Bioresource Technology 98 (2007) 2207–2211

InXuence of temperature and pH on biomass production andprotein biosynthesis in a putative Spirulina sp.

Kemka H. Ogbonda 1, Rebecca E. Aminigo, Gideon O. Abu ¤

Department of Microbiology, University of Port Harcourt, PMB 5323, Port Harcourt, Nigeria

Received 8 March 2004; received in revised form 11 July 2006; accepted 31 August 2006Available online 1 November 2006

Abstract

The inXuence of temperature and pH on biomass production and protein biosynthesis in a Spirulina sp. isolated from an oil-pollutedbrackish water environment in the Niger Delta was studied. The isolated organism was identiWed on the basis of its phenotypic character-istics such as nature and direction of helix, temperature, pH and salt tolerance ranges. Biomass concentration in the culture media wascalculated as cell dry weight. The combination of 30 °C and pH 9.0 gave the highest values of 4.9 mg/ml and 48.2 g/100 g for biomass andtotal crude protein, respectively. The eVect of pH was modulated by temperature and vice versa during biomass production. This nativeisolate of Spirulina sp. oVers a good source of natural protein that could be easily accepted by rural communities as single cell protein inthe form of feed, food and health supplement when properly processed.© 2006 Published by Elsevier Ltd.

Keywords: Spirulina sp. temperature; pH; Biomass; Protein; Amino acids

1. Introduction

Spirulina is known to be useful to man in various ways.For instance, as food, it provides a rich source of proteincomprising all the essential amino acids (Santillan, 1982;Fox, 1986; Richmond, 1986b; Silva and Moe, 1992).Pigments for food colouring can be extracted from theorganism. Spirulina has also been used as a health food toalleviate malnutrition, in wound treatment, in growth stim-ulation through thyroid hormone synthesis, in protectionagainst cancer (Richmond, 1986a) and to enhance milksecretion in mothers experiencing lactation problems. Stud-ies of environmental factors that aVect biomass productionand metabolic end-products synthesis by microorganismsare essential since they contribute to understanding thecontrol of metabolic activities and the optimising of yields

* Corresponding author. Tel.: +2348033423726.E-mail address: gideonabu1@yahoo.com (G.O. Abu).

1 Current address: Department of Biology, Rivers State College ofEducation, Rumuolumeni, PMB 5047, Port Harcourt, Nigeria.

0960-8524/$ - see front matter © 2006 Published by Elsevier Ltd.doi:10.1016/j.biortech.2006.08.028

of metabolic end-products of interest. Temperature and pHare among the environmental factors that play a signiWcantrole in the metabolic activities of microalgae (Richmond,1986b; Vonshak et al., 1982; Bhatia and Srivastava, 1995;RaWqul et al., 2005). Protein sources experience diVerentlevels of success in acceptance by diVerent communities.The aim of the present study was to examine the inXuenceof temperature and pH on biomass production and proteinbiosynthesis of an isolate of Spirulina sp. The isolate, whichhas very high percentage protein, could be easily acceptableto local communities when used as feed and/or as foodsupplement.

2. Methods

2.1. Species isolation and characterization

The Spirulina species used in this study was isolatedfrom an oil-polluted Xame pit by the single cell technique,using Bangladesh No. 3 (Bd. 3) medium (Khatum et al.,1994). The water sample contained a large number of

2208 K.H. Ogbonda et al. / Bioresource Technology 98 (2007) 2207–2211

Oscillatoria Wlaments as well. This made isolating the Spiru-lina sp. more demanding. The number of Oscillatoria Wla-ments was, however, reduced by Wrst Wltering the watersample through a Whatman No. 1 Wlter paper using a vac-uum pump and collecting the Wltrate. The Wltrate containedfewer Oscillatoria Wlaments. A syringe Wtted with a hypo-dermic needle was used to draw the Spirulina Wlaments,which were transferred to a watch-glass containing 1 ml ofsterile medium. This process was continued until a unialgalculture of Spirulina was obtained. The content of thewatch-glass (3–4 strands) was then transferred into a 150 mlscrew-capped test tube containing 20 ml of the sterilemedium. The culture was incubated in a lighted chamber(with two 4 ft white Xuorescent tubes placed at 30 cm fromthe bench top, giving a light output of ca 15�E m¡2 s¡1 pertube) for the organism to grow and multiply. This was rep-licated into several lots. Aeration was provided by an aera-tor, which was used to pump air at 150 bubbles per minutethrough a drip set (plastic tubing) Wtted with a regulator(Anaga and Abu, 1996). This set up also provided mixing.The average temperature of the water body (the isolationsite) over a period of three months was determined using asimple glass thermometer. Water samples taken from thesite were used to determine the pH of the water body usinga Jenway 3015 pH meter.

2.2. Determination of the eVect of temperature and pH on biomass concentration of the organism

To determine the eVect of temperature, 250 ml Erlen-meyer conical Xasks were used, each containing 50 ml ofthe growth medium. Each Xask was inoculated with 10 ml(0.05 mg) of the pure culture of the organism as preparedabove. Each temperature regime was in triplicate. The cul-tures were incubated in water baths at the appropriate tem-perature (25, 30, 35 and 40 °C) and periodically replenishedwith the growth medium to prevent drying up of the cul-tures. Lighting was provided through a bank of Xuorescenttubes (Anaga and Abu, 1996) while mixing was providedthrough the aerator as stated above.

To determine the eVect of pH, 250 ml Erlenmeyer conicalXasks were used, each containing 50 ml of the growthmedium. Each Xask was inoculated with 10 ml (0.05 mg) ofthe pure culture of the organism. Each pH (8.5, 9.0, 9.5 and10.0) was in triplicate. Lighting was provided through abank of Xuorescent tubes (Anaga and Abu, 1996).

2.3. Interaction between temperature and pH

The interaction between temperature and pH on bio-mass production of the Spirulina isolate was studied usingthe 4£ 4 contingency cross matching with the followingselection of parameters, pH 8.5, 9.0, 9.5 and 10.0; andtemperatures 25, 30, 35 and 40 °C. The treatments were intriplicates at each pH and temperature level.

The eVect of temperature and pH on protein biosynthe-sis was also studied. The experiments were set up as

described previously for biomass production. However, inestimating the protein and amino acid contents of theorganism, it was necessary to deal with cultures free fromcontaminating bacteria. It has been noted that antibioticsmay aVect blue green algae (which include Spirulina spe-cies) in a similar manner to bacteria (Middlebrook andBowmann, 1964; Brock, 1974), hence bacterial contamina-tion was eliminated by a method involving treatment with amixture of detergent and phenol (McDaniel et al., 1962).

2.4. Biomass analysis

Biomass concentration in the culture suspension wasdetermined as cell dry weight by the method of Vonshaket al. (1982).

2.5. Statistical analysis

Test of independence was done using a 4£ 4 contingencytable and �2 statistics to Wnd out the association betweenpH and temperature in determining the biomass (dryweight) production of the organism.

2.6. Proximal composition

Moisture and ash were determined by the air ovenmethod (AOAC, 1984). Crude fat was determined by theSoxhlet extraction method of Egan et al. (1981). Crudeprotein was determined by the Micro–Kjeldahl method(AOAC, 1984) and the conversion factor from nitrogen toprotein was 6.25. Total available carbohydrate was deter-mined using the Anthrone method (Osborne and Voogt,1978). Crude Wbre was calculated by diVerence.

2.7. Amino acid composition

The amino acids were estimated by paper chromatogra-phy, employing the methods of Allen et al. (1984). Concen-trations of the amino acids are expressed as g/16 g N(approximately 100 g protein).

3. Results and discussion

The characteristics of the isolate compared with thosedescribed in Holt et al. (1994) are shown in Table 1. Theisolate might be identiWed as Spirulina sp. based on thenature and direction of the helix, pH and temperature toler-ance ranges. The putative Spirulina sp. was adapted to fourdiVerent temperatures during Wve culture generations.Fig. 1 gives the biomass concentration over a range of tem-peratures. As can be seen, the optimum temperature for theorganism was 30 °C, at which the highest biomass concen-tration of 4.4 mg/ml was obtained in 35 days. No limitingeVect of lighting, which is a major parameter, was observedat this point. Tables 2 and 3 show proximal compositionsof the organism at various temperatures and pH, respec-tively. The highest amount of protein (46.4 g/100 g) and a

K.H. Ogbonda et al. / Bioresource Technology 98 (2007) 2207–2211 2209

sis showed signiWcant diVerence in the amounts of protein

Table 1Characteristics of a Spirulina sp. isolated from an oil-polluted Xame pit in the Niger Delta

Symbols: +, present; ¡, negative; NS, not stated; ND, not determined; M, marine; B, brackish water; F, fresh water.

Parameter Spirulina maxima Spirulina platensis Spirulina labyrinthiformis Spirulina isolate

Habitat M/B/F M/B/F M/B/F BNature of helix Tight Open spiral Tight Open spiralDirection of helix RH LH RH LHTrichome width <1–5 �m 3–12 �m <1–5 �m NDDiameter of spiral 12 �m 35–60 �m NS NDG+C content (%) 54 44.3 NS NDGas vacuole Present Present Present PresentSulphide + – + NDNitrogenase Present Absent Present NDpH tolerance NS 11.0 NS 8.5–10.0Temperature tolerance Mesophile Mesophile Thermophile Mesophile

5.00

corresponding amino acid content of 76.1 g/16 g N (Table 4)were recovered at 30 °C and a pH of 9.0. This showed thatfor this isolate, 30 °C is the optimum temperature for bio-mass production and protein biosynthesis. Statistical analy-

Fig. 1. EVect of temperature on biomass production in a Spirulina sp.isolated from an oil-polluted Xame pit.

0.000.501.001.502.002.503.003.504.004.50

0 10 20 30 40 50

Temperature (oC)

Bio

mas

s d

ry w

eigh

t (m

g/m

l)

produced at 5% conWdence limit at the various tempera-tures. Richmond (1986b) reported an optimum growthtemperature for Spirulina of 35–37 °C under laboratoryconditions. Cultivation of the organism outdoors has anoptimum growth temperature of about 39 °C. Thermo-tolerant strains of Spirulina have been cultivated between35 and 40 °C. Such a property has the advantage of elimi-nating microbial mesophiles (Vonshak et al., 1982).

The organism was adapted to four pH regimes. The pro-Wle for biomass production is shown in Fig. 2, and as can beseen the optimum pH was 9.0, at which the highest biomassconcentration of 4.9 mg/ml on a dry weight basis wasobtained. Tables 4 and 5 show the amino acids synthesizedby the organism at various pH and temperature valuesrespectively. The highest amounts of protein (48.2 g/100 g,Table 3) and amino acid (78.7 g/16 g N, Table 5) wereobtained at pH 9.0 and a temperature of 30 °C. A full com-plement of amino acids was produced under the conditionstested. This indicates the vitality of the Spirulina isolate for

Table 2Proximate composition of a Spirulina sp. isolated from an oil-polluted Xame pit grown at diVerent temperatures for 35 days, pH 9.0

a Mean of three determinations + SD.

Temperature (°C) Proximate Composition in g/100 g dry weighta

Moisture Ash Carbohydrate Lipids Crude Wbre Crude protein

25 8.30§ 1.1 10.20 § 0.1 14.31 § 0.11 5.40 § 0.7 20.10 § 03 41.90 § 0.3030 7.11§ 0.31 13.13 § 0.28 16.26 § 1.1 4.38 § 061 13.60 § 0.31 46.39 § 0.0435 6.91§ 1.01 11.26 § 0.81 18.96 § 0.93 6.51 § 0.26 12.34 § 0.13 44.89 § 0.140 5.63§ 0.78 14.58 § 0.34 20.69 § 0.66 6.31 § 0.49 14.96 § 0.28 38.54 § 0.03

Table 3Proximate composition of a Spirulina sp. isolated from an oil-polluted Xame pit grown at diVerent pH regimes for 35 days, temperature 30 °C

a Mean of three determinations + SD.

pH Proximate composition in g/100 g dry weighta

Moisture Ash Carbohydrate Lipids Crude Wbre Crude protein

8.5 11.62 § 1.3 12.31§ 0.86 20.11§ 1.2 9.28 § 0.48 13.11 § 0.48 34.10§ 0.339.0 11.19 § 0.83 10.11§ 0.36 14.23§ 0.71 5.32 § 0.71 8.21 § 0.22 48.23§ 1.19.5 10.28 § 1.1 12.31§ 0.11 15.63§ 0.9 8.21 § 0.18 9.19 § 0.38 45.11§ 0.93

10.0 19.18 § 1.2 11.28§ 0.23 21.66§ 1.1 6.33 § 1.8 14.36 § 1.2 36.31§ 0.47

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single cell protein. This observation is noteworthy becausenew technologies including biotechnology are usuallyaccepted with reservations even by well informed societies.This isolate therefore, which has very high percentageprotein, could be easily accepted by local communitieswhen used as feed and/or as food supplement.

SigniWcant diVerence was found in the amounts of pro-tein synthesized at 5% conWdence limit at the various pHlevels. The result of the eVect of temperature and pH takentogether on the biomass production of the organism isshown in Table 6. pH values had a modulating eVect onbiomass production temperature. At pH 8.5, the highestbiomass of the organism was produced at a temperature of35 °C, whereas at pH values of 9.5 and 10.0 the highest bio-

Fig. 2. EVect of pH on biomass production in a Spirulina sp. isolated froman oil-polluted Xame pit.

0.00

1.00

2.00

3.00

4.00

5.00

6.00

8 8.5 9 9.5 10 10.5pH

Bio

mas

s dr

y w

eigh

t (m

g/m

l)

mass production occurred at 30 °C. The organism producedthe highest biomass at a temperature of 30 °C and a pH of9.0 (Figs. 1 and 2). RaWqul et al. (2005) reported 32 °C andpH 9.0 as optimal conditions for biomass production bySpirulina platensis. For Spirulina fusiformis, these authorsreported 37 °C and pH 10.0 as optimal conditions. The sol-ubility of CO2 and other mineral compounds is aVected bypH. For Spirulina, high alkalinity is a requisite for optimalgrowth. For maximum biomass production with the isolatedescribed in the present paper, a shift in the pH of thegrowth medium may require a corresponding change intemperature. The isolate also has potential for biomass pro-duction using petroleum hydrocarbons, since it was isolatedfrom an oil-polluted environment which was its habitat.The ability to metabolize hydrocarbons would furtherenhance the potential of the isolate for bioremediation ofimpacted media such as soil and water bodies.

In conclusion, in the present work, the optimal tempera-ture and pH conditions for biomass production and proteinbiosynthesis were demonstrated for a Spirulina sp. that wasisolated from an oil-polluted Xame pit. These environmen-

Table 6Interaction of pH and temperature in biomass production by Spirulina sp.isolated from an oil-polluted Xame pit in the Niger Delta

pH Temperature (°C)

25 30 35 40 Total Proportion

Biomass g/L8.5 2.93 3.20 3.60 1.90 11.63 0.239.0 3.10 4.60 4.14 3.67 15.51 0.309.5 2.82 4.10 3.20 2.30 12.42 0.24

10.0 3.03 3.98 2.46 2.04 11.51 0.23

Total 11.88 15.88 13.40 9.91 51.07 1.0

Table 4Amino acid composition of a Spirulina sp. isolated from an oil-polluted Xame pit grown at diVerent temperatures and pH 9.0

Temp, Temperature.a Not determined.b Essential amino acid.

Temp °C Amino acid (g/16 g N)

Ser Metb Lysb Asp Trya,b Tyr Asn Glu Lleb Cysa Gln Hisb Arg Leub Phe Ala Thrb Valb Pro Gly Total

25 1.10 1.62 0.45 1.32 ND 5.95 0.78 7.94 2.20 ND 6.31 0.99 8.32 1.10 0.93 6.55 0.83 0.78 0.54 2.31 50.0030 1.91 2.23 0.86 2.66 ND 8.11 1.14 9.26 2.70 ND 7.11 1.28 0.29 2.84 3.11 8.26 4.18 3.45 1.89 5.81 76.0935 1.56 1.98 0.79 1.41 ND 6.64 1.86 8.88 2.61 ND 6.83 1.13 9.88 1.79 2.56 7.92 3.76 3.11 1.22 5.33 69.2640 1.32 1.87 0.64 1.62 ND 2.73 0.83 6.59 1.15 ND 6.62 1.18 7.58 2.20 1.87 7.13 2.09 1.98 0.73 4.72 52.85

Table 5Amino acid composition of a Spirulina sp. isolated from an oil-polluted Xame pit grown at diVerent pH values and 30 °C

a Not determined.b Essential amino acid.

pH Amino acid (g/16 g N)

Ser Metb Lysb Asp Trya,b Tyr Asn Glu Ile Cysa Gln Hisb Arg Leub Pheb Ala Thrb Valb Pro Gly Total

8.5 2.23 0.99 3.28 1.88 ND 6.11 1.91 6.26 1.82 ND 4.18 0.45 7.13 0.98 0.84 5.39 2.18 3.41 0.88 3.44 53.369.0 5.58 1.73 4.13 2.89 ND 7.23 2.66 8.13 2.93 ND 5.23 1.81 9.28 2.11 2.11 7.21 3.44 5.28 2.10 4.89 78.749.5 4.64 1.33 3.86 2.27 ND 6.86 2.48 7.90 2.11 ND 5.10 1.22 8.16 1.93 1.66 6.99 3.12 4.11 1.88 3.56 69.22

10.0 3.11 1.10 2.38 1.68 ND 6.42 2.23 6.81 1.91 ND 4.63 0.92 7.94 1.24 1.13 5.86 2.81 3.68 1.12 3.48 58.45

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tal growth conditions, namely pH and temperature, areamong the critical factors (Bhatia and Srivastava, 1995;RaWqul et al., 2005) for algal biotechnological processes.

Acknowledgements

We acknowledge the cooperation and assistance ofthe staV and management of TotalFinal Elf (Nig.) Ltd., PortHarcourt in collecting the samples and Weld data for thestudy.

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