Scriningul si optimizarea mediului heterotrofic necesar cultivarii microalgelor pentru productia de biodiesel.pdf

8/10/2019 Scriningul si optimizarea mediului heterotrofic necesar cultivarii microalgelor pentru productia de biodiesel.pdf

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Screening, Growth Medium Optimisation

and Heterotrophic Cultivation of Microalgaefor Biodiesel Production

Zongchao Jia &Ying Liu &Maurycy Daroch &Shu Geng &

Jay J. Cheng

Received: 14 February 2014 /Accepted: 6 May 2014 /

Published online: 21 May 2014# Springer Science+Business Media New York 2014

Abstract This article presents a study on screening of microalgal strains from the

Peking University Algae Collection and heterotrophic cultivation for biodiesel produc-

tion of a selected microalgal strain. Among 89 strains, only five were capable of

growing under heterotrophic conditions in liquid cultures and Chlorella sp. PKUAC

102 was found the best for the production of heterotrophic algal biodiesel. Compo-

sition of the growth medium was optimised using response surface methodology and

optimised growth conditions were successfully used for cultivation of the strain in afermentor. Conversion of algal lipids to fatty acid methyl esters (FAMEs) showed that

the lipid profile of the heterotrophically cultivated Chlorella sp. PKUAC 102 contains

fatty acids suitable for biodiesel production.

Keywords Microalgae . Heterotrophic cultivation . Oil accumulation . Algal biodiesel

Abbreviations

PKUAC Peking University Algae Collection

FAME Fatty acid methyl estersRSM Response surface methodology

Appl Biochem Biotechnol (2014) 173:16671679

DOI 10.1007/s12010-014-0954-7

Z. Jia:Y. Liu :M. Daroch (*) :S. Geng :J. J. Cheng

Shenzhen Engineering Laboratory for Algal Biofuel Technology Development and Application, School of

Environment and Energy, Peking University-Shenzhen Graduate School, Shenzhen 518055, China

e-mail: [email protected]

J. J. Cheng (*)

Department of Biological and Agricultural Engineering, North Carolina State University, Raleigh, NC

27695, USA

e-mail: [email protected]

S. Geng

Department of Plant Sciences, University of California, Davis, Davis, CA 95616, USA


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Introduction

Owing to increasingly diminishing oil reserves and aggravation of the environmental conse-

quences caused by the combustion of fossil fuels, the production of renewable energy has

attracted considerable attention during the recent decades. Biodiesel, as a biodegradable,renewable and eco-friendly fuel, has become an important research field in recent years [1].

Currently, most of biodiesel fuel is primarily derived from the soybean oil, corn oil, palm oil,

rapeseed oil, waste cooking oil and animal fats. Despite the variety of feedstocks used,

biodiesel can still cover only a small percentage of global demand for fuels [2]. Microalgae

has been considered as one of the most promising and sustainable feedstocks for biodiesel

production in the last several years, due to their short growth cycle, high oil content, strong

adversity resistance and no land competition with food [2,3].

Microalgae are a very diverse group of organisms that can exhibit different metabolisms.

The most common method of microalgae cultivation is autotrophic growth utilizing CO2as the

carbon source and sunlight as the energy supply. However, this growth mode results in

biomass productivity not higher than 89 % of the total incident solar radiation set by

maximum photosynthetic efficiency [4]. These values translate to an average productivity of

14.31 gDW m2 day1 globally [4]. Areas of higher than average irradiance exhibit higher

productivity but values above 30 gDW m2 day1 are unlikely to be achieved in long-term

commercial cultivation year-round [5]. In addition to physiological constraints, high density

algal cultures result in decreased light penetration and mutual shading of cells, further

decreasing the intensity of solar radiation available for photosynthesis [6]. Furthermore, a

number of technical constraints, like contamination, will further limit actual productivities of

microalgae grown autotrophically [4]. It has been also shown that autotrophic cultivation resultin low to moderate at most accumulation of lipids under optimal growth conditions, and

common methods of increasing the lipid content, i.e. nutrient starvation further decrease

biomass productivity [7].

Compared to the autotrophic cultivation of algae, heterotrophic cultivation using an external

carbon source can enhance both biomass production and lipid accumulation dramatically [8].

Much higher biomass and lipid productivities are common when algae are grown in hetero-

trophy under optimised cultivation conditions. The biomass concentration of heterotrophic

algal cultures ofChlorella protothecoides has been reported to reach 15.5 g L1 under fed-

batch cultivation in a laboratory-scale fermentor [9], and a concentration of 14.2 g L1 has

been reported for 10,000 L industrial fermentors [10]. These values translate to lipid produc-tivities of 7.15 and 6.36 g L1, respectively, which surpasses usual volumetric productivities of

autotrophic cultures by a factor of 10 to 20 [11,12]. Due to high productivity of heterotrophic

cultures, in recent years, there have been significant interests in identifying microalgal strains

that yield high lipid content under optimised heterotrophic growth conditions [13,14].

The composition of growth medium, especially carbon and nitrogen sources, is one of the

most vital factors for the microalgae cultivation [13,15]. The concentration of nitrogen has an

important effect on lipid accumulation in both autotrophic and heterotrophic modes of

cultivation. Reports have shown that lower nitrogen content in the medium results in higher

lipid content in microalgal cells [13]. The lipid content of the C. protothecoides heterotrophi-cally cultured in the medium with the addition of carbon source and the reduction of the

nitrogen source could reach up to 55.2 % of the dry weight, which was about four times that of

autotrophic cultivation of the same strain [8]. Thus, to achieve a maximum performance of the

biomass and lipid productivities, it is of crucial importance to find the appropriate carbon and

nitrogen sources for a particular algal strain as well as their optimal concentrations in medium

composition.

1668 Appl Biochem Biotechnol (2014) 173:16671679


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Here, a specific microalgal strain, Chlorella sp. PKUAC 102, originally isolated from

Shenzhen offshore area, has been screened from Peking University Algae Collection and

assessed for its applicability for biodiesel production. Response surface methodology (RSM)

was conducted for the optimisation of medium composition with the purpose of enhancing the

biomass and lipid productivities.

Materials and Methods

Peking University Algae Collection

Algae specimens were collected as described by Guo et al. [16], Shao et al. [17] and Daroch

et al. [7] at five different sites in January and July of 2011. Redundant strains were excluded by

a combination of morphological analysis under bright field microscope and molecular methods

as described previously [7,17] to yield 89 unique isolatesPeking University Algae Collec-

tion (PKUAC). The collection is stored at 4 C with limited lighting conditions using BG11

growth medium prepared as described by Stanier [18] and subcultured bi-monthly.

Heterotrophic Growth Media

The Shihira-Ishikawa Kase (S-IK) medium was used as a basal medium for the heterotrophic

cultivation of the alga in this study. The medium was composed of the following components,

per litre: KH2PO4, 0.7 g; K2HPO4, 0.3 g; MgSO4 7H2O, 0.3 g; FeSO47H2O, 3 mg; thiamine

hydrochloride, 10 g; glucose, 10 g; glycine, 0.1 g; Arnons A5 solution, 1 mL; pH 6.3.

Arnons A5 solution contained, per litre: H3BO3, 2.9 g; MnCl24H2O, 1.8 g; ZnSO4 7H2O,

0.22 g; CuSO4 5H2O, 0.08 g; MnO3, 0.018 g.

In modified S-IK medium, three different kinds of carbon sources (glucose 10 g L1; sucrose

9.5 g L1; and lactose 9.5 g L1) and four different types of nitrogen sources (glycine 0.1 g L1,

yeast extract 0.166 g L1; potassium nitrate 0.135 g L

1; and ammonium nitrate 0.053 g L

1)

were tested for heterotrophic growth of the algae. Solid growth media were supplemented with

15 g L1 agar. Chloromycetin (0.01 g L1) was added to the medium after sterilization to

prevent contamination. All chemicals used for growth media preparation were of analytical

grade. Growth medium and the apparatus used were sterilised at 121 C, 0.12 MPa for 30 min.

Determination of Algal Growth and Dry Weight

Cellular growth of the algae was monitored by measuring the optical density at 540 nm

(OD540) using a UVVis spectrophotometer Nanophotometer P300 (Implen, Germany).

Estimation of dry biomass weight was carried out in a following manner: 100 mL of algal

culture was filtered through a pre-weighted glass fibre filter GF/C (Whatman, USA) and

washed twice with deionised water. Resultant filter was oven dried at 65 C for 12 h until the

consistent weight was reached.

Cultivation of Isolated Algal Strains

Plate Screening of Peking University Algae Collection for Heterotrophic Algae

Eighty-nine unique algal strains from Peking University Algae Collection isolated from Pearl

River Delta during previous studies [16,7,17] were streaked onto S-IK medium plates for the

Appl Biochem Biotechnol (2014) 173:16671679 1669


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primary screening of heterotrophic algae. The plates were then incubated at 28 C at dark for

2 weeks to test the potential of growth under fully heterotrophic conditions.

Shake Flask Cultivation

The sterilised flasks were inoculated with the pre-cultured alga in the exponential phase until

the initial optical density (OD540) of each flask reached 0.05 which corresponds to an algal

cell density of 0.09 gDW L1. Algal cultures in triplicates were cultivated in 150 mL Erlen-

meyer flasks containing 100 mL S-IK medium for 8 days so that the cultures reached the early

stationary phase. Cultures were incubated in dark in a shaking incubator (JCLAB, China) at

28 C, 80 rpm. Algal growth was monitored daily by measuring optical density at 540 nm.

Lipid accumulation was monitored every other day under fluorescence microscope (OLYM-

PUS BX53, Japan) using Nile Red staining essentially as described previously [7].

Fermentation in 7.5 L Bioreactor

Fermentor-scale cultivation of microalgae Chlorella sp. PKU AC102 was performed using

BIOFCO & GELLIGEN 310 fermentor (New Brunswick Scientific, USA) in optimised

growth medium containing glucose as carbon source (12.37 g L1) and potassium nitrate as

nitrogen source (0.43 g L1). Five litres of sterilised growth medium was inoculated with

exponentially grown Chlorella sp. PKU AC102 to the initial OD540 at 0.05. The agitation

speed and the aeration rate were set at 200 rpm and 180 L h1 (1:1; v/v), respectively.

Temperature was controlled at 28 C. KOH solution (0.5 M) was batch-fed to keep the pH

value at 6.30.3. Dissolved oxygen (DO) concentration was maintained over 25 % airsaturation. The organicsilicon 0.2 (v/v) was added to prevent foaming.

Experimental Design and Optimisation Using Response Surface Methodology

Three carbon sources and four nitrogen sources were initially tested among five heterotrophic

strains. Two of these independent variables (glucose as the carbon source and potassium nitrate

as the nitrogen source) were optimised for biomass production and the lipid accumulation of

the algae Chlorella sp. PKU AC102 during the shake flask cultivation. Response surface

methodology was then carried out to determine optimal growth medium composition. Opti-

misation of the effect of these two variables on biomass growth and lipid productivity wascarried out by central composite design (CCD). The CCD experimental design matrix with two

factors at five levels and the experimental results are presented in Table 1. The cell concen-

tration and the lipid content (FAMEs) content were used as the responses. A two-factor CCD,

with three central points, two factorial points, and six axial points (=1), was conducted for

the medium optimisation, resulting in a total of 11 runs performed in triplicates (Table 1). In

order to predict the optimum point, a quadratic function, calculated with software Design

Expert 8.0.6.1 trial (Stat-Ease, USA), was derived to correlate the relationship between the

response and the variables with following equation:

Y B0 B1X1 B2X2 B12X1X2 B11X12 B22X22

Where Y is the predicted response, X1 and X2 are independent variables that

represent glucose and potassium nitrate concentration, respectively, B0 is a constant,



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B1 and B2 are linear coefficients, B11 and B22 are quadratic coefficients, and B12 is the

interaction effect coefficients. The Design Expert was further used to analyse the

following CCD results: quadratic model significance, the relationship between the

variables and the corresponding responses and analysis of variance (ANOVA) to

identify the effects of the individual variables.

Biodiesel Production and FAME Determination

In situ transesterification of algal lipids was performed with modified protocol ofJohnson and Wen [19] essentially as described by Daroch [7]. Freeze-dried algal

biomass (0.02 g) was placed in a glass test tube and mixed with 1.7 mL of methanol,

0.3 mL of sulphuric acid and 2 mL of hexane. The reaction mixture was heated at

90 C for 1 h and the samples were well-mixed during heating. After the reaction, the

tubes were allowed to cool to room temperature, and 2 mL distilled water was added,

vortexed and centrifuged for 30 min at 3,220g. The hexane layer that contained

FAMEs was collected and transferred to a pre-weighed glass vial. The solvent was

evaporated using N2, and the mass of FAMEs was determined gravimetrically. FAME

profiles were determined using a gas chromatography (Agilent 7890A, Agilent, USA)with auto sampler, flame ion detector (FID) and HP-INNOWAX column (60 m

320 m0.25 m). The column oven was set to 160 C equilibration for 6.0 min

and then 20 C min1 increase to 200 C, 5 C min1 to 235 C and then held for

20 min, and 0.5 C min1 increase to 240 C. The injection was splitless and the

injection temperature was set to 250 C. Helium was used as the carrier gas [7].

Table 1 Coded factors and experimental results of the central composite design ofChlorellasp. PKUAC 102

cultivated in shake flask incubator. Predictedoptimal conditions predicted by the model; Exp. SF experi-

mental results for shake flasks cultivation; Exp. Fer.experimental results in 7.5 L fermenter

Run Codes values Actual values Cell

concentration[g L1]

FAMEs

content (%)

Productivity

[g L

1

]

C/N

ratioGlucose Potassium

nitrate

Glucose

[g L1]

Potassium

nitrate [g L1]

1 0 1.414 10.00 0.60 3.41 29.3 1.00 56

2 1 1 12.50 0.27 2.33 53.2 1.24 156

3 0 0 10.00 0.40 2.92 46.8 1.37 84

4 1.414 0 13.54 0.40 3.04 46.2 1.40 114

5 1.414 0 6.46 0.40 2.02 43.4 0.88 54

6 1 1 7.50 0.54 2.92 25.9 0.76 47

7 1 1 12.50 0.54 3.32 32.1 1.07 788 0 0 10.00 0.40 2.84 46.6 1.32 84

9 1 1 7.50 0.27 2.24 48.6 1.09 94

10 0 0 10.00 0.40 2.82 46.9 1.32 84

11 0 1.414 10.00 0.21 2.04 51.0 1.04 160

Predicted 12.37 0.43 3.04 46.5 1.41 83

Exp. SF 12.37 0.43 3.18 49.7 1.58 83

Exp. Fer 12.37 0.43 3.25 52.8 1.76 83



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Results and Discussion

Screening of Algal Strains for the Ability of Heterotrophic Growth

A total of 89 unique algal strains from Peking University Algae Collection were platescreened for their ability to grow in heterotrophic conditions using different carbon

and nitrogen sources that were modified from S-IK medium. Altogether nine strains

from the collection, all initially isolated with Kuhl medium [20] from Shenzhen

offshore area [7, 17], were capable of growing under heterotrophic conditions on a

plate (Table 2). Only five of these strains were capable of growth in liquid cultures

under the heterotrophic conditions tested. As a result, five strains, i.e. PKUAC 102,

105, 115, 118, and 154, were selected to the second round of screening using shake

flask cultures containing different carbon (glucose, sucrose or lactose) and nitrogen

(glycine, yeast extract, potassium nitrate or ammonium nitrate) sources. Four out of

five reported strains belong to genus Chlorella, which confirms previous findings

regarding high applicability of Chlorella strains for heterotrophic cultivation [8, 13,

10]. The sole non-Chlorella strain was Scenedesmus sp. PKUAC 118. Scendemaceae

family has been sparingly reported for heterotrophic production of algal oils, but the

reported strain fell below Chlorelaceae in terms of biomass and lipid productivities

under all conditions tested (Table 3).

To investigate the effect of different nitrogen sources on the cell growth and the lipid

accumulation, glucose was used as the carbon source; otherwise, glycine was used as the

nitrogen source in order to study the influence of different carbon sources on the cell growth

and lipid accumulation. The concentration of carbon and nitrogen was kept constant withdifferent carbon and nitrogen sources based on the standard S-IK medium. The selected strains

were monitored for growth rate by the measurements of: optical density, dry weight and lipid

accumulation using Nile Red staining (Table 3). The results have shown that Chlorella sp.

PKUAC 102 grown in full heterotrophy using glucose as a nitrogen source and potassium

nitrate as a nitrogen source is the best strain from Peking University Algae Collection for

heterotrophic cultivation. Fluorescence microscopy coupled with Nile Red staining indicated

that the reported strain is the only strain capable of accumulating large content of lipids under

heterotrophic growth conditions and to achieve cell concentrations surpassing 1 gDW L1 in

shake flask cultivation. Even in non-optimised growth medium, the biomass productivity

significantly surpasses the cellular densities of the same strain under phototrophic conditions(0.31 gDWL

1).

When compared with otherChlorellastrains grown under heterotrophic conditions in shake

flask cultures, Chlorella sp. PKUAC 102 shows moderate productivities. Other studies have

reported productivity values of 31.3 gDW L1 [13], 9.7 gDW L

1[21], 4.5 gDW L1 [22], 3.74

gDWL1 [9] and 1.2 gDWL

1 [23] using different growth media and concentrations of glucose

ranging from 40 g L1 [13] to 10 g L1 [23]. The most meaningful comparison can be made

with the study of Xu et al. [9] as both algal strains have been cultivated in modified S-IK

medium containing 10 g L1 glucose. Chlorella sp. PKUAC 102 shows lower biomass

productivity than C. protothecoides from their study (3.74 gDW L

1

). In unmodified S-IKmedium containing 0.1 g L1 glycine as a nitrogen source,Chlorellasp. PKUAC 102 showed

sevenfold lower productivity than reportedC. protothecoidesstrain. Replacing glycine with an

inorganic nitrogen source, potassium nitrate resulted in an increase of biomass productivity

from 0.58 to 1.04 g L1 with a simultaneous increase of lipid content measured with Nile Red

(Table3), showing that optimisation of growth medium composition can significantly increase

productivity of heterotrophically grownChlorellasp. PKUAC 102.



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Table2

InitialscreeningofalgalstrainsfromPekingUniversityAlgaeCollectionfortheircapabilityofheterotrophicgrowthonS

-IKmediumsupplementedwithglu

coseandglycine.

Resultsafter14daysofgrowth.

(+)capableofgrowth

inthemedium;()incapableofg

rowthinthemedium

Algaestrainno.

Genus

Family

Isolatedon

Location

Growthonsolid

S-IK

medium

Growthinliquid

S-IK

medium

PKU

AC101

Micractiniumsp.

Micractiniaceae

18July2011

223100N,1135959E

+

PKU

AC102

Chlorellasp.

Chlorellaceae

18July2011

222930N,1135701E

+

+

PKU

AC103

Chlorellasp.

Chlorellaceae

18July2011

223100N,1135959E

+

PKU

AC105

Chlorellasp.

Chlorellaceae

18July2011

223100N,1135959E

+

+

PKU

AC109

Ourococcussp.

Cocc

omyxaceae

18July2011

222930N,1135701E

+

PKU

AC115

Chlorellasp.

Chlorellaceae

18July2011

223100N,1135959E

+

+

PKU

AC118

Scenedesmussp.

Scenedesmaceae

18July2011

223100N,1135959E

+

+

PKU

AC154

Chlorellasp.

Chlorellaceae

18July2011

222930N,1135701E

+

+

PKU

AC155

Chlorellasp.

Chlorellaceae

18July2011

223100N,1135959E

+



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Table3

Screeningof

algalstrainsfromPekingUniversity

AlgaeCollectionfortheircapabilityofheterotrophicgrowthondifferentcarbonandnitrogensources.Resu

ltsafter8daysof

growth.

CScarbons

ource,

NSnitrogensource;NileR

edqualitativescreening:()noflu

orescence,(+)low

fluorescence,(+

+)medium

fluorescence,(+++)h

ighfluorescence

Algaestrainno.

CellconcentrationwithSIK

medium

[gL1

]

Cellconcentrationw

ithmodifiedSIK

medium

[gL1]

CS:glucoseNS:glycine

NS:glycine

CS:Glucose

CS:sucrose

CS

:lactose

NS:yeastextract

NS:potassium

nitrate

NS:a

mmonium

nitrate

PKU

AC102

0.580.05

0.230.02

0.280.03

0.230.00

1.040.06

0.28

0.02

PKU

AC105

1.460.10

0.150.02

0.150.00

1.220.04

1.200.08

1.22

0.02

PKU

AC115

0.350.01

0.120.00

0.140.01

0.140.01

0.150.01

0.19

0.00

PKU

AC118

0.110.00

0.120.00

0.110.00

0.200.03

0.240.05

0.16

0.00

PKU

AC154

0.460.03

0.300.01

0.300.00

0.290.01

1.040.02

0.88

0.03

Algaestrainno.

NileRedlipidcontent

NileRedlipidconte

nt

CS:glucoseNS:glycine

NS:glycine

CS:glucose

CS:sucrose

CS

:lactose

NS:yeastextract

NS:potassium

nitrate

NS:a

mmonium

nitrate

PKU

AC102

+++

+++

+++

+

PKU

AC105

+

PKU

AC115

+

PKU

AC118

+

++

PKU

AC154

+

+++



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Optimisation of Growth Medium Composition Using Response Surface Methodology

To maximise biomass production and lipid accumulation under heterotrophic growth condi-

tions, RSM was used for growth medium optimisation. Experimental matrix was designed

with two factors at five levels (Table 1) and concentrations of carbon (glucose) and nitrogen(potassium nitrate) were optimised during the process. Cell concentration (as dry biomass

weight per litre after 8 days of growth) and lipid content (as FAMEs) were used as responses.

Two-factor central composite design with three central points, two factorial points and six axial

points (=1) was conducted, resulting in a total of 11 runs carried out randomly to minimise

the effects of the uncontrolled factors (Table1). Experimental results obtained during the study

indicate the importance of medium optimisation for heterotrophic production of algal oils. Cell

concentration varied from 2 to 3.4 g L1 depending on medium composition and the content of

lipids also varied significantly from approximately 26 % to over 53 % depending on carbon to

nitrogen ratio (C/N) (from 54 to 160). Overall lipid productivity was the highest for modified

growth medium containing 12.16 g L1glucose and 0.27 g L1 potassium nitrate among all

media compositions tested.

ANOVA was used to determine following parameters of the model: sum of squares, degrees

of freedom (df), mean squares, F values, and p values (Table 4). Results show that the

coefficient of determination was 0.9421 and 0.9506 for responses as cell concentration and

FAMEs content, respectively, indicating that the models fit well with the experimental data.

Moreover, both models were found significant (pvalue F

Cell concentrationa

Model 2.23 5 0.45 16.28 0.0041

Residual 0.14 5 0.027

Lack of fit 0.13 3 0.044 15.52 0.0611

Pure error 5.633E003 2 2.816E003

Corr. total 2.36 10

FAME contentb

Model 829.81 5 165.96 19.22 0.0028

Residual 43.17 5 8.63 Lack of fit 43.13 3 14.38 734.10 0.0014

Pure error 0.039 2 0.020

Corr. total 872.98 10

aSD = 0.17;R2 =0.9421;R2 Adj=0.8843b SD=2.94;R2 =0.9506;R2 Adj=0.9011



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positive impact on lipid productivity (Fig.1a). On the other hand, when lipid content is used as

a response, it is observed that there is a positive relationship between an increased concentra-

tion of carbon source and lipid content and a negative relationship between the concentration

of nitrogen and lipid yield (Fig.1b). It is therefore important to find the right balance between

the concentrations of these two components for optimal heterotrophic cultivation ofChlorella

sp. PKUAC 102 for algal oil production. These findings are in line with other studies that

confirm that the nitrogen concentration in the growth medium largely determines whether the

carbon flux is directed for cellular growth or for production of storage materials like lipids or

carbohydrates [12].

On the basis of RSM, it has been found that growth medium containing 12.37 g L1 glucose

and 0.43 g L

1

potassium nitrate yielding the C/N ratio of 83 (Table1) is optimal for lipidproduction using heterotrophic cultivation ofChlorellasp. PKUAC 102. The ratio of carbon to

nitrogen is very important for directing the metabolic flux towards lipid synthesis. Comparison

of growth medium carbon to nitrogen ratios optimal for cultivation ofChlorellasp. PKUAC

102 with similar studies suggests that the reported strain requires higher ratio of carbon to

nitrogen for channelling the carbon flux to lipid metabolism. Other reports suggest that C/N

ratios in a range of 3570 are normally used forChlorellastrains [2427], whereas ratios as

high as 278 were reported for microalgae Neochloris oleoabundans producing 52 % (w/w)

lipids. Optimised growth medium composition was subsequently used to cultivate Chlorella

sp. PKUAC 102 in the shake flask yielding cellular density of 3.18 g L1 and lipid content of

49.7 % (Table1). These values were very close to predicted 3.04 g L1 and 46.5 % (Table1).Cultivation of the strain was performed in 7.5 L fermentor to test the feasibility of producing

biomass in the bioreactor.

Fermentor-Scale Batch Cultivation ofChlorellasp. PKUAC 102

After optimal medium composition has been determined using response surface

methodology, attempts were made to cultivate the strain in 7.5 L fermentor. Hetero-

trophically cultivated microalga Chlorella sp. PKUAC 102 exhibited cellular density

of 3.25 g L1 after 8 days of growth, a slight improvement over shake flask cultures

which achieved cellular density of 3.18 g L1 and tenfold over the same strain

cultivated in autotrophic conditions (0.31 g L1). Lipid content in the fermentor was

estimated to be 52.8 % as FAMEs which was higher than the value obtained during

shake flask cultivation (49.7 %) in the same medium. These values translate into lipid

productivity of 0.22 g L1 day1, very close to 0.247 g L1 day1, result obtained by

Xie et al. [25] in a similar study using an isolated Chlorella sp. LAM-H.

Fig. 1 Response surface and contour plots representing the mutual effect of glucose and potassium nitrate

concentration on the cell concentration (a) and lipid content (FAMEs, b)



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The course of fermentation is presented in Fig. 2. The carbon source under optimised

growth conditions has been depleted after 5 days of cultivation, whereas nitrogen after 4 days.

Nitrogen depletion coincided with an increase of lipid content monitored with Nile Red

staining and acidification of the growth medium that resulted in an increased consumption

of KOH to maintain the pH at 6.5. It suggests that in addition to lipid synthesis, additional

products of fermentation, most likely organic acids were produced during heterotrophic

cultivation of Chlorella sp. PKUAC 102 to generate ATP needed for growth and lipid

synthesis.

Derivatisation of algal lipids to FAMEs (Table5) showed that the lipid profile ofChlorella

sp. PKUAC 102 cultivated heterotrophically in the fermentor contained predominantely C16

and C18 fatty acids suitable for biodiesel production. Moreover, content of unsaturated fatty

Fig. 2 Course of fermentation ofChlorellasp. PKUAC 102 cultivated in 7.5 L fermentor. Cell concentration in

gram per litre (squares); glucose concentration in gram per litre (circles); potassium nitrate in gram per liter g L1

(closed triangles); total KOH utiligsation milliliters (open triangles)

Table 5 Fatty acid methyl esterprofiles ofChlorellasp. PKUAC

102 cultivated in 7.5 L fermentor

aCorresponding to chain lengths

C16 (~3 %) and C19 (~5 %)

FAMEs profile

C16:0 28.6 %

C16:1 3.7 %

C17:1 3.8 %

C18:0 1.9 %

C18:1 12.2 %

C18:2 32.6 %C18:3 8.6 %

Uncharacteriseda 8.6 %

Saturated (Sat) 30.5 %

Unsaturated (Un) 60.9 %

Un/Sat 1.99



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acid -linolenic acid (18:3) is below the limit of 12 % set by EN14214 biodiesel standard, and

the lipid profile shows lack of polyunsaturated fatty acids that may seriously compromise

oxidative stability of resultant biodiesel fuel [28]. The favourable lipid profiles combined with

higher lipid yield than those from phototrophically grown cells make Chlorella sp. PKUAC

102 a promising strain for heterotrophic production of algal oils for biodiesel fuel.

Conclusions

Microalgal strains from PKUAC were screened for their capability of growth under hetero-

trophic conditions. Among 89 collected strains, only five were capable of growth under

heterotrophic conditions in liquid cultures andChlorellasp. PKUAC 102 was found the best

for the production of heterotrophic algal biodiesel. Growth medium composition was

optimised using response surface methodology and optimised growth conditions were suc-

cessfully used for cultivation of the strain in the fermentor. Derivatisation of algal lipids to

FAMEs showed that lipid profile of the heterotrophically cultivatedChlorellasp. PKUAC 102

contained fatty acids suitable for biodiesel production.

Acknowledgments This project was predominantly funded by a Shenzhen Development and Reform Com-

mission grant [2011] 835 and partially co-funded from start-up grant of Peking University Shenzhen Graduate

School number 0068 to MD and National Research Foundation and Economic Development Board of Singapore

(SPORE, COY-15-EWI-RCFSA/N197-1) to ZCJ. Authors would like to acknowledge Fei Zhang and Weilin Yi

for their lab assistance.

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Scriningul si optimizarea mediului heterotrofic necesar cultivarii microalgelor pentru productia de biodiesel.pdf