Effect of Conway Medium and f/2 Medium on the growth of six genera of South China Sea marine microalgae Fathurrahman Lananan a , Ahmad Jusoh a,b,⇑ , Nora’aini Ali a , Su Shiung Lam a , Azizah Endut c a Faculty of Science and Technology, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Malaysia b Institute of Tropical Aquaculture, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Malaysia c Faculty of Agriculture and Biotechnology, Universiti Sultan Zainal Abidin, Gong Badak Campus, 21300 Kuala Terengganu, Malaysia highlights Growth of microalgae from South China Sea were tested in Conway and f/2 media. Parameters including SGR, density and mother–daughter intercept were investigated. Late mother–daughter intercept showed the high-tolerance on nutrient depletion. Chlorella sp. found to be the most resilient among microalgae genera tested. Cultivation medium selection should be based on the application of the microalgae. article info Article history: Available online 14 March 2013 Keywords: Microalgae Conway Medium f/2 Medium Cell density Growth performance abstract A study was performed to determine the effect of Conway and f/2 media on the growth of microalgae genera. Genera of Chlorella sp., Dunaliella sp., Isochrysis sp., Chaetoceros sp., Pavlova sp. and Tetraselmis sp. were isolated from the South China Sea. During the cultivation period, the density of cells were deter- mined using Syringe Liquid Sampler Particle Measuring System (SLS-PMS) that also generated the popu- lation distribution curve based on the size of the cells. The population of the microalgae genera is thought to consist of mother and daughter generations since these microalgae genera reproduce by releasing small non-motile reproductive cells (autospores). It was found that the reproduction of Tetraselmis sp., Dunaliella sp. and Pavlova sp. could be sustained longer in f/2 Medium. Higher cell density was achieved by genus Dunaliella, Chlorella and Isochrysis in Conway Medium. Different genera of microalgae had a preference for different types of cultivation media. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Microalgae cultures are commonly used as live feed for fish larvae, crustacean larvae and mollusks in all growth stages (Meireles et al., 2003). In addition, microalgae-based processes have been receiving increasing interest due to their key character- istics such as their use of solar light as the main energy source and their ecological nature in phytoremediation (Xue et al., 2011; Yoshida et al., 2006). Microalgae has been proposed as one of the biofuel feedstock to solve global energy crisis due to its high pho- tosynthetic rate of more than 6.9 10 4 cells mL 1 h 1 (Suali and Sarbatly, 2012). The growth performance of microalgae is mostly determined by the quality of medium used for their cultivation (Lam and Lee, 2012; Li et al., 2012; Prathima Devi et al., 2012). Agricultural fertilizers derived mainly from mineral sources are widely used for the preparation of cultivation media for microalgae in outdoor cultures for commercial aquaculture. However, the pro- duction of mineral fertilizer implies the use of energy derived from fossil fuels and is thus deemed as an unsustainable long-term prac- tice. Lam and Lee (2012) and Alvarado et al. (2008) had suggested the use of organic fertilizer produced from derivative of food waste and composted sea weed as an alternative nutrient for microalgae cultivation. However, the culture of pure strains microalgae de- mands a more stringent composition of nutrient in its cultivation media. Special formulation of media such as Conway and f/2 media are normally used to produce microalgae monoculture in labora- tory scale. Sufficient supplement of nutrients for the growth of microalgae is a key step to produce bulk quantity of high quality microalgae biomass (Xin et al., 2010). Hence, the present study was performed to evaluate the effect of two media, i.e. Conway Medium and f/2 Medium, on the growth performance of microal- gae. Six genera of microalgae namely Chlorella sp., Dunaliella sp., Isochrysis sp., Chaetoceros sp., Pavlova sp. and Tetraselmis sp. were 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.03.006 ⇑ Corresponding author at: Faculty of Science and Technology, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Malaysia. Tel.: +60 9 6683344; fax: +60 9 6694660. E-mail address: [email protected](A. Jusoh). Bioresource Technology 141 (2013) 75–82 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Transcript
Bioresource Technology 141 (2013) 75–82
Contents lists available at SciVerse ScienceDirect
Fathurrahman Lananan a, Ahmad Jusoh a,b,⇑, Nora’aini Ali a, Su Shiung Lam a, Azizah Endut c
a Faculty of Science and Technology, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Malaysiab Institute of Tropical Aquaculture, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Malaysiac Faculty of Agriculture and Biotechnology, Universiti Sultan Zainal Abidin, Gong Badak Campus, 21300 Kuala Terengganu, Malaysia
h i g h l i g h t s
� Growth of microalgae from South China Sea were tested in Conway and f/2 media.� Parameters including SGR, density and mother–daughter intercept were investigated.� Late mother–daughter intercept showed the high-tolerance on nutrient depletion.� Chlorella sp. found to be the most resilient among microalgae genera tested.� Cultivation medium selection should be based on the application of the microalgae.
A study was performed to determine the effect of Conway and f/2 media on the growth of microalgaegenera. Genera of Chlorella sp., Dunaliella sp., Isochrysis sp., Chaetoceros sp., Pavlova sp. and Tetraselmissp. were isolated from the South China Sea. During the cultivation period, the density of cells were deter-mined using Syringe Liquid Sampler Particle Measuring System (SLS-PMS) that also generated the popu-lation distribution curve based on the size of the cells. The population of the microalgae genera is thoughtto consist of mother and daughter generations since these microalgae genera reproduce by releasingsmall non-motile reproductive cells (autospores). It was found that the reproduction of Tetraselmis sp.,Dunaliella sp. and Pavlova sp. could be sustained longer in f/2 Medium. Higher cell density was achievedby genus Dunaliella, Chlorella and Isochrysis in Conway Medium. Different genera of microalgae had apreference for different types of cultivation media.
� 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Microalgae cultures are commonly used as live feed for fishlarvae, crustacean larvae and mollusks in all growth stages(Meireles et al., 2003). In addition, microalgae-based processeshave been receiving increasing interest due to their key character-istics such as their use of solar light as the main energy source andtheir ecological nature in phytoremediation (Xue et al., 2011;Yoshida et al., 2006). Microalgae has been proposed as one of thebiofuel feedstock to solve global energy crisis due to its high pho-tosynthetic rate of more than 6.9 � 104 cells mL�1 h�1 (Suali andSarbatly, 2012). The growth performance of microalgae is mostlydetermined by the quality of medium used for their cultivation(Lam and Lee, 2012; Li et al., 2012; Prathima Devi et al., 2012).
Agricultural fertilizers derived mainly from mineral sources arewidely used for the preparation of cultivation media for microalgaein outdoor cultures for commercial aquaculture. However, the pro-duction of mineral fertilizer implies the use of energy derived fromfossil fuels and is thus deemed as an unsustainable long-term prac-tice. Lam and Lee (2012) and Alvarado et al. (2008) had suggestedthe use of organic fertilizer produced from derivative of food wasteand composted sea weed as an alternative nutrient for microalgaecultivation. However, the culture of pure strains microalgae de-mands a more stringent composition of nutrient in its cultivationmedia. Special formulation of media such as Conway and f/2 mediaare normally used to produce microalgae monoculture in labora-tory scale. Sufficient supplement of nutrients for the growth ofmicroalgae is a key step to produce bulk quantity of high qualitymicroalgae biomass (Xin et al., 2010). Hence, the present studywas performed to evaluate the effect of two media, i.e. ConwayMedium and f/2 Medium, on the growth performance of microal-gae. Six genera of microalgae namely Chlorella sp., Dunaliella sp.,Isochrysis sp., Chaetoceros sp., Pavlova sp. and Tetraselmis sp. were
Table 1Taxonomic class, essential pigments and cell size of six microalgae genera isolated from the South China Sea.
Microalgae genera Taxonomic class Essential pigments Average cell size (lm)
Dunaliella sp. Chlorophyceae (Assunção et al., 2012) b-carotene (Lamers et al., 2012) 9.5Tetraselmis sp. Prasinophyceae (Zhang and Hu, 2002) Loroxanthin (Garrido et al., 2009) 8.5Chlorella sp. Trebouxiophyceae (Hanagata et al., 1998) Violoaxanthin (De-Bashan et al., 2002) 4.5Pavlova sp. Haptophyceae (Bendif et al., 2011) Fucoxanthin (Hiller et al., 1988) 2.5Isochrysis sp. Haptophyceae (Lacour et al., 2012) b-carotene (Bai et al., 2011) 2.5Chaetoceros sp. Bacillariophyceae (Hernández-Becerril, 2009) Fucoxanthin (Ikeda et al., 2008) 2.5
76 F. Lananan et al. / Bioresource Technology 141 (2013) 75–82
isolated from marine water of the South China Sea. Taxonomicclass and essential pigments of the microalgae genera were tabu-lated in Table 1. Since the microalgae genera were locally isolated,standard medium were used in order to facilitate the comparisonwith other genera. Various culture parameters, e.g. microalgaegrowth throughout the cultivation period, maximum cell density,mother–daughter cell intercept, time to reach a peak density, andspecific growth rate were investigated. All experiments, exceptfor the seed culture, were performed using one-liter Erlenmeyerflask and filtered-autoclaved seawater (FASW). This approachwas selected to prevent contamination and minimize the experi-mental error that may occur in the examination of culture param-eter during the cultivation period.
2. Methods
2.1. Algae genera and growth conditions
Chlorella sp., Dunaliella sp., Chaetoceros sp., Tetraselmis sp.,Pavlova sp., and Isochrysis sp. were isolated from the South ChinaSea and maintained in the Live Feed Culture Laboratory of Instituteof Tropical Aquaculture (AKUATROP), Universiti Malaysia Tereng-ganu. The population of these genera were scaled up in test tubesusing 10 mL mono-specific seed culture. These genera were grownin Conway Medium (Tompkins et al., 1995) and f/2 Medium(Guillard, 1975). The composition of nitrate, phosphate, trace met-als, and vitamins used in the cultivation medium are presented inTable 2.
2.2. Cultivation of microalgae in Conway and f/2 media
A two-step cultivation was used for the introduction of growthsubstrate in this study. The algal cells were first divided into twogroups followed by the addition of Conway Medium (Tompkinset al., 1995) or f/2 Medium (Guillard, 1975) as the nutrient supple-ment. Cultivation was performed for a period of 7 days in order toreach a growth plateau, and the biomass was then collected bycentrifugation at 550 rad s�1 (Li et al., 2012). Then, the cultures
Table 2Chemical composition of Conway Medium and f/2 Medium.
Conway Medium (Tompkins et
Nitrate KNO3 (100 g m�3)Phosphate Na3PO4 (20 g m�3)
Trace metal Na2H2EDTA�2H2O (45 g m�3)FeCl3�6H2O (1.3 g m�3)MnCl2�4H2O (0.36 g m�3)ZnCl2 (4.2 g m�3)CoCl2�6H2O (4.0 g m�3)CuSO4�5H2O (4.0 g m�3)(NH4)6Mo7O24�4H2O (1.8 g m�3)H3BO3 (33.4 g m�3)
were maintained with the respective growth substrate in one-litersterile Erlenmeyer flasks for 30 days under illumination in an air-conditioned room at a temperature of 25 ± 2 �C before being sub-jected to the desired experimental condition.
Erlenmeyer flask of 1 L was filled with 250 mL of microalgaeinoculation (25% v/v inoculation), 750 mL of filtered autoclavedseawater (FASW), and 250 lL of growth substrate of either ConwayMedium or f/2 Medium. FASW filtration was then performed usingglass vacuum filtration apparatus (47 mm filtration assembledwith Standard Taper 40/35 Joint Connection, P811613, FAVORIT).FASW was then autoclaved at 121 �C and 15 psi for 20 min priorto inoculation of microalgae and media injection. Erlenmeyer flaskcontaining the microalgae culture was then stoppered with sterilenon-absorbent cotton wools wrapped with sterile cotton gauze.
Liquid samples of 15 mL were drawn daily and stored in pre-treated centrifuge tubes (15 mL, FALCON) according to APHA(2012). Microalgae cell density was determined from 1 mL aliquotusing Syringe Liquid Sampler Particle Measuring System (SLS-PMS,Model SLS-2000, PMeasuring Inc.).
2.3. Microalgae growth analysis
SLS-PMS was used to determine the cell density. The commontechniques used for biomass determination, such as Chlorophyll-a, turbidostat and haemacytometer, can only be used to determinethe total cell density. In contrast, SLS-PMS can be used to distin-guish the cell density based on the cell diameter. This techniqueis capable of differentiating mother cells and daughter cells andin turn generates the growth kinetic curve. The growth kineticcurve of mother–daughter cell provides additional informationon the reproduction and mortality of microalgae genera within aspecified period of time. A predetermined capillary column sizewas configured at a width of 2–16 lm using the SamplerSight™software. The determination of cell density was computed usingMicrosoft Office Excel™ and Originlab 8.6™ software in order togenerate the growth curve, the mother–daughter intercept, thetime to reach a maximum cell density, and the specific growth rate.One-way ANOVA in Minitab 16™ software was used to identify thesignificance of the microalgae growth performance between the
al., 1995) f/2 Medium (Guillard, 1975)
NaNO3 (75 g m�3)NaH2PO4 (5 g m�3)
Na2H2EDTA (4.36 g m�3)FeCl3�6H2O (3.15 g m�3)MnCl2�4H2O (180 mg m�3)ZnSO4 (22.0 mg m�3)CoCl2�6H2O (10.0 mg m�3)CuSO4�5H2O (9.8 mg m�3)Na2MoO4�2H2O (6.3 mg m�3)Na2SiO3 (30 g m�3)
F. Lananan et al. / Bioresource Technology 141 (2013) 75–82 77
different cultivation medium. Furthermore, growth evolutioncurve and scatter plot with standard deviations were establishedto depict the result graphically.
2.4. Measurement of nitrate content in medium
Nitrate concentration was determined according to the Ultravi-olet Spectrophotometric Screening Method (APHA, 2012). Superna-tant of 1 mL was collected and optical density was measured at275 nm and 220 nm using a spectrophotometer (Shimadzu DualBeam UV–Vis, UV 1800). Then, the absorbance reading at 220 nmwas subtracted two times from the reading at 275 nm which wasthe absorption of organic matter, in order to obtain the actualabsorbance representing the concentration of nitrate. Dry potas-sium nitrate (KNO3) of different concentrations was used for cali-bration purposes.
3. Results and discussion
3.1. Time to reach the peak of growth and the maximum cell density
Sufficient supplement of nutrient is a crucial factor to the per-formance of the growth of microalgae (Xin et al., 2010). One of
Fig. 1. Maximum cell density and period to reach maximum cell density of six genera insubstrate were in the group of low cell density (<10,000,000 cells mL�1). The maximum ce
the most important growth parameter is the maximum growthor cell density. High cell density would enable the possibility forthe application of mass culture such as for biodiesel productionand pharmaceutical purpose (Chen et al., 2011; Quinn et al.,2012). As shown in Fig. 1, the maximum cell density between thegenera could be divided into two significantly different groups(P = 2.97 � 10�7, a = 0.05) – genera group with high cell density,and genera group with low cell density. The genera with high celldensity consists of Chlorella sp., Pavlova sp., and Isochrysis sp.,whereas the genera with low cell density consist of Dunaliella sp.,Tetraselmis sp., and Chaetoceros sp. Table 3 lists the maximumaverage cell density for six genera in both cultivation media.Microalgae genera is categorized into different subsets showingsignificantly different means with 95% confidence interval. Dunal-iella sp. in Conway and f/2 media, and Tetraselmis sp. and Chaetoc-eros sp. in Conway Medium were in the lowest density subset.Intermediate density subset consists of Tetraselmis sp. and Chaetoc-eros sp. in f/2 Medium, whereas the other genera forms the highestdensity subset. In particular, Isochrysis sp. grown in Conway Med-ium showed the highest value of maximum cell density with22,584,430 cells mL�1, whereas Dunaliella sp. in f/2 Mediumshowed the lowest value of maximum cell density (i.e.7,517,478 cells mL�1). The cultivation media was found to have
Conway and f/2 media. Dunaliella, Tetraselmis and Chaetoceros regardless of growthll density for Chlorella, Pavlova and Isochrysis were more than 20,000,000 cells mL�1.
Table 3Taxonomic class, cell size, time to reach mother–daughter intercept, maximum average cell density, specific growth rate and essential pigments of microalgae genera cultivated inConway and f/2 media. Statistically different maximum cell density with 95% confidence interval is grouped in subsets represented with superscript.
Time to reach (day) Maximum cell density(cells mL�1)
SGR Nitrate
Microalgae genera Intercept Max. density (1 � 10�6 cellsmL�1 d�1)
78 F. Lananan et al. / Bioresource Technology 141 (2013) 75–82
no influence on the maximum concentration of microalgae generaas each genera exhibits little difference in the results obtained withthe different types of cultivation media used. However, the prefer-ence of cultivation medium as shown by the maximum cell densitycould be observed between the genera. Chaetoceros sp., Dunaliellasp., and Chlorella sp. and Isochrysis sp. demonstrated a slightlyhigher maximum cell density in Conway Medium (11.90%, 8.00%,1.49% and 1.45%, respectively), in contrast, Tetraselmis sp. recordeda higher maximum cell density (i.e. 7.90%) in f/2 Medium.
However, it should be noted that the maximum cell density isonly one of the properties in determining the growth performanceof microalgae. Some commercial and industrial applications (e.g. inwastewater treatment) prefer microalgae genera with fast growthrate as compared to those with the maximum density. The effectsof cultivation media on the time taken to reach the maximum celldensity could clearly be observed in Chlorella sp. This genus re-quired only 14 days to reach a maximum cell density in ConwayMedium compared to 20 days in f/2 Medium. There is 6 days differ-ence between the utilization of the different types of cultivationmedium. Chaetoceros sp. grown in f/2 Medium took 23 days toreach its peak compared to 27 days in Conway Medium. The culti-vation medium in these two cases was found to have effects on thetime to achieve maximum cell density. However, other genera
-
5
10
15
20
25
0 2 4 6
Max
imum
cel
l den
sity
(106
cells
mL-
1 )
Cell size (µm)
Fig. 2. Maximum average cell density and cell size of microalgae genera. Dotted line inincrease of microalgae genera cell size. Conway and f/2 media used for each genera is r
were found to have preference for different cultivation medium(i.e. Conway Medium for Chlorella sp. and f/2 Medium for Chaetoc-eros sp.).
The variation in the maximum cell density among the generahas also been reported to be influenced by the cell size (Lamerset al., 2012). Dunaliella sp. with the size of 9–10 lm are much lar-ger compared to Isochrysis sp. with the size of 2–3 lm (Table 2).Fig. 2 shows the proposed relationship between cell size andmaximum average cell density. The increase of cell size from thesmaller to larger genera of Isochrysis sp., Pavlova sp., Chlorella sp.,Tetraselmis sp. to Dunaliella sp. caused the decrease in maximumcell density. However, Chaetoceros sp. did not follow thiscorrelation, and this is likely due to its different phylum of Hetero-kontophyta, as the other five genera belong to the phylum of Chlo-rophyta (Hernández-Becerril, 2009).
3.2. Mother–daughter cell intercept
Mother–daughter cell intercept provides new insights into thegrowth performance of microalgae. Reproduction rate is postulatedto be lesser than the mortality rate in certain microalgae generawhen the mother cell growth intercepts the daughter cell. The timetaken for each microalgae genus to reach the mother–daughter
8 10
Dunaliella (f/2)
Dunaliella (CM)
Tetraselmis (f/2)
Tetraselmis (CM)
Chlorella (f/2)
Chlorella (CM)
Pavlova (f/2)
Pavlova (CM)
Isochrysis (f/2)
Isochrysis (CM)
Chaetoceros (f/2)
Chaetocetos (CM)
the chart suggested that the number of maximum cell density correlated with theepresented with symbols (j and N, respectively).
Fig. 3. Growth kinetics of Dunaliella sp. in Conway and f/2 media, Tetraselmis sp. and Chlorella sp. throughout the 30-days cultivation period. Total microalgae cell density,mother cell density and daughter cell density are represented with symbols (j, N and 4, respectively). Base-line data was corrected with equation C = Ch � C0.
F. Lananan et al. / Bioresource Technology 141 (2013) 75–82 79
Fig. 4. Growth kinetics of Pavlova sp. in Conway and f/2 media, Isochrysis sp. and Chaetoceros sp. throughout the 30-days cultivation period. Total microalgae cell density,mother cell density and daughter cell density are represented with symbols (j, N and 4, respectively). Base-line data was corrected with equation C = Ch � C0.
80 F. Lananan et al. / Bioresource Technology 141 (2013) 75–82
F. Lananan et al. / Bioresource Technology 141 (2013) 75–82 81
intercept was tabulated and presented in Table 3. It was found thateach microalgae genera had their own unique mother–daughterintercept signature. As shown in Fig. 3 and Fig. 4, it could beobserved that mother–daughter interception occurred within thestationary phase of Pavlova sp., Dunaliella sp., Isochrysis sp. andChaetoceros sp. in regardless of the cultivation medium used.However, mother–daughter interception for Tetraselmis sp. inConway Medium occurred at the late log phase. Chlorella sp. wasreported as having late mother–daughter interception in decliningphase. The late mother–daughter interception depict thecharacteristic of Chlorella sp. which is significantly robust andhigh-tolerance with nutrient depletion as compared to other mic-roalgae genera.
In addition, it was found that different genus showed differentlength of period in maintaining their plateau growth phase. Itwas observed that larger cell size genera such as Dunaliella sp.and Tetraselmis sp. had earlier mother–daughter intercept com-pared to those of the other genera. Mother–daughter intercept inDunaliella sp. in both cultivation media were found inconsistentand occurred within the log phase and it could only maintain thestationary growth for a maximum of 2–3 days as compared tothe longer period (i.e. 7–10 days) and a more stable plateau ob-served in another five smaller-cell-size genera. Tetraselmis sp.maintained a more stable growth plateau as compared to Dunaliel-la sp., however, this genera presented an earlier mother–daughterinterception compared to other genera. Due to the large cell size ofDunaliella sp. and Tetraselmis sp., higher concentration of nutrientwould be required for the reproduction through the formation ofautospores (Assunção et al., 2012). At this point, it could be postu-lated that the nutrient supplied by both cultivation medium werealmost depleted. Depletion of nutrient in the cultivation mediumwould cause starvation, stress, and stunted reproduction of micro-algae (Li et al., 2012; Sun and Li, 2012). Thus, this explains theshorter plateau exhibited by Dunaliella sp. as compared to othermicroalgae genera used in this study.
Chlorella sp. showed the longest time (i.e. 36–42 days) requiredfor the mother–daughter interception to occur. In terms of cell size,Chlorella sp. with an average size of 4.5 lm was larger as comparedto Pavlova sp. with a size of 2.5 lm, Isochrysis sp., and Chaetocerossp. However, it is likely that Chlorella sp. had maintained itsreproduction due to its high tolerance to high range of nutrientconcentrations, temperature, pH, and illumination conditions(González-Fernández et al., 2011; Xin et al., 2010). Mother–daughter intercept of Chlorella sp. was observed more than the to-tal time because it was not occurring within 30 days, e.g. 36 days(f/2 Medium) and 42 days (Conway Medium). In fact, latemother–daughter interception is better than early mother–daughter interception since microalgae having late mother–daughter interception maintained its reproduction longer ascompared to those having early mother–daughter interception.This is due to the mother–daughter interception indicate the pointwhere cell mortality exceed reproduction rate of microalgae.Mother–daughter interception has been reported to be genera-spe-cific since each genera had different endurance against starvationand nutrient depletion (Collet et al., 2011). Pavlova sp. served asan example of genera with intermediate periods to reachmother–daughter intercept; this also applies to Isochrysis sp. andChaetoceros sp. Mother–daughter interception in these genera oc-curred in the middle of the plateau growth phase. Thus, these gen-era could be considered as an average microalgae in terms ofadaptability and endurance with limited nutrient availability.Hernández-Becerril (2009) and Thomas et al. (1984) also reportedthat the conditions suitable for the growth of Chaetoceros sp. andTetraselmis sp. were in the intermediate and narrow range of nutri-ent availability, illumination and salinity. Tetraselmis sp. grown inConway Medium had the shortest period before the mother–
daughter interception. This is followed by Dunaliella sp., Pavlovasp., Isochrysis sp. and Chlorella sp. However, no significant differ-ence were found on the effect of cultivation medium on mother–daughter interception between the genera.
3.3. Specific growth rate
Specific growth rate (SGR) is one important parameter toexpress the relative ecological success of microalgae genera inadapting to the experimental environment imposed upon it(Levasseur et al., 1993). SGR is calculated in the log phase ofmicroalgae growth within the total time of 30 days. SGR of mic-roalgae genera were found affected by the utilization of cultiva-tion medium. As shown in Table 3, SGR of Dunaliella sp.,Chlorella sp., Chaetoceros sp. and Tetraselmis sp. were enhancedin f/2 Medium (72.00%, 40.36%, 22.40% and 4.13%, respectively)whereas Pavlova sp. and Isochrysis sp. thrived in Conway Medium(16.39% and 4.64%, respectively). Dunaliella sp. exhibited highestgrowth rate but with relatively lower cell density due to its largecell size (9.5 lm) as compared to the other microalgae generasuch as Tetraselmis sp. (8.5 lm), Chlorella sp. (4.5 lm), Pavlovasp. (2.5 lm), Isochrysis sp. (2.5 lm) and Chaetoceros sp. (2.5 lm).Larger cell required more nutrient for growth and reproduction.Due to limited nutrient supply, Dunaliella sp. had significantlylower cell density (P < 0.01, a < 0.05) as compared to smaller mic-roalgae genera such as Chlorella sp., Pavlova sp., Isochrysis sp. andChaetoceros sp. The response of microalgae genera on the utiliza-tion of the cultivation medium were based on its specific biolog-ical requirement and thus it differed between genera (Colletet al., 2011).
3.4. Nitrate reduction rate and maximum removal percentage
Removal of nitrate from aquaculture wastewater is importantsince nitrate is mostly associated with eutrophication of waterbodies (Gregory et al., 2012). Microalgae had been proven in sev-eral laboratory studies as the most suitable environment-friendlysolution in treating high nutrient content wastewater (Arbibet al., 2012; Di Termini et al., 2011). As shown in Table 3, Tetrasel-mis sp., Pavlova sp., Chlorella sp., and Isochrysis sp. showed highermaximum nitrate removal (6.5%, 6.3%, 5.4% and 3.5%, respectively)in f/2 Medium as compared to Conway Medium. However, Dunal-iella sp. showed higher nitrate removal in Conway Medium (13.4%),whereas nitrate removal for Chaetoceros sp. was found insignificantbetween the two cultivation media. Among the microalgae generastudied, Pavlova sp. in Conway Medium showed the highest nitratereduction rate (i.e. k of 1.609), whereas the lowest reduction rate (kof 1.147) of all microalgae genera was observed for Dunaliella sp. inConway Medium. Dunaliella sp. in both cultivation media showedthe lowest nitrate reduction rate compared to other microalgaegenera. This is due to the large cell size of Dunaliella sp. (with anaverage diameter of 9.5 lm) which would produce lower surfacearea per volume of as compared to smaller microalgae genera suchas Pavlova sp., Isochrysis sp. and Chaetoceros sp., which had an aver-age diameter of 2.5 lm (Eldridge et al., 2012). Small surface areaper volume would limit the absorption of nitrate and thus contrib-ute to lower nutrient reduction rate. The absorbed nitrate would beused for the growth of microalgae and the production of biomass interms of specific growth rate and maximum average cell density.The higher nitrate removal percentage and maximum cell densitysuggest their preference for the cultivation media between thetwo genera. Other microalgae genera did not show an obvious dif-ference in maximum cell density between cultivation medium,thus the correlation with maximum nitrate removal could not beobserved.
82 F. Lananan et al. / Bioresource Technology 141 (2013) 75–82
4. Conclusion
This study assessed the effect of two growth substrate, i.e. Con-way Medium and f/2 Medium, on the growth of six genera of mic-roalgae found in the South China Sea. The results from the batchcultivation confirmed that the growth performance of the microal-gae genera were influenced by the utilization of Conway and f/2media in terms of the SGR, nitrate removal percentage, nitratereduction rate and maximum cell density. It is proposed that theselection of a suitable cultivation medium should be performedbased on the application of microalgae genera (e.g. wastewatertreatment, biodiesel production, live feed culture, etc.), and alsothe consideration of factors either the shortest time to reach peakof growth or higher maximum cell density.
Acknowledgements
The authors acknowledges the financial assistance by theMalaysian Ministry of Higher Education’s through the Fundamen-tal Research Grant Scheme (FRGS 59209), and the research plat-form provided by the Institute of Tropical Aquaculture, UniversitiMalaysia Terengganu.
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