Online Version ISSN: 1314-412XVolume 5, Number 2

June 2013

2013

Scope and policy of the journalAgricultural Science and Technology /AST/ – an International Scientific Journal of Agricultural and Technology Sciences is published in English in one volume of 4 issues per year, as a printed journal and in electronic form. The policy of the journal is to publish original papers, reviews and short communications covering the aspects of agriculture related with life sciences and modern technologies. It will offer opportunities to address the global needs relating to food and environment, health, exploit the technology to provide innovative products and sustainable development. Papers will be considered in aspects of both fundamental and applied science in the areas of Genetics and Breeding, Nutrition and Physiology, Production Systems, Agriculture and Environment and Product Quality and Safety. Other categories closely related to the above topics could be considered by the editors. The detailed information of the journal is available at the website. Proceedings of scientific meetings and conference reports will be considered for special issues.

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Editor-in-Chief

Tsanko YablanskiFaculty of AgricultureTrakia University, Stara ZagoraBulgaria

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Editors and Sections

Genetics and Breading

Atanas Atanasov (Bulgaria)Ihsan Soysal (Turkey)Max Rothschild (USA)Stoicho Metodiev (Bulgaria)

Nutrition and Physiology

Nikolai Todorov (Bulgaria)Peter Surai (UK)Zervas Georgios (Greece)Ivan Varlyakov (Bulgaria)

Production Systems

Dimitar Pavlov (Bulgaria)Dimitar Panaiotov (Bulgaria)Banko Banev (Bulgaria)Georgy Zhelyazkov (Bulgaria)

Agriculture and Environment

Georgi Petkov (Bulgaria)Ramesh Kanwar (USA)

Product Quality and Safety

Marin Kabakchiev (Bulgaria)Stefan Denev (Bulgaria)Vasil Atanasov (Bulgaria)

English Editor

Yanka Ivanova (Bulgaria)

2013

ISSN 1313 - 8820 Volume 5, Number 2June 2013

Production Systems

Cultivation of Scenedesmus dimorphus strain for biofuel production

K. Velichkova*, I. Sirakov, G. Georgiev

1Department of Biology and Aquaculture, Faculty of Agriculture, Trakia University, 6000 Stara Zagora, Bulgaria

Abstract. Microalgae have several advantages, including higher photosynthetic efficiency as well as higher growth rates and higher biomass production compared to other energy crops. The Scenedesmus dimorphus strain was studied by using two media – BBM and 3N-BBM, and its potential for biofuel production was established. The temperature varied between 25 – 27ºC during the experiment. Fluorescent light was used to assure optimal light condition and a photoperiod of 15/9h light and dark cycle was maintained.The duration of the experiment was 25 days. Dry weight, optical density, chlorophyll, carotenoids and total lipids were measured for the biomass evaluation. The received results showed that the maximum vegetative growth was reached after approximately

-1 -116 days of incubation. The maximum growth rate during this period was 1.690 mg.l dry weight in 3N-BBM medium, and in BBM medium – 0.960 mg.l . The lipid content which we received from the examined strain was 21.6% in BBM medium, and in 3N-BBM – 18.5%.

Keywords: biofuel, biomass, media, Scenedesmus dimorphus

AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 5, No 2, pp , 2013181 - 185

Introduction environmental factors, cultivation conditions and growth phases may affect the fatty acid composition. The main effect on growth enhancement could be attributed to the initial content of some macro Biofuel can be produced from a variety of sources, including and micro-nutrients especially carbon and nitrogen.plants (bio-ethanol from corn, canola), bacteria (bio-hydrogen from

The aim of our research was to compare the growth and lipid microbial fuel cells using Geobacter sp.), microalgae (biodiesel, bio-content of Scenedesmus dimorphus (SKU: AC-1002) strain oil and bio-hydrogen from Chlorella, Scenedesmus and cultivated in two nutrition media in connection of its use for biofuel Botryococcus) and solid wastes (biogas from anaerobic digestion). production. Among these, microalgae look promising as a versatile,

environmentally friendly and economically sustainable solution (Hutchinson, 2007).

Microalgae can provide feedstock for several different types of Materials and methodsrenewable fuels such as biodiesel, methane, hydrogen, ethanol, among others. Algae biodiesel contains no sulfur and performs as

Microalgae strain and mediumwell as petroleum diesel, while reducing emissions of particulate Scenedesmus dimorphus (SKU: AC-1002) was purchased matter, CO and hydrocarbons (Delucchi, 2003).

from Algae depot – USA (www.algaedepot.com). S. dimorphus was High lipid contents are usually produced under environmental grown on two types of media: BBM medium (http://www.ccap.ac.uk/ stress, typical nutrient limitation, which is often associated with media/documents/BB_000.pdf): NaNO – 10.0 g, MgSO .7H O – 3.0 3 4 2relatively low biomass productivities and, therefore, low overall lipid g, NaCl – 1.0 g, K HPO – 3.0 g, KH PO .3H O – 7.0 g, CaCl .2H O – 2 4 2 4 2 2 2productivity (Li et al., 2008). The lipid content of microalgae could be 1.0 g (stocks per 400 ml); ZnSO .7H O – 8.82 g, MnCl .4H O – 1.44 4 2 2 2increased by various cultivation strategies, such as nitrogen g, MoO – 0.71 g, CuSO .5H O – 1.57 g, Co(NO ) .6H O – 0.49 g depletion (Li et al., 2008), phosphate limitation (Reitan et al., 1994), 3 4 2 3 2 2

(trace elements solution per litre); EDTANa – 5.0 g, FeSO .7H O – high salinity (Rao et al., 2007), and high iron concentration (Liu et al., 2 4 2

2008). Micro algae have several advantages, including higher 4.98 g.photosynthetic efficiency as well as higher growth rates and higher 3N-Bold's basal medium with added vitamins according to the biomass production compared to other energy crops. Several micro recipe provided on the CCAP website (http://www.ccap.ac.uk/ algae strains have been reported (Pulz and Gross, 2004; Rodolfi et media/documents/3N_BBM_V_000.pdf): NaNO – 25.0 g, 3

al., 2009; Radakovits et al., 2011; Mc Donald, 2011) to have the MgSO .7H O – 7.5 g, NaCl – 1.0 g, K HPO .3H O – 7.5 g, KH PO 4 2 2 4 2 2 4

ability to accumulate large quantities of lipids: Scenedesmus – 17.5 g, NaCl – 2.5 g (stocks per 1000 ml); ZnCl – 5.0 mg, 2

dimorphus, Botryococcus braunii, Nannochloropsis oculata, FeCl .6H O – 97.0 mg, MnCl .4H O – 41.0 mg, 3 2 2 2Phaeodactylum tricornutum, Chlorella protothecoides. According to Becker (1994) S. dimorphus contains 16–40% lipid on dry weight basis. In light of this meaning, the potential biotechnological applications of microalgae growth and lipid production characteristics need to be explored. Different nutritional and

Na MoO .2H O – 4. 2 4 2

mg, CoCl .6H O – 2.0 mg (trace elements solution per litre); 2 2

EDTANa – 0.75 g, vitamins B – 0.12 g and B – 0.1 g.2 1 12

CultivationThe cells in exponential period were inoculated (10%, v/v) in a

181

* e-mail: genova@abv.bg

182

liquid medium. Cultivation was initiated in a 500ml Erlenmeyer flask Lipid contentcontaining 400ml medium. The cultures were kept at room The total lipids were extracted from microalgae biomass using a temperature (25–27ºC) in fluorescent light with a light:dark modified method of Bligh and Dyer (1959). The lipids were extracted photoperiod of 15 h: 9 h. Sterile-air containing 2% (v/v) CO was using a mixture of chloroform/methanol (1:2 v/v). The quantity of lipid 2

residue was measured gravimetrically and expressed as dry weight aerated into the flask through an air sparger at the bottom of the percentage.flask. The strains were checked for 25 days growth period. All

Data analyses were conducted by using ANOVA (MS Office, experiments were conducted in duplicates (BBM medium – bb and 2010).bb1; 3N-BBM medium – 3N and 3N1).

Growth measurementsThe growth of S. dimorphus was measured via Results and discussion

spectrophotometry (DR 2800) and biomass dry weight. Optical density for biomass factor was determined at wavelength 550 nm.

Like other microalgae, S. dimorphus culture requires water, One ml of the sample was appropriately diluted with deionized water

light, CO , and inorganic nutrients. Culture productivity is affected by 2and the absorbance of the sample was read at 550 nm. factors such as pH, CO , irradiance, salinity, and temperature 2The cultures were determined gravimetrically and growth was (Banerjee et al., 2002). According to Lupi et al. (1991) the optimum expressed in terms of dry weight (mg/l) (Rao et al., 2007). The temperature for growth is 25°C. The temperature was held between cultures were harvested by centrifugation at 3.000 x g for 10 min and 25–27°C during the experiment. the cells were washed with distilled water. Then the pellet was freeze

In our study as we expected the cultures grown in the BBM dried. The dry weight of algal biomass was determined medium have lower values of optical density than the cultures grown gravimetrically and growth was expressed in terms of dry weight

-1 in the medium with three times more nitrates (3N-BBM). The (mg.l ). maximum values of the optical density at S. dimorphus grown in 3N-BBM medium was 1.89 compared with BBM medium where it was Chlorophyll and carotenoid content1.53 (Figure 1). The medium enriched with higher nitrates content The isolation of pigments from algae cells included the following showed better algae's culture growth compared with BBM. procedures: harvesting 2 ml of microalgae cells by centrifugation at According to Ilavarasi et al. (2011) Scenedesmus sp. (NTAI03) 10000 rpm, two times for 3 min and discarding the supernatant, showed maximum growth in Bold's Basal medium, based on the suspension of cells in 2 ml methanol/water 90:10 v/v and mixing of optical density measurement.Vortex for 1 min, heating of the suspension for half an hour in a water

-1Maximum dry biomass (1.690 mg.l ) of S. dimorphus was bath at 60ºC, cooling the samples at room temperature, obtained in medium enriched with nitrates (Figure 2), in comparison centrifugating the suspension (10000 rpm for 3 min) and discarding

-1with its dry weight in BBM medium (0.960 mg.l ) (Table 1). The the supernatant with dissolved pigments. The absorbance of the received results showed that the maximum vegetative growth was pigment extract (665, 652 nm for chlorophyll content (a+b) and 470, reached after approximately 16 days of incubation. According to 666nm for carotenoids content) was recorded by using a

-1 Goswami and Kalita (2011) the maximum increase in biomass per spectrophotometer. The chlorophyll content was computed (mg.l ) day for Scenedesmus dimorphus was found to be 1.523 mg/l/day. according to Porra et al. (1989) and carotenoid content was

-1 Varsharani and Getta (2011) reported dry biomass concentration of computed (mg.l ) according to Lichtenthaler (1987).-11.200 mg.l .

Figure 1. Growth response of S. dimorphus (at 550nm) for 25 days under different media

Opt

ical

den

sity

2 S.dimorphus bb

S.dimorphus bb1

S.dimorphus 3N

S.dimorphus 3N1

1.8

1.6

1.4

1.2

1

0.8

0.6

0.4

0.2

0

3 7 10 13

Days

16 21 24

183

In the accounting for chlorophyll of S. dimorphus again higher carotenoid accumulation by Scenedesmus, Chlorella and -1 Haematococcus showed that the addition of at least 10% of nutrients values (9.6 mg.l ) occurred in cultures grown in 3N-BBM medium

mainly nitrogen are required to overcome the dry weight (Figure 3) (Table 1). Varsharani and Geeta (2011) reported -1 accumulation failure (El-Shafey et al., 1999). The main reason could chlorophyll (a+b) concentration - 11.5 mg.l in S. dimorphus.

be ascribed to the presence of organic carbon that allows the fast Prabakaran and Ravindran (2012) received higher values of carotenoid accumulation (El-Sayed, 2010). In our study the chlorophyll in S. dimorphus grown in nitrogen sources and carbon carotenoids of S. dimorphus were again with higher values (2.3 mg/l) source in different concentrations.in cultures grown in medium enriched with nitrates (Figure 4).Different hypothesis on carotenoid accumulation by green

According to Goswami and Kalita (2011) the maximum increase microalgae were mentioned by scientists. Previous studies on

-1Figure 2. Dry weight (mg.l ) of S. dimorphus for 25 days grown in different media

-1D

ry w

eigh

t (m

g.l

)

S.dimorphus bb

S.dimorphus bb1

S.dimorphus 3N

S.dimorphus 3N1

1800

1600

1400

1200

1000

800

600

400

200

0

5 10 15 20

Days

25

-1Figure 3. Chlorophyll (mg.l ) of S. dimorphus for 25 days grown in different media

-1C

hlor

ophy

ll a+

b (m

g.l

)

S.dimorphus bb

S.dimorphus bb1

S.dimorphus 3N

S.dimorphus 3N1

12

10

8

6

4

2

0

5 10 15 20

Days

25

Table 1. Optical density, dry weight, chlorophyll and carotenoid of S. dimorphus grown of different media (BBM, 3N-BBM)

*–P≤0.05

ParametersMin Max MEAN ± SEM Min Max MEAN ± SEM

S.dimorphus BBM S.dimorphus 3N-BBM

Optical density-1Dry weight (mg.l )

-1Chlorophyll a+b (mg.l )-1Carotenoid (mg.l )

0.23 1.53 0.21 1.89 1.17 ± 0.22

942.40 ± 3.52*

5.28 ± 10.30*

1.15 ± 0.57

1690

9.60

2.30

260

0.45

0.10

0.96 ± 0.15

558.22 ± 9.12

2.91 ± 3.49

0.74 ± 0.26

920

5.50

1.60

116

0.40

0.12

184

in biomass per day and lipid content for S. dimorphus was found to to accumulate spare products in the form of fatty acids and glycerol, be 1.523 mg/l/day and 34%. Our results about total lipids were: for S. which were connected by triglyceride. In the 3N-BBM medium dimorphus grown in BBM – 21.6%; S. dimorphus grown in 3N-BBM nutrients were still not the limiting factor for cell division, and the – 18.5%. This was probably due to the fact that in these media there energy and building blocks were redirected in the direction of growth was depletion of nutrients at much earlier stage compared to the and division of cells rather than the accumulation of spare products.media enriched with nitrogen. The cells stopped dividing and began

-1Figure 4. Carotenoid (mg.l ) of S. dimorphus for 25 days grown in different media

-1C

arot

enoi

d (m

g.l

)

S.dimorphus bb

S.dimorphus bb1

S.dimorphus 3N

S.dimorphus 3N1

2.5

2

1.5

1

0.5

0

5 10 15 20

Days

25

Goswami R and Kalita M, 2011. Scenedesmus dimorphus and ConclusionScenedesmus quadricauda: two potent indigenous microalgae strains for biomass production and CO2 mitigation - A study on their The obtained results showed that the researched strain of S. growth behavior and lipid productivity under different concentration dimorphus developed better in 3N-BBM, as larger values were of urea as nitrogen source. Journal of Algal Biomass Utilization, 2, 4, observed in the biomass, but the percentage of lipids was better in 42- 49.BBM. Chlorophyll content in all cultures followed the dynamics of Hutchinson H, 2007.Growing interest in algae. Mechanical variation of the curves of growth. Carotenoid content had the same Engineering, 129, 8, 19-20. character, and it was five times less than chlorophyll.Ilavarasi A, Mubarakali D, Praveenkumar R, Baldev E and Thajuddin N, 2011. Optimization of Various Growth Media to Freshwater Microalgae for Biomass Production. Biotechnology, 10,

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Review

Genetics and Breeding

Nutrition and Physiology

Production Systems

Trends in battery cage husbandry systems for laying hens. Enriched cages for housing laying hens H. Lukanov, D. Alexieva

Influence of environments on the amount and stability of grain yield in modern winter wheat cultivars I. Interaction and degree of variabilityN. Tsenov, D. Atanasova

Variation of yield components in coriander (Coriandrum Sativum L.)N. Dyulgerov, B. Dyulgerova

Plant cell walls fiber component analysis and digestibility of birdsfoot trefoil (Lotus corniculatus L) in the vegetationY. Naydenova, A. Kyuchukova, D. Pavlov

Functional properties of maltitolV. Dobreva, M. Hadjikinova, A. Slavov, D.Hadjikinov, G. Dobrev, B. Zhekova

Food spectrum of grey mullet (Mugil cephalus L.) along the Bulgarian Black Sea coastR. Bekova, G. Raikova-Petrova, D. Gerdzhikov, E. Petrova, V. Vachkova, D. Klisarova

Metabolic and enzymatic profile of sheep fed on forage treated with the synthetic pyrethroid Supersect 10 ECR. Ivanova

Cultivation of Scenedesmus dimorphus strain for biofuel productionK. Velichkova, I. Sirakov, G. Georgiev

Study of the effect of soil trampling on the structural elements of yield and productivity of soybeanV. Sabev, S. Raykov, V. Arnaudov

Stability of herbicides and herbicide tank-mixtures at winter oilseed canola by influence of different meteorological conditionsG. Delchev

Screening of plant protection products against downy mildew on cucumbers (Pseudoperonospora Cubensis (Berkeley & M. A. Curtis) Rostovzev) in cultivation facilitiesS. Masheva, N. Velkov, N. Valchev, V. Yankova

CONTENTS 1 / 2

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Efficacy and selectivity of vegetation-applied herbicides and their mixtures with growth stimulator Amalgerol premium at oil-bearing sunflower grown by conventional, Clearfield and ExpressSun technologiesG. Delchev

V. Atanasov, E. Valkova, G. Kostadinova, G. Petkov, Ts. Yablanski, P. Valkova, D. Dermendjieva

Seasonal and vertical dynamics of the water temperature and oxygen content in Kardzhali reservoir, BulgariaI. Iliev, L. Hadjinikolova

Condition and changes in types of natural pasture swards in the Sakar mountain under the influence of climatic and geographic factorsV. Vateva, K Stoеva, D. Pavlov

Comparative studies on the gross composition of White brined cheese and its imitations, marketed in the town of Stara ZagoraN. Naydenova, T. Iliev, G. Mihaylova, S. Atanasova

Effect of the environment on the quality of flour from common winter wheat cultivarsI. Stoeva, E. Penchev

Agriculture and Environment

Product Quality and Safety

Manganese levels in water, sediment and algae from waterbodies with high anthropogenic impact

CONTENTS 2 / 2

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thmance in dairy cows,IX International Conference on Production Diseases in Farm Animals, Sept.11 – 14, Berlin, Germany, p. 302 (Abstr.).Thesis:Penkov D, 2008. Estimation of metabolic energy and true digestibility of amino acids of some feeds in experiments with muscus duck (Carina moshata, L). Thesis for DSc. Agrarian University, Plovdiv, 314 pp.

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Volume 5, Number 2June 2013