The removal of sodium (Na) from seawater using two photosynthetic bacteria was investigated using Rhodobacter sphaeroides SSI (SSI) and Rhodovulum sp. which is a marine photosynthetic bacterium. Both Rhodovulum sp. and acclimated SSI were shown to grow well in a 3% NaCl supplemented glutamate-malate medium. The maximum rate of Na removal was 39.3% by SSI and 64.9% by Rhodovulum sp. after two days cultivation under static light conditions. However, Na was re-released back into the medium after two to three days. When a nutrient-supplemented seawater medium (3.3% NaCl, 13.10 gNa/l) was used, the maximum Na removal rates were 30.3% (9.05 gNa/l) by SSI and 48.9% (6.69 gNa/l) by Rhodovulum sp., under static light conditions. Similar growth and Na removal rates were found under aerobic dark cultivation. In this case, no re-release of Na was observed with either bacterium. Two stages culturing was conducted first, with Rhodovulum sp. and then with SSI replacement. The Na concentration was reduced to 0.79 gNa/l (94.0% removal) after cultivation for eight days under aerobic dark conditions. The supernatant was applied successfully as a liquid fertilizer in the cultivation of Japanese radish.
Photosynthetic bacteria that produce extracellular polymeric substances (EPS) on the cell surface adsorb cationic metals such as Cd, Pb, Cr, Hg, Cu and As at rates between 85% and 100% using the negative charge of the EPS [
The Great East Japan Earthquake occurred in March 2011. It released large amounts of radioactive materials, mainly Cs, from the Daiichi Nuclear Power Plant in Fukushima which then began to spread throughout the local and re- gional environment. In a previous study, we investigated the removal of radioac- tive Cs from the water and sediment mud of a swimming pool Fukushima City, demonstrating removal rates of almost 100% and 93% from the water and sediment mud of a swimming pool, respectively. This was achieved in four days using aerobic treatment with SSI immobilized alginate beads [
Panwichian et al. reported removal rates using newly isolated photosynthetic bacteria of the NW16 and the KMS 24 strains of 39% for Pb, 20% for Cu, 7% for Cd, 5% for Zn, and 31% for Na. These stains were cultured in a 3% NaCl sea- water medium and exhibited Na reduction under microaerobic light (static light) and aerobic dark conditions. However, the precise function of Na reduction wa- sunclear [
This study focused on the Na removal using two photosynthetic bacterial strains using a synthetic medium and a nutrient supplemented seawater medium (NSSW medium). Practical Na removal was demonstrated using both strains.
The marine photosynthetic bacterium used was Rhodovulum sp. [
Two NaCl supplemented glutamate-malate media (3% NaCl and 1% NaCl) were used for growth and Na removal. The composition of both GM media was as follows (g/l): sodium-L glutamate 3.8, DL-Malic acid 2.7, (NH4)2HPO4 0.8, Yeast extract 1.0, KH2PO4 0.5, K2HPO4 0.5, MgSO4∙6H2O 0.2, CaCl2∙2H2O 0.05, Thiamine-HCl 1 × 10−3, Nicotinic acid 1 × 10−3, Biotin 0.01 × 10−3. This GM medium was modified from GM supplemented medium by Lascelles [
An NSSW medium with 3.3% NaCl, was prepared by nutrients the addition of 5.0 g/l of glucose and 2.0 g/l of peptone. Seawater was taken from Hiroshima Bay, Japan. The initial pH of the medium was adjusted to 7.0 ± 0.1 using a HH4OH solution or 6N-HCl. Media were sterilized by autoclave (at 121˚C, 20 min.).
To improve the growth of SSI in the 3% NaCl GM medium, acclimation was performed. First, the SSI was cultured in a 0.5% NaCl GM medium under static light conditions for two to three weeks. Slow growth was observed. The culture broth was then transferred to a 1% NaCl GM medium. Again, slow growth was observed. Cultivation was then performed three to five times in a 2% NaCl GM medium, and three to five times in 3% NaCl GM medium. The acclimated strain of SSI was then placed on agar plate of 3% NaCl GM medium. Cultivation of all strains was conducted at 30 ± 0.5˚C.
Pre-culturing was performed in a 300 ml conical flask (100 ml medium) under micro-aerobic condition with static light for three days. Illumination was sup- plied by a tungsten bubble at 5 klux (80 μE/m2∙sec) onto the surface of the flask. For the main culture, a 300 ml conical flask (200 ml medium) was held under the same conditions for five days. An aerobic dark culture was prepared in a 300 ml conical flask (medium 150 ml) placed on rotary shaker (120 rpm) for five days. Inoculation of the pre-cultured broth was conducted at 10% (v/v).
Culture replacement was performed in two stages. For Rhodovulum sp., cul- turing was first performed using the NSSW medium under both static light and aerobic dark conditions. After four days, the broth was centrifuged at 10,000 xg for 20 min to isolate the supernatant. Centrifugation was performed at room temperature (23˚C - 30˚C). This was sterilized using Millipore filter (0.45 μm pore size) and placed in a sterilized flask. In the second stage, SSI pre-cultured broth was inoculated at 10% (v/v) and cultivated for further four days under static light or aerobic dark conditions. On day eight, centrifugation at 10,000xg 20 min. was performed at room temperature (23˚C - 30˚C). In the next stage the experiment, the supernatant obtained was used as liquid fertilizer. Static light and aerobic dark culture were performed with 200 ml and 150 ml culture broth, respectively, in 300 ml flask. Cultivation temperature of Rhodovulum sp. and SSI was 30 ± 0.5˚C.
During the cultivation of both strains, 10 ml of culture broth was pulled out and measured OD660 for the growth evaluation every day. And then, culture broth was centrifuged (10,000 xg, 20 min.) at 4˚C. From the supernatant, Na concentration was measured.
Sprout of Japanese radish (Raphanas sativas) were cultured in the polyethylene container (10 × 20 × 5 cm) on 2 cm layer of cotton(put on the bottom) with a supply of deionized water to have wet condition. The container was placed near a window under indirect sunlight at roomtemperature (23˚C - 30˚C).
As a control, sprouts were sprayed with a commercial liquid fertilizer, diluted 1000 times HYPONeX stock solution (HYPONeX Japan Co. Ltd.) at one to three days intervals. A second container, supernatant as liquid fertilizer was sprayed in the same manner as the control. Growth and damages were observed for about one month.
The growth of the photosynthetic bacteria was observed at OD660 using a spec- trophotometer (Shimadzu, UV-1700). The Na concentration was measured us- ing anatomic adsorption spectrophotometer (Shimadzu, AA-6200). Experimental errors of OD660 and Na were the mean value ± 5% and ± 10%, respectively. Data were the mean values from triplet experiments.
In contrast, Rhodovulum sp. showed more rapid growth and greater Nareduc- tion. The maximum Na removal rate of 4.25 gNa/l (64.9% removal) was record- ed after one day under the static light conditions (
It is well known that the energy status of the cells of photosynthetic bacteria affects the uptake of Na. Under high energy conditions, Na is incorporated, whereas, under low energy conditions Na is exported [
of growth, energy harvesting fell because of the limitations on carbon, light and oxygen, so that Na might be exported again into the medium.
As shown in
The growth of Rhodovulum sp in the 1% NaCl GM medium (
In the removal of Na by SSI, the negative charge of the EPS on the cell surface appear to function in the same way the adsorption of cationic heavy metals and radionuclide [
The SSI strain successfully grew in the NSSW medium removing up to 30.3 % of the Na after four days under both the static light conditions (
the aerobic dark conditions (
When Rhodovulum sp. was grown in the NSSW medium under the static light (
Both SSI and Rhodovulum sp. were able to remove Na from the NSSW me- dium under the aerobic dark conditions. This suggests that the use of photosyn- thetic bacteria offers a convenient, low-cost approach for the desalinization of seawater. In other desalinization processes that use photosynthetic bacteria, for example, cyanobacteria, large ponds are required to provide sunlight for growth of cells [
We observed relatively high Na reduction by Rhodovulum sp. and SSI in the NSSW medium and almost nore-export of Na into the broth. Na removal was performed in two stages. As can be seen from
The minimum Na concentration of 0.79 gNa/l (0.2% NaCl concentration) was reached after four days of the second stage (total culture time of eight days) un- der both conditions. This represented a 94.0% removal of Na from the seawater. It must be emphasized that it was high level desalinization establishment using the two stages aerobic culture. For example, Panwichian et al. reported that re- duction to 31% of Na removal was observed using photosynthetic bacteriacul-
ture from the seawater [
The supernatant obtained by centrifugation was used as a liquid fertilizer for the cultivation of Japanese radish. The growth and the appearance of sprouts were similar to the samples grown using the conventional liquid fertilizer of HYPO- NeX solution for one month (data not shown).
The supernatant contained photosynthetic bacterial metabolites such as ami- no acids, organic acids and extracellular products. These might support vegeta- ble growth at a low Na concentration. For example, a photosynthetic bacterial supernatant usually contains 10 - 30 μmole/I of 5-aminolevulinic acid (ALA). This extracellular product supports the growth of rice seedling [
This study suggests the possibility of producing liquid fertilizer from seawater after two stage treatment using photosynthetic bacteria. This would support agriculture in regions where fresh water is scarce, such as islands and coastal areas of Japan. However, further investigation and long term observation of this liquid fertilizer (to assess its safety) will be needed before applying it to the cul- tivation of vegetables, plants or flowers.
Based on our results, we propose the practical process of Na reduction and bio- conversion of seawater into liquid fertilizer as shown in
Photosynthetic bacteria can grow without large contamination under low oxygen level with a dissolved oxygen level below 1.0 mg/l. In Japan, practical wastewater treatment plants handling food processing wastewater mainly use ac- tivated sludge treatment. Photosynthetic bacteria are usually dominant under the low oxygen level in this aerobic treatment [
After cultivation of Rhodovulum sp., chitosan sedimentation is performed to separate the cells. Chitosan is a good coagulant and for photosynthetic bacteria and can easily sediment the bacteria. As chitosan is a natural material, the sedi- mented cells can also be used as a feedstock for production of pet food, animal feed and fish feed. After three to four days of SSI culture, the cells are again se- dimented and separate using chitosan treatment. The supernatant obtained can be used as a liquid fertilizer or sprinkling water for vegetables, flowers and water source for soilless culture or hydroponic culture.
In this study, we demonstrated possibility of the bioconversion of seawater into liquid fertilized by removal of Na using two strains of photosynthetic bacteria. The comparative cost of this alternative approach is currently being investigated.
The removal of Na by photosynthetic bacteria was investigated, and the following results were obtained.
1) Acclimated SSI was shown to grow well in a 3% NaClGM medium (11.8 gNa/l). The maximum Na removal was 39.3% (7.16 gNa/l) under static light conditions and 36.7% (7.47 gNa/l) under aerobic dark conditions.
2) Rhodovulum sp. also grew well in the 3% NaCl GM medium maximum Na removal of 64.9% (4.2 gNa/l) was observed after one day of culture under both conditions but Na began to be released back into the broth after two days.
3) In the NSSW medium (3.3% NaCl, 13.1 gNa/l), SSI was shown to remove up to 30.3% of the Na (9.05 gNa/l) after four days under the static light condi- tions, and Rhodovulum sp. up to 48.9% (6.69 gNa/l). In this case, almost no Na was later exported. Similar results were observed under the aerobic dark conditions.
4) Tow stage culturing first, with Rhodovulum sp. then with an SSI replace- ment culture was performed under the aerobic dark conditions using the NSSW medium. Finally, the Na concentration was reduced to 0.79 gNa/l (94% removal) after eight days. This represented a high level of desalinization.
5) The supernatant (0.2% NaCl) was used in the cultivation of Japanese ra- dish, demonstrating its use as a liquid fertilizer.
6) Our study demonstrated a practical approach to the bioconversion of sea- water into liquid fertilizer using Rhodovulum sp. and SSI.
Sasaki, K., Hosokawa, Y., Takeno, K. and Sasaki, K. (2017) Removal of Sodium from Seawater Medium Using Photosynthetic Bacteria. Journal of Agricultural Chemistry and Environment, 6, 133-143. https://doi.org/10.4236/jacen.2017.63008