag Berlin Heidelberg 2014

Abstract Filamentous oleaginous microalgae Tribonema

minus have advantages in relatively easy harvesting and

grazers resistance in mass cultivation due to its filaments in

previous study. To evaluate whether the genus Tribonema

is a valuable candidate for use in biofuel production, the

morphology, growth, biochemical composition and fatty

acid profile of six filamentous microalgae strains Tribo-

nema sp. were investigated. All the strains are unbranched

filament in single row of elongated cylinder, attaining

0.5–3 mm in length. The growth rates of tested strains were

0.35–0.42 g L-1 d-1. Generally, for all strains, decrease in

protein content was followed by a slight increase in lipid

and significant increase in carbohydrate in early phase,

afterwards, lipid increased constantly inversely to decrease

in carbohydrate content. After 15-day cultivation, total

lipid contents of tested strains ranged from 38–61 %, of

which TAG were the majority and palmitic acid (C16:0)

and palmitoleic acid (C16:1) were the dominant

components. The study confirmed that the genus Tribo-

nema is the potential for biodiesel and bioethanol produc-

tion upon culture time.

Keywords Tribonema sp. � Morphology � Biochemical

composition � Lipid � Fatty acid

Introduction

Limited fossil fuel reserves of the earth have been rapidly

depleted and there is an increasing need for renewable

energy sources, especially biofuels [1]. Microalgae, which

grow fast in variety of water environments to accumulate

lipids and polysaccharides, exhibit advantages to be the

potential source for biodiesel and/or bioethanol production

when compared with terrestrial plants [2, 3]. Many

researches have been tried to set up the microalgae biofuel

process, however, no commercial system has succeeded in

economic viability, mainly due to the technical and cost

problems related with the microalgae biomass production

[4, 5]. Microalgae strains and mass cultivation are thought

to be the core technologies of microalgae biofuel indus-

trialization. Besides the biological behaviors of fast growth

and high lipid content, more industrial properties including

robustness to contamination, ease to harvest, etc., for ole-

aginous microalgae specie should be more emphasized to

ensure the success of mass cultivation and low cost [6].

To date, studies on microalgae are mainly concentrated

in the unicellular microalgae with the size \30 lm [7, 8].

However, in the outdoor scale culture, such nutrient-rich

algal cells with tiny size are usually palatable for grazers

(ciliate, amoeba and rotifera) to promote their massive

proliferation during cultivation and as a result, the mass

cultivation of microalgae usually crashed down. In

W. Hui and J. Bei contributed equally to this work.

H. Wang � B. Ji � J. Wang � F. Guo � W. Zhou � L. Gao �T. Z. Liu (&)

Key Laboratory of Biofuels, Qingdao Institute of Bioenergy

and Bioprocess Technology, Chinese Academy of Sciences,

Qingdao, Shandong 266101, People’s Republic of China

e-mail: liutz@qibebt.ac.cn

B. Ji � F. Guo

University of Chinese Academy of Sciences,

Beijing 100049, People’s Republic of China

B. Ji

College of Chemical and Environmental Engineering,

Shandong University of Science and Technology, Qingdao,

Shandong 266590, People’s Republic of China

123

Bioprocess Biosyst Eng

DOI 10.1007/s00449-014-1238-x

addition, the harvesting of unicellular microalgae with tiny

size from culture medium is another tough work which

related with the power consumption and biomass quality

and cost [9]. Centrifuging is the usual way to harvest the

unicellular microalgae, but it is time consuming and costly

[10]. Filtration, gravitational sedimentation and dissolved-

air flotation are the other options to harvest microalgae

from medium; however, their harvesting efficiency is

usually dominated by the formation of floccule of tiny

unicellular algae cells induced by flocculent additives. The

flocculant residuals in both algal biomass and harvested

water are not only negative for later processing, but also

disadvantages for culture medium recycling [11].

Kim et al. [12] reported that filamentous microalgae

Spirulina platensis could be harvested economically by

filtration or flotation without any flocculants. Moreover,

Wang et al. [13] introduced that microalgae cells with

bigger size might have grazer resistance. It is reasonable

to believe that those filamentous species with large size

could overcome above puzzling problems. Therefore,

selection of filamentous microalgae with high lipid con-

tent is the key to the future success of algal biofuels [6].

Fortunately, a filamentous microalgal strain Tribonema

minus was proved as a potential candidate for bidodiesl

production in our preliminary evaluation, due to fast

growth rate, high lipid content, bigger size, grazers

resistance and relatively easy harvesting [14]. And an

integrated process of biodiesel production from Tribo-

nema minus was successfully built up.

It is known that the filamentous microalgal genus

Tribonema are common in freshwater environments

worldwide [15], and the group includes about 100 genera.

Whether other Tribonema genera have the potential to be

the biofuel algal strains? Here, six Tribonema sp. strains

were investigated from their morphologies, biomass and

biochemical compositions. It is expected to provide more

options of microalgae species and evaluate the feasibility

of Tribonema genus as the feedstock for biofuel

production.

Materials and methods

Algae and culture conditions

The filamentous microalgae T. aequale 200.80, T. aequale

880-1, T. minus 880-3, T. ulotrichoides 21.94, T. utricu-

losum 22.94, T. vulgare 24.94 used in this study was pur-

chased from the Culture Collection of Algae of Gottingen

University. Column photo bioreactor (40 cm height, 2 cm

width) with 200 mL of medium settled as suspended cul-

tures to cultivate these filamentous microalgae. Cultures

were grown under 100 lmol photons m-2 s-1 of artificial

light and bubbled with compressed air containing 1.5 %

CO2 (v/v).

Nitrogen utilization in tested microalgal strain culture

For nitrogen analyses, 10 mL of culture were filtered

(0.2 lm), the filtrate were analyzed for concentrations

using nutrient autoanalyser (Vario EL cube, Elementary

Inc., Germany).

Description of cell sizes and morphologies

A 20 lL sample was taken from each column of strain and

placed in a slide, and then covered with cover glass. The

slide was placed under microscope and the cellular mor-

phologies (BX51, Olympus, Germany), especially the sizes

of tested strains were detected. Each sample was analyzed

in triplicate.

Determination of biomass and biochemical composition

A certain volume (V) of algal culture was filtered onto a pre-

weighed 0.45 lm GF/C filter membrane (Whatman, W0).

The membrane was oven dried at 105 �C overnight and then

weighted (W1). The biomass density of culture was calcu-

lated as (W1 - W0)/V. The total nitrogen content of the

microalgae was first detected by an elemental analyzer, and

then the crude protein concentration was obtained according

to the correlation reported in the literature (i.e., protein

concentration = nitrogen content 9 6.25). The carbohy-

drate content and composition in microalgae were deter-

mined using the modified quantitative saccharification (QS)

method reported by the National Renewable Energy Labo-

ratory (NREL), USA [16]. The total lipid content was

determined by gravimetric analysis according to modified

Bligh and Dyer’s method [17]. Approximately 50 mg of

dried algal pellet was ground with quart firstly and then

mixed with 7.5 ml chloroform/methanol (1:2, v/v) at 37 �C

overnight and then centrifuged. The supernatant was col-

lected and residual biomass was extracted twice. The

supernatants were combined, and chloroform and 1 %

sodium chloride solution were added to a final volume ratio

of 1:1:0.9 (chloroform:methanol:water).

Analysis of lipid composition and fatty acid profiles

Lipid composition was analyzed using a thin-layer chro-

matography (TLC) system (TLC-FID, MK-6, Iatron Lab-

oratories, Inc., Japan) [18]. Samples were dissolved in

chloroform to a concentration of 5–8 mg ml-1 and spotted

onto Chromarod S-III silica coated quartz rods held in a

frame. The rods were developed in a solvent system of

benzene:chloroform:methanol (150:60:2, v/v/v) for the first

Bioprocess Biosyst Eng

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migration to 7 cm, followed by a solution of ben-

zene:hexane (50:50, v/v) for the second migration to

10 cm. The individual lipid components were identified by

co-chromatography with pure standards (SE; FAME; FFA;

TAG; DAG; MAG; PL, purchased from Sigma, St. Louis,

MO, USA). The quantity of TAG was estimated from the

peak areas of pure standards.

Fatty acid profiles in microalgae cells were determined

post-conversion to fatty acid methyl esters (FAMEs) [19].

And then a 0.5-mg sample was dissolved in 1 mL heptane

containing 50 lg nonadecanoic acid as internal standard for

FAME analysis on a Varian 450GC (Varian Inc., USA)

equipped with a flame ionization detector (FID) and Agilent

HP-5 GC Capillary Column (30 m 9 0.25 mm 9 0.25 lm).

The individual FAMEs were identified by chromatographic

comparison with authentic standards (Sigma). The quantities

of individual FAMEs were calculated as W = ms 9 fi 9 Ai/

(m 9 As) 9 100 %, where W is the relative content of each

fatty acid, presented in a percentage of total fatty acid; ms is

the mass of internal standard, fi is the coefficient value of

section i, Ai is the peak area of section i, m is the weight of

sample and As is the area of standard.

Results

Cellular morphologies of tested strains

As shown in Fig. 1, all tested strains have unbranched

filament composed of a single row of elongated, cylindrical

cells, as described by Ivan [20]. The cells of T. vulgare

24.94 are with 7–10 lm wide and 16–21 lm long while the

cells of T. aequale 200.80 are with 4–7 lm wide and

8–17 lm long (Fig. 1a, b). The width and length of T. u-

lotrichoides 21.94 cells are 6–10 and 10–20 lm, which is

slight bigger than T. aequale 880-1 which is with 3–5 lm

wide and 7–13 lm long (Fig. 1c, d). The cells of T. utri-

culosum 22.94 are with 9–15 lm wide and 17–29 lm long,

and that of T. minus 880-3 are with 6–8 lm wide and

13–16 lm long.

Data above revealed that even single cell of Tribonema

sp. (3–15 lm wide and 7–20 lm long) is bigger than

common unicellular microalgae, such as Chlorella sp.

(3–8 lm) and Nannochloropsis sp. (2–4 lm). Moreover,

Tribonema is relatively large, attaining sizes of 0.5–3 mm

filament in length, which is longer than the size of

main grazers, such as ciliate (5–200 lm) and rotifer

(0.01–0.5 mm). The longer size perhaps makes the strains

unpalatable to grazers to contribute to their pest resistance.

In a previous study, cells of Tribonema minus were suc-

cessfully harvested by dissolved-air flotation (DAF) with-

out any flocculants [14]. The similar size of tested strains

indicated a good flotation activity of filamentous microal-

gae Tribonema sp.

Growth of algae in column photobioreactor

Growth curve of tested microalgae in terms of biomass was

presented in Fig. 2. The tested microalgal strains revealed

the similar biomass concentration during 15 days. From

inoculation concentration of ca. 0.093 g L-1, high biomass

(g L-1) of 5.792, 6.422, 5.319, 5.248, 5.543, 5.853 were

Fig. 1 Cellular morphologies of tested strains. a T. vulgare 24.94, b T. aequale 200.80, c T. aequale 880-1, d T. ulotrichoides 21.94, e T.

utriculosum 22.94, f T. minus 880-3

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found for T. vulgare 24.94, T. aequale 200.80, T. aequale

880-1, T. ulotrichoides 21.94, T. utriculosum 22.94,

T. minus 880-3, respectively. Most microalgal strains had

biomass productivity, i.e., 0.344–0.380 g L-1 d-1, except

T. aequale 200.80 had higher biomass productivity of

0.422 g L-1 d-1. In addition, most strains had a growth

cycle of 12 days to stationary phase.

The biomass productivities of tested microalgae strains

were ca. 0.35–0.42 g L-1 d-1, which was lower than those of

unicellular microalgae such as Chlorella sp. (0.44–

0.62 g L-1 d-1), Scenedesmus sp. (0.59–0.95 g L-1 d-1),

Nannochloropsis sp. (0.42–0.55 g L-1 d-1) in similar culture

scale and light condition [21, 22]. Light supply the energy

makes it is an important parameter in the growth and lipid

accumulation of microalgae [23]. During the cultivation, cells

of unicellular microalgae were more easily mixed than fila-

mentous microalgae, resulting in the different uniformities of

light absorption, which means although external energy sup-

ply is equal, the rates of energy absorption and utilization

between single-cell microalgae and filamentous microalgae

are different. Therefore, improving the external energy supply

might be an effective way to speed up the growth rate of

filamentous microalgae.

Changes in protein, total lipid and carbohydrate

The biochemical compositions of tested microalgal strains

were measured by day 7 and day 14, respectively (Fig. 3).

Total protein in the algal samples decreased in different

degrees in response to the culture time. From initial con-

tents of ca. 22.3–30.9 % of dry weight (Fig. 3a), 30.1, 42.1,

53.6, 39.4, 55.4 and 44.4 % decreases in protein contents

were observed after 7-day culture (Fig. 3b) and decreases

of 38.6, 37.3, 54.2, 58.1, 70.3 and 63.2 % were observed by

day 14 (Fig. 3c) in T. vulgare 24.94, T. aequale 200.80, T.

aequale 880-1, T. ulotrichoides 21.94, T. utriculosum 22.94

and T. minus 880-3, respectively.

Corresponding methods were adopted to detect the

contents and compositions of carbohydrate in tested mic-

roalgal strains. No starch was found in any tested algae,

because the cytoplasm of genus Tribonema contains no

pyrenoid [24]. The carbohydrates of Tribonema include

small amount of soluble sugar and large amount of insol-

uble polysaccharides because the cell wall of genus

Tribonema filament is mainly made up of cellulose. Thus,

the carbohydrate content shown in Fig. 3 was the sum of

the contents of soluble sugar and cellulose. As shown in

Fig. 3, there were slight increases in carbohydrate in most

tested strains by day 7 (Fig. 3b) from an initial value of

40.45–53.23 % of dry weight (Fig. 3a), subsequently,

the carbohydrate contents declined linearly to ca.

25.52–39.07 % of dry weight inversely to the increase in

lipid content by day 14 (Fig. 3c).

Based on methanol–chloroform extract and gravimetric

method, the initial total lipid contents were ca.

Fig. 2 Growth curve of tested microalgal strains

Fig. 3 Biochemical composition contents of tested microalgae. a day

0, b day 7, c day 14

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16.5–20.5 % of dry weight (Fig. 3a). There were no

obvious increases in lipid contents of dry weight in

T. vulgare 24.94, T. aequale 200.80, T. aequale 880-1 and

T. ulotrichoides 21.94 by day 7 (Fig. 3b); however, the

lipid contents of dry weight in T. utriculosum 22.94 and T.

minus 880-3 increased to 26.1 and 35.6 % from 18.6 and

20.5 %, respectively. After 14-day cultivation, the lipid

contents of dry weight in T. vulgare 24.94, T. aequale

200.80, T. aequale 880-1, T. ulotrichoides 21.94, T. utri-

culosum 22.94 and T. minus 880-3 achieved to 53.2, 48.8,

38.9, 46.8, 52.9 and 61.8 %, respectively (Fig. 3c).

In general, decrease in protein content of dry weight was

followed by a simultaneous increase in carbohydrate and

lipid contents in all tested strains during the early phase of

cultivation. Moreover, carbohydrate content increment was

faster and higher than increase in lipid content of dry

weight. In the later phase of cultivation, the protein content

of dry weight decreased constantly, meanwhile, lipid con-

tent increased dramatically while carbohydrate decreased

by day 14. This may indicate that Tribonema sp. first

accumulate carbohydrate and then induced lipid synthesis,

similar condition also appeared in Chlamydomonas rein-

hardtii reported by [25]. The difference was that C. rein-

hardtii first accumulated starch and then lipid under

nitrogen starvation. However, nitrogen was always suffi-

cient during 15-day cultivation in this study (Table 1). Why

the carbohydrate decreased while lipid increased in later

phase of cultivation under nitrogen-replete condition will be

observed in further study. On the other hand, potential

competition between synthesis of lipid and carbohydrates is

an important factor when deciding optimal biofuel pro-

duction strategies. Microalgae lipid is used for biodiesel

production while carbohydrate are excellent substrate for

bioethanol production [26]. Tribonema sp. based carbohy-

drates are mainly in the form of cellulose (with the absence

of lignin) are thus much easier to convert to monosaccha-

rides when compared with lignocellulosic materials [27].

Therefore, Tribonema sp. biomass contains abundant car-

bohydrate could be considered for bioethanol fermentation,

while biomass contains lipid for biodiesel production.

Lipid composition and fatty acid profiles

Triacylglycerols (TAG) are stored in specialized cytosolic

oil bodies and function as energy reserve. Therefore,

quantities and fatty acid compositions of triacylglycerols

(TAG) in tested stains are important factors to evaluate

whether they can serve as feedstock for biodiesel produc-

tion [28]. Lipid compositions of tested microalgae strains

cultured by day 14 were analyzed using TLC-FID (Fig. 4).

Sterol esters (SE), triacylglycerols (TAG), digalactosyl

diacyglycerol (DAG), and phospholipid (PL) were pre-

sented in tested strains. TAGs accounted for 66.2, 78.9,

71.1, 75.1, 81.3 and 81.4 % of dry weights in T. vulgare

24.94, T. aequale 200.80, T. aequale 880-1, T. ulotricho-

ides 21.94, T. utriculosum 22.94 and T. minus 880-3,

respectively. Obviously, TAG was the majority ingredient

in total lipid in all tested strains under normal conditions,

revealing the specie of Tribonema could be a potential raw

material for biodiesel production.

Phospholipids were the second ingredient in total lipids

from tested strains, 15.1, 5.7, 22.2, 18.4, 15.4 and 12.5 %

of phospholipids were detected in T. vulgare 24.94, T.

aequale 200.80, T. aequale 880-1, T. ulotrichoides 21.94,

T. utriculosum 22.94, T. minus 880-3, respectively. The

lowest ratio of TAG/PL approached to 3.2 presented in T.

aequale 880-1 yet the highest one accounted for 6.52 in T.

minus 880-3. Such higher TAG/PL lipid predicts a rela-

tively easier and better biodiesel conversion.

Fatty acids in tested strains were primarily esterified and

the major fatty acid composition of each was determined

using GC–MS analysis (Table 2). The major fatty acids in

the six strains were myristic acid (C14:0), palmitic acid

(C16:0), palmitoleic acid (C16:1), oleic acid (C18:1)

comprising 6.9–12.1 %, 19.25–33.68 %, 38.49–51.21 %

and 2.96–7.28 % of the total fatty acids, respectively,

whereas myristoleic (C14:1), pentadecanoic (C15:0), hex-

adecadioneic acid (C16:2) and linoleic acid (C18:2) existed

as minor fatty acids. Table 2 also revealed that saturated

Table 1 N–NO3- concentration in the medium after 15-day cultivation of six strains

Strains T.v 24.94 T.a 200.80 T.a 880-1 T.u 21.94 T.u 22.94 T.m 880-3

N–NO3- concentration (mg/L) 126.06 ± 7.5 174.82 ± 4.8 155.61 ± 5.2 162.5 ± 6.7 148.43 ± 2.9 169.7 ± 7.3

Fig. 4 Lipid compositions of tested microalgal strains cultured for

14 days

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and monounsaturated fatty acids were the dominant com-

ponents, comprising 88.28–90.4 % of the total fatty acids

in the biodiesels from the tested algae. In addition, eico-

sapentaenoic acid (C20:5) was the main component of

polyunsaturated fatty acids in all tested strains.

Biodiesel reduces emissions of CO2, CO, hydrocarbons

and particle emissions; however, such biodiesel also suffers

from several performance-related problems including poor

cold flow properties and insufficient oxidative stability [29].

The most common fatty acid methyl esters present in bio-

diesel are palmitic acid (16:0), steric acid (18:0), oleic acid

(18:1), linoleic acid (18:2) and linolenic acid (18:3) [30].

However, palmitoleic acid (16:1) instead of linolenic acid

(18:3) presented in the fatty acid profiles from six Tribonema

strains. The monosaturated fatty acid was the dominant

component, comprising 43.15–54.19 % of the total fatty

acids in lipid from six strains, which was considered to be

better than polyunsaturated fatty methyl esters for improving

CN and oxidative stability without any concomitant adverse

effect on the cold properties of the diesel [31].

Conclusion

In short, all strains of genus Tribonema are unbranched

filament composed of a single row of elongated, cylindrical

cells, attaining 0.5–3 mm in length. The biomass produc-

tivities of all tested Tribonema sp. strains in bubbled

column are ca. 0.35–0.42 g L-1 d-1, similar to those of

unicellular microalgae in same condition. The genus

Tribonema mainly accumulates carbohydrate in early

phase and then induced lipid synthesis in later phase of

cultivation under nitrogen repletion. The highest total lipid

contents of tested strains ranged from 38 to 61 %, of which

TAG was the majority ingredient. Besides the dominant

components of palmitic acid (C16:0) and palmitoleic acid

(C16:1), eicosapentaenoic acid (C20:5) was also found in

all tested strains. Analyses of the present results suggest

that genus Tribonema is promising feedstock for bioethanol

and biodiesel production and such filamentous microalgae

should be paid more attention.

Acknowledgments This work was supported by the Solar Energy

Initiative Plan (KGCX2-EW-309) of Chinese Academy of Sciences,

Director Innovation Foundation of Qingdao Institute of Bioenergy

and Bioprocess Technology, CAS (Y37204110E) and the Science

and Technology Development Planning of Shandong Province

(2013GGF01008).

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Table 2 Fatty acid composition of tested microalgal strains (% of

total FAME)

Properties Strains

T.v

24.94

T.a

200.80

T.a

880-1

T.u

21.94

T.u

22.94

T.m

880-3

C14:0 10.73 9.31 9.09 12.03 11.97 6.85

C14:1 0.62 nd nd 0.54 0.98 nd

C15:0 0.46 nd nd 0.86 1.07 0.56

C16:0 26.32 24.53 27.65 33.68 19.25 28.35

C16:1 42.5 51.1 45.93 38.49 51.21 50.65

C16:1 nd nd 0.50 nd nd nd

C16:2 nd 0.72 0.58 0.68 nd 0.67

C16:2 nd nd nd 0.79 0.01 0.88

C18:0 1.72 0.66 1.06 1.2 0.99 1.02

C18:1 7.28 4.71 4.54 3.12 6.92 2.96

C18:2 0.63 0.55 0.65 nd 0.54 0.71

C20:3 1.82 1.56 1.75 1.43 1.74 1.20

C20:4 2.53 1.34 2.11 3.02 3.17 3.02

C20:5 5.32 5.45 6.12 4.15 2.15 3.14

SFA 39.23 35.56 37.81 46.78 33.29 36.78

MUFA 50.38 54.19 50.47 42.15 57.11 52.51

PUFA 10.3 9.62 11.21 10.7 7.61 ± 1.3 9.61

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