Spirulina sp. Production in Brine Wastewater from Pickle Factory

P. Duangsri Department of Chemical Engineering

Faculty of Engineering, Chulalongkorn University Bangkok 10330, Thailand P.duangsri@gmail.com

C.Satirapipathkul Department of Chemical Engineering

Faculty of Engineering, Chulalongkorn University Bangkok 10330, Thailand chutimon.s@chula.ac.th

Abstract—Spirulina sp. cells, growing photoautotrophically in complex media containing pickling wastewater were exposed to different light intensities and salinities. Evaluation of growth and chemical composition of Spirulina platensis in batch cultures was undertaken in laboratory batch experiments. Biomass concentration (as dry weight) and protein content of cultivation in complex medium were lower to those observed in the inorganic Zarrouk medium, regardless of the light intensity. Increasing salinity of the medium reduced biomass and protein concentration, but considerably increased low molecular weight carbohydrate concentration. Moreover, biomass from the complex medium was enriched in total lipid, when cultures were exposed to the lower light intensity. Scanning electron microscope illustrated the morphological change of the stressed cells increased with the increase in the wastewater salinity.

Keywords-brine wastewater; pickle factory; Spirulina sp.; morphology; scanning electron microscope

I. INTRODUCTION Commercial production of pickling vegetables in

Thailand has increased continuously due to rising export demands, as well as domestic consumption. At present, the actual amount of wastewater generated in the manufacture of pickling has not been investigated. However, based on annual pickling vegetable production of 100 thousand tons, it is estimated that approximately 50-70 thousand tons of wastewater are produced. In practice, the brine wastewater from pickling industry is released without treatment, and this is a serious potential source of pollution. Moreover, the effluent from storage pond would cause environmental problem if discharged directly into the waterways [1, 2].

Currently, the commercial production of Spirulina in many areas of Thailand is oriented mainly towards the health food market. Considerable interest has been invested in outdoor cultivation of Spirulina sp. for commercial biomass production because of its potential source of protein and valuable chemicals [3]. Spirulina sp. is capable of adapting to high NaCl concentrations. Such adaptation is associated with an increase in respiratory activity and a partial recovery of photosynthetic activity in the Spirulina cells after the initial decline due to exposure to high salinity. [4-6]. The culture medium is an important factor in the production cost of the algal product, massive cultivation of Spirulina in wastewater media could improve the prospects for industrial production. Our previous

results showed that the wastewater from pickling factory can be used as the complex media for Spirulina production [3]. However, the adaptation of Spirulina sp. to salinity stress is a complex process. Yet the site of damage induced by the salinity stress and biochemical change was not identified.

The objective of the present study was to investigate the effect of salinity and light intensity stress on Spirulina sp. We also observed the biochemical component and morphological changes in an attempt to understand how salinity stress affects growth of Spirulina sp.

II. 1BMATERIAL AND METHOD

A. 4BMicroorganisms Spirulina sp. was locally isolated strain from the storage

pond of the pickling factory. The culture was maintained as an axenic culture in the modified Zarrouk liquid medium.

B. 5BPreparation of inoculum Subcultures of Spirulina sp. in which 10% of the volume

remained as inoculum were performed in modified Zarrouk's medium at 35°C with an initial pH of 9.0. Cultivation was done in glass containers subjected to a moderate mixing provided by a small air pump operating at a rate of 0.046 vvm (volumetric flow rate of air per volume of liquid per minute). Cultures were exposed to tungsten lamps (39 W), to provide either 120 or 200 µmol photon m-2s-1, depending on the kind of experiment in which they were used. The white lamps operated in cycles of 12 h of light and 12 h of darkness. The light intensity was measured by a digital light meter (Luxtron LX-101).

C. 6BCulture medium Wastewater and the effluent were collected from local

pickling factory. In the laboratory they are stored at 4°C in the cold room. The wastewater is filtered through coarse cloth gauze to remove the fibers and other debris and subsequently diluted in the different proportions with fresh water. The sample of the solid fraction of effluent (after being left to settle during 24 h) from storage pond was added to the diluted wastewater. The NaCl content, nitrogen content, phosphorus content, biological oxygen demand (BOD) and pH of wastewater and effluent sample were measured.

Wastewater was diluted with fresh water. A 1% (v/v) sample of the effluent was added to the batch culture of

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2011 International Conference on Bioscience, Biochemistry and Bioinformatics IPCBEE vol.5 (2011) © (2011) IACSIT Press, Singapore

Spirulina. Sodium bicarbonate was added to provide 2 g l-1 in the final mixture. pH of the medium was adjusted to pH 8.5 with NH4OH at time zero of cultivation.

Control medium was prepared according to Zarrouk (1966), except for the lack of three trace elements (Va, Ti and Ni which were not available), and it was also utilized to preserve the strain.

D. 7BCulture conditions Experiments were carried out at 35°C and exposed to

different light intensities (120 and 200 µmol photon m-2s-1) and salinity (0-16 ppt). Cultivations were done in the control medium and the complex medium. The initial chl concentration of the culture was 1.5 mg ml-1. These conditions were maintained for all subsequent batch studies. Analytical methods

Total solids, total N and phosphorus were determined according to Standard Methods as published by American Public Health Association [7]. Biomass was expressed in mg L−1 dry wt based on chl-acontent, while the specific growth rate was calculated [8]. Protein was determined by the modified biuret method Chlorophyll was estimated spectrophotometrically after extraction in cold methanol in the dark. Carbohydrates were extracted by the method of Dubois [9]. Total lipids were extracted with Soxhlet equipment, utilizing a mixture of chloroform and methanol (2:1) and were quantified spectrophotometrically at 628nm by the reaction with sulfophosphovanillin [10]. All the results concerning biomass concentration and chemical composition are averages of three experiments.

E. 8BMorphplogical observations by a scanning electron microscope (SEM) Small pieces of Spirulina culture grown in control and

complex medium were and post-fixed in 0.2% OsO4 in distilled water for 1 h. The samples were dehydrated by passage through an acetone-water series (25-50-75-100-100% acetone) and dried at the critical point in liquid CO2. Special care was taken to purge all the acetone with liquid CO2 from these relatively large SEM samples. All samples were viewed on the scanning electron microscope.

III. 2BRESULT AND DISCUSSION

A. 9BWastewater and effluent from storage pond composition The wastewater collected from the local pickling factory

contained a high concentration of salt (Fig.1). It consisted of 82 g/l total soluble solid, 11 g/l total sugar, 0.9g/l total acidity, 0.2g/l nitrogen, 1.6g/l phosphorus, 64 g/l NaCl and 10,800 mg/l BOD with a pH of 4.8. The effluent from storage pond consisted of 623 g/l total nitrogen, 101 g/l total phosphorus, 237g/l NaCl and 20, 8763mg/l BOD with a pH of 4.2 (Fig.2). C: N:P ratio of wastewater is high and pH is acidic. The nitrogen concentration is the most important limiting factor for intensive growth of algae, but wastewater seemed to be not the good source. Supplementation with solid effluent from the storage pond improved biomass production of Spirulina sp.

Figure 1. Brine wastewater and Effluents from storage pond

B. 10BEffect of salinity In this work, the final salinity of the complex medium is

lower than 20 ppt. The response of Spirulina sp. was investigated when grown in different salinlty of complex medium (0-16 ppt). The growth rate of Spirulina sp. significantly inhibited in salinity stress compared to the control (Table 1). The growth rate was lower and inversely correlated with the degree of salinity i.e. the more was salinity, the slower was growth rate. High salinity of complex medium reduced the specific growth rate. This is due to the inhibition of photosynthetic and respiratory system after exposed to high salt concentration (16 ppt). Decrease in growth rate was observed in the high salinity. It has been suggested that the inhibition of photosynthesis arising from rapid entry of sodium, might be the result of the detachment of phycobilisomes from the thylakoid membranes [11].

The change in growth response of salt-stressed cultures to light reflects a change in their photosynthetic capacity. Biomass and protein content (% of dry weight) were significantly decreased when cultivated in complex medium, while carbohydrate and lipid content were remarkably increased. An increase in carbohydrate content reflected the need to increase the intracellular osmoticum in order to balance the higher osmotic of the medium. Most of those osmoregulants are identified as amino acids or carbohydrates. Protein content varied from 30.9 to 54.9% when the salinity of media varied from 0 to 16 ppt. Carbohydrate content varied from 16.1 to 31.7 % and lipid content varied from 6.8 to 16.4%. At 16 ppt a marked increase in lipid of 16.4 was observed which were significantly higher compared to the control. Stress cells have a lower protein synthesis capacity increasing carbohydrate and lipid metabolism.

TABLE I. GROWTH AND BIOCHEMICAL CONSTITUENTS OF SPIRULINA SP. GROWN IN DIFFFERENT SALINITY USING COMPLEX MEDIA

Salinity Parameter

Specific growth rate

(d-1)

Protein (% Dw)

Carbohydrate (% Dw)

Lipid (% Dw)

0 0.13 54.9 16.1 6.8

2 0.08 38.6 20.1 10.3

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Salinity Parameter

Specific growth rate

(d-1)

Protein (% Dw)

Carbohydrate (% Dw)

Lipid (% Dw)

4 0.07 34.8 26.8 12.0

8 0.04 31.2 29.3 14.7

16 0.03 30.9 31.7 16.1

C. 11BEffect of light intensity One of the major advantages of Spirulina cultivation

utilizing effluent from the storage pond is that the macro- and micro-nutrients required for its growth are mainly provided by this kind of low-cost raw material. However, this effluents needs to be diluted before preparing culture medium for avoid an excess of ammonia nitrogen, turbidity and salinity. When Spirulina cultures are grown at optimum temperature and pH condition in control and complex medium (8 ppt) supplemented with 1 % effluent (Fig.2). An Increase in the biomass production of Spirulina sp. growing in control medium is observed with increasing light intensities from 120 to 200µmol photon m-2s-1 (Fig.3). . The dry weight increased from 2.3 to 4.8 g l-1.

The growth obtained at supplementation with increasing concentration of effluent was higher. The phosphorous in effluent gave better growth. At optimum light intensity (120 µmol photon m-2s-1) and temperature (35 °C), the Spirulina biomass increased from 1.3 to 2.7 g/l as the solid effluent concentration increased from 1 to 5 %. However, increasing light intensities decreases dry weight production from 1.3 to 0.8 g l-1. The protein, carbohydrate and lipid content of Spirulina sp. were significantly changed when grown in both control and complex medium.

Figure 2. Chemical composition of Spirulina sp. grown in the control and

complex medium at 120 µmol photon m-2s-1

1% e

Figure 3. Chemical composition of Spirulina sp. grown in the control

and complex medium at 200 µmol photon m-2s-1.

D. 12BMorphological change Spirulina sp. isolated from wastewater pond consists of

straight filaments. Though the typical helical shape of Spirulina sp. was maintained in the cultures grown with different salinity of complex medium (8-16 ppt) and Zarrouk’ s medium, there were some difference in the length of trichomes, degree of helicity, color and amount of cellular content. Fig. 4 showed scanning electron microscope view of Spirulina grown in three medium. Increase in salinity above the basal level inhibits the growth of helicoidal morphome, while the straight morphome’s growth behavior remains unaffected. Usually long trichomes, sometimes forming aggregates, were observed in control medium whereas short trichomes were dominating in high saline medium (Fig. 4a and 4b). Very lax helices were distinctive for cultures grown in complex medium with the highest salinity (Fig. 4c). The change in the morphology of the trichome (from the helicoidal to the straight form) as modifications of physiological behavior of Spirulina sp. in response to the increase in salinity in complex media. Microscopic observation showed that pigmentation and cellular contents of algae were lower in complex medium than in control medium.

IV. 3BCONCLUSION The present work shows that the response of Spirulina sp.

to salinity-stress consisted of an adaptation process. The data showed that the light at which algae are grown affects the ability of cells to response and adapt to salinity-stress. Stressed cell showed higher lipid and carbohydrate content than non stressed cells. However, brine wastewater and the

5% Effluent Control

1% Effluent Control

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effluent generated during the production of pickling vegetables could serve as a medium for algal cultivation to produce biomass and chemical composition. The information developed in this study should be applied to a large scale operation to further evaluate its reuse potential.

13BACKNOWLEDGMENT This work was financial supported by Thailand research

fund.

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Figure 4. Scanning electron microscopic view of Spirulina sp. grown in three medium; a. Zarrouk; b. complex media (8 ppt); c .complex media (16 ppt)

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