Using waste mineral water from RO column to culture Chlorella vulgaris algae biomass

Trinh Thi Lan, Nguyen Thi Thuy Hang, Nguyen Tran Thien Khanh, Nguyen Thi Bich Hanh, Nguyen Hieu Nhan, Nguyen Thi Bao Thoa, Nguyen Ngoc Phuong Trang and Nguyen Huu Yen Nhi

An giang University, Vietnam National University Ho Chi Minh City, Vietnam
ttlan@agu.edu.vn

Abstract

A trial to evaluate the ability of using waste mineral water from the RO filter column of the SM bottled mineral water factory on cultivating Chlorella algae biomass as feed for aquaculture was carried out from June to October 2021. The experiment was arranged in a completely randomized design with 5 treatments and 3 replicates to evaluate the rate of waste mineral water suitable for algae biomass culture. The percentages of waste mineral water include 100%, 75%, 50%, 20% and 0% (as a control treatment). The environmental factors were monitored and adjusted to get suitable levels for the growth of Chlorella vulgaris. The results of the experiment showed that algae growth peaked at day 5, preferably in the 100% mineral water treatment, and there was a significant difference between treatments. Therefore, it is possible to use 100% of waste mineral water directly from the RO column to grow Chlorella vulgaris algae biomass in order to reduce the amount of water discharged into the environment, save underground mineral water and increase biological productivity for human life and in aquaculture.

Keywords: algae biomass, chlorella vulgaris, mineral, wastewater

Introduction

Chlorella is a fast-growing alga and is considered a valuable source of nutrients in the future with a high protein content of about 50% and contains most of the essential amino acids such as lysine, methionine, tryptophan, arginine, histidine, the lipid of algae varies from 10-20% with the majority of unsaturated fatty acids. Chlorella contains most of vitamins: A, B1, B2, B6, B12, C, D, K, nicotinic acid, pantotenic acid especially rich in vitamin C. In nature, algae is an important linkage in the feed chain. In aquaculture, Chlorella is often used as a suitable feed source for rotifers, Moina, fish larvae Besides, Chlorella is also used in green water systems to rear shrimp and fish larvae. It is used to stabilize the water environment, prevent the formation of toxic compounds. Especially, Chlorella has the ability to produce chlorellin, a compound from fatty acids capable of preventing the growth of some bacteria (Pratt 1948). In addition, in Chlorella also contains CGF (Chlorella growth factor), CGF is a peptide nucleotide chain produced and helps Chlorella to increase photosynthesis, grow quickly, effectively stimulate growth, and help people recovery after illness (Chen 1996; Lee 1997) so it is well known in medicine, food in powder or tablet form to provide essential nutrients to the body. Chlorella is also used as a functional food because it has anti-cancer, antioxidant and skin-lightening properties, and as well as a source of raw materials for bio-oil production (Xu et al 2006; Xiong et al 2008). Therefore, to serve these purposes, Chlorella has been cultured in many different environments and systems. In the previous study, different water environments such as biodigester effluent or wastewater from biogas digesters (Lan et al 2012; Nguyen Minh Tuan et al 2012) wastewater from fish ponds (Tran Chan Bac et al 2015) and domestic wastewater (Nguyen Tran Thien Khanh et al 2017) were done to grow Chlorella algae.

In livestock and aquaculture, Chlorella is an important feed for rotifers because of its ability to make rotifers grow rapidly and reach maximum density. In addition, Chlorella also plays an important role in seed production, rearing larvae of giant freshwater prawns, sea crabs and marine fish in green water models... The addition of Chlorella in the nursery tank helps to increase the quantity feed as well as dissolved microelements needed when there is not enough food in the tank. On the other hand, Chlorella has the ability to limit the growth of pathogenic bacteria when there is a high growth of algae in the nursery tank and can be used as a protein source to replace animal and plant protein sources in feed for aquaculture as well as livestock.

Currently, in the process of producing purified water and bottled mineral water, water plants have discharged a large amount of water into the environment from the RO water filter column. For mineral water sources, in addition to the amount of water that is filtered when passing through the RO filter core of the water purifier, the RO system also has the amount of water that cannot pass through the 4th filter called wastewater. The input water, after passing through the first three filters, it has been treated with all suspended solids > 1 micrometer (0.001 mm) and odor. When it reaches the RO membrane, the purified water passes through the membrane into the tank and the rest carry the micro-minerals out by the wastewater. This is a source of waste minerals that can be absorbed very well by algae, so it has the potential to be utilized to grow Chlorella algae, increasing the quality of algae.

Objectives

The objective of the study is to reuse the wastewater from the RO column in the process of producing mineral water, to save underground mineral water and increase biological productivity, which is to create a feed source for shrimp larvae and fry fish in aquaculture.

Materials and methods

Prepare research materials

Waste mineral water were collected at the wastewater discharged from RO filter column of SM bottled mineral water production company, then transferred to the laboratory of An Giang University.

Chlorella vulgaris samples were collected and isolated from the catfish ponds. The medium for isolation used is the Walne environment. To make the solid medium, 10 g/L (1%) agarose was added to the liquid medium. Microalgae samples were diluted by stratified dilution in the respective medium. Stratified dilutions were performed aseptically and obtained at 10^-2, 10^-3 and 10^-4 dilutions. A total of 5 drops of sample from each dilution level were applied to the surface of the sterile solid medium and spread evenly. The isolates were incubated at room temperature (23-25 ​​°C) with a light:dark cycle of 24:0 for 7-14 days. Isolated colonies grown on this medium were transferred to fresh sterile solid medium. The selection of isolates was based on colony morphology, colony color and cell morphological differences. The process is carried out continuously until pure strains are obtained. Culture of pure strain isolates on liquid medium was performed by daily aeration at room temperature (23-25˚C), with a light:dark cycle of 24:0 for 14 days (Ermavitalini et al 2021). After that, the biomass was increased in Walne medium to have enough density for experimental layout.

Photo 1. Microalgae culture isolates	Photo 2. Colony morphology of Chlorella microalgae	Photo 3. Pure Chlorella vulgaris isolated

Photo 4. Morphology of microalgae Chlorella vulgaris	Photo 5. Algae proliferation Chlorella vulgaris

Experimental layout

The experiment was arranged in a completely randomized design with 5 treatments and 3 replications.

• 100% of wastewater discharged from RO filter column
• 75% wastewater discharged from RO filter column + 25% tap water
• 50% wastewater discharged from RO filter column + 50% tap water
• 25% wastewater discharged from RO filter column + 75% tap water
• 100% tap water

All treatments were supplemented 2ml of Walne's medium for 1 liter of algae culture water with role as nutrients and microelements. The experimental period was 15 days.

All treatments; were conducted in colloidal glass containers (5 liters/ container). The input algae will account for 10% of the culture water volume. These media ratios was be premixed and put into each container following the dose of each treatment.

All colloidal glass must be aerated, illuminated continuously 24/24 during the experiment and covered tightly to avoid the invasion of other algae.

Collect samples on days 0, 1, 3, 5, 7, 9,11, 13 and 15 at 8:00 am – 9:00 am to calculate density and analyze indicators of NO₂^-, NH₄⁺, PO₄^3- and chlorophyll a.

Samples for physicochemical analysis and chlorophyll a were collected into 110 ml plastic bottles and stored cold at 4oC in the laboratory's refrigerator, samples were collected for 50 ml of algae density and fixed with 4% formalin.

Measure pH, temperature, and DO indicators every day with a handheld meter.

Measurement

Chlorella density was determined by the method of Coutteu (1996). Density of Phytoplankton was counted in a Sedgewick Rafter counting chamber according to the formula:

M (Individuals/ml) = (T*1000*V_cd)/(A*N*V_mt)

In which: T: Total number of individuals counted

A: area of the cell count

N: number of cells counted

V_cd: the volume of water condensed

V_mt: the volume of sample collected

Water quality including NO₂^-, NH₄ ⁺, PO₄^3- were collected and analyzed according to APHA (1995).

Determination of algae biomass by spectrophotometric method Nush.

- Collect 100 ml of sample into a 125 ml glass bottle.

- Put 3 ml of 1% MgCO3 on filter paper when placed in the suction machine (before filtration).

- Pass 100 ml of the filter sample through filter paper until the water runs out.

Roll filter paper into a test tube 18 ml Ethanol 90% (alcohol 90^o).

- Boil at 78^oC for about 15 minutes.

- Cool, centrifuge at 3000/min for 5 minutes.

- Compare colors at 665nm and 750nm.

- Take 8 ml of the solution and compare the color of acidified with 1 drop of 2N HCl.

- spectrophotometric at wavelength at 665nm and 750nm.

- Record the results and calculate according to the formula:

Chlorophyll a (μg/L) = [(E665 – E750) – (E665a – E750a)] *(V1.D/V2.d) * 1000 * 29.6

- V1: Alcohol volume 90^o (18 ml).

- V2: Volume of filtered water.

- D: Number of dilutions.

- d: Length of light passing through Cuvet (1 cm).

-E: colorimetric value measured at wavelengths

Biomass B (μg/L). = Chlorophyll- a * 67

Statistical analysis

The data were analysed by use a Minitab program with general linear model (GLM) option (version 16.0) ANOVA software (Minitab 2016). Analyse variation are treatments and error.

Results and discussion

Water quality of waste mineral water

The waste mineral water (after going through the RO column) has the appropriate criteria according to the standard QCVN 06-1: 2010/BYT (Table 1). The composition of this waste mineral water has a rather large content of K and Na, P are very suitable for algae culture because these are the 3 trace elements necessary for the growth of algae (Sunda 2005). Besides, the content of HCO₃^- is also necessary for the growth of microalgae, so this is a favorable condition to be able to use this water source in the cultivation of microalgae biomass. Therefore, if this amount of waste mineral water is discharged into the environment every day, it will cause a significant waste of mineral water.

Environmental parameters during the experiment

The effect of temperature on photosynthesis of Chlorella cells varies from species to species (Oh-Hama and Miyachi 1986). Temperature affects not only the metabolism directly or indirectly, but also the cellular structure (Payer et al 1980). Each species of algae has a different suitable temperature range. But in general, the optimal temperature for algae culture ranges from 23-30^oC depending on the species (Truong Sy Ky 2004). However, the suitable temperature for Chlorella algae is 25-35^oC, but the algae can tolerate 37^oC (Liao et al 1983).

The pH limit for the growth of algae is from 7-9, but suitable in the range of 8.2-8.7. If the pH change is large, it can cause the algae to die out (Nguyen Thanh Phuong 2003). In the case of high density algae culture, CO ₂ addition to stabilize the pH below 9 during the algae growth is necessary, the pH suitable for Chlorella to grow is best from 8-9 (Tran Thi Thuy 2008).

Environmental factors in the experiment such as pH and temperature in the experiment were in the appropriate range for the growth of Chlorella algae. The temperature fluctuates in the range of 24.5 - 24.9^oC; The pH ranges from 7.8 to 8.3.

NH­₄⁺ ranged from 0.01 to 0.08 mg/L, the highest was in the 100% mineral wastewater treatment and the lowest was in the 50% mineral wastewater and 0% mineral wastewater treatment. Although NH ₄⁺ fluctuated between treatments, it was not significant and did not affect the growth of algae. Similar to NH₄⁺, NO₂^- and PO₄ ^3- also fluctuated between treatments but did not affect the growth of algae (Table 2).

Variation of algae density over time in the experiment

Variation of algae density between treatments during the experimental period was shown in Table 3 and Figure 6.).

Algae fluctuations in all treatments followed a similar trend, and the density continuously increased and began to decrease according to the algae growth cycle.

On the first day, the density of algae in all treatments was relatively the same and was not statistically significant difference between treatments. This shows similarity in the input density of the treatments (Table 3).

Figure 1. Density growth of Chlorella vulgaris in different treatments

Chlorella algae density reached the highest value on day 5 of all treatments, at this time treatment 1 reached the highest value (6,689,629 individual/mL) because this treatment was used 100% mineral water waste from RO filter column, mineral content (microelements) is relatively higher, especially elements such as K, which are essential for cell division of Chlorella algae. Treatment 5 had the lowest value (2,427,407 individual/mL) which was the treatment without waste mineral water, the medium of this treatment was 100% tap water, and however, the density of algae in this treatment was difference compared to 75 and 100. The density of algae in the treatments between the days was statistically significant (P<0.05) (Table 3).

In the first five days, the density of algae in all treatments increased quite rapidly, then after the 7th day, all of them decreased simultaneously. This is also consistent with the time progression of growth and development of algae (FAO 1996) including 5 stages as lag phase, exponential phase, declining relative growth phase, stationary phase and death/lysis phase.

It means that If the Chlorella grow to a peak density without harvesting, the density was decrease due to the lack of suitable environment, space and conditions for the chlorella to grow and chlorella die and easy to be contamination of others algae. Therefore, in production practice, farmers should harvest Chlorella when the density of Chlorella is at the peak for feeding to zooplankton and larvae fish. Chlorella need to be renewed by new batches because the extended time of culturing Chlorella is susceptible to contamination, which reduces the density.

By day 7th and later, the algae density in the all treatments decreased quite quickly and there was a statistically significant difference in all treatments. Especially, on the 11th day, the density of algae in the treatment 100% and 75% mineral wastewater did not have statistical significance different (Table 3). There by, it is possible to use the 100% of mineral wastewater to raise algae, which give the highest density.

Biomass of algae in the experiment

Biomass and density of algae are related to each other, biomass is determined based on the value of chlorophyll_a in algae. Therefore, under the same conditions, an algae species increase in density is also a certain increase in biomass and vice versa.

Besides, chlorophyll_a is also the main pigment involved in the photosynthesis of algae. The chlorophyll_a content in algae usually accounts for about 1-2% of the dry weight of the algae (Dang Thi Sy 2005).

Variation of algal biomass between treatments during the experimental is shown in Table 4 and Figure 2.

Figure 2. Biomass of Chlorella vulgaris

Conclusion

In general, from the first day to the end of the experiment, the algal biomass of each treatment had a difference over time and this difference was statistically significant. Through the results of the density and biomass of Chlorella vulgaris, it is possible to use 100% of the mineral wastewater to grow algae to limit the amount of mineral water discharged into the environment, save water and provide natural food for larvae, fry in aquaculture. In addition, in production practice, farmers should harvest Chlorella when the density of Chlorella is at the peak and new batches of Chlorella need to be cultured Chlorella to avoid contamination and reduces density.

Acknowledgement

This research is funded by Vietnam National University HoChiMinh City (VNU-HCM) under grant number “C2021-16-07”.

References

APHA (American Public Health Association) 1995 Standard Methods for the Examination of Water and Waste Water. 18th Edn., Washington, DC, USA., pp: 69.

Chen F 1996 High cell density culture of microalgae in heterotrophic growth. Trends in biotechnology 14: 412-426.

Countteau P 1996 « Micro-algae », Manual on the production and use of live food for aquaculture, Patrick Laven and Patrick Sorgeloos (Eds). Published by Food and Agriculture Organization of the United Nations.

Dang Thi Sy 2005 Algal studies. Hanoi National University Publishing House.

Ermavitalini D, Rukhmana S Y, Meidina T, Baskoro L P D C, Saputro T B and Purnobasuki H 2021 Astaxanthin-Producing Microalgae Identification Using 18S rRNA: Isolates from Bangkalan Mangrove Waters and Sowan Tuban Northern Waters, East Java, Indonesia. Journal of Tropical Biodiversity and Biotechnology, 6(3), 64882

FAO 1996 Manual on the Production and Use of Live Food for Aquaculture. Food and Agriculture Organization of the United Nations, Viale delle Terme di Caracalla, 00100 Rome, Italy. https://www.fao.org/3/w3732e/w3732e00.htm#Contents

Lan T T, Nhi N H Y and Khoa L T M 2012 Utilization of biodigester effluent for production of Chlorella. Livestock Research for Rural Development, 24(8).2012.

Lee Y K 1997 Commercial production of microalgae in the Asia-Pacific rim. Journal of Applied Phycology 9: 403-411.

Liao I C, H M Su and I H Lin 1983 Larval foods for penaeus prawns, in: SRC. Handbook of marineculture, VI: Crustacean Aquaculture, Jame, P. (Eds): 43-69.

Nguyen Minh Tuan, Le Thi Bich Yen, Nguyen Phuoc Hai, Nguyen Thi Thanh Xuan, Dang Kim Hoang, Nguyen Hoang Minh, Nguyen Ngoc Tuan 2012 Studying the effects of farming conditions on growth and yield of oil collection of Chlorella vulgaris algae to make biodiesel production materials. Collection of reports of the 8th Scientific Research Student Conference, Da Nang University in 2012.

Nguyen Thanh Phuong 2003 Principles and techniques of giant freshwater shrimp production. Agriculture Publishing House, Ho Chi Minh City.

Nguyen Tran Thien Khanh, Vo Thi Dao Chi and Nguyen Thi Phuong Dung 2017 Studying the ability to treat nitrogen and phosphorus in domestic wastewater with Chlorella sp. Science journal of An Giang University. Volume 15 (3) 49-60.

Oh-Hama T and S Myjachi 1986 “Chlorella” Micro-algal Biotechnology. Michael A.Borowitzka and Lesley J. Borow itzka (Eds), Cambridge University press, pp.3-26.

Payer H D, Y Chiemvichak, K Hosakul, C Kong-Panichkul, L Kraidej, M Nguirtagul, S Reungmanipytoon and P Buri 1980 Temperature as an important climictic factor duringmass production of microscopic algae, Algae biomass. G. Shelef and C.J. Soeder (Eds). Elseveier/North-Holland Biomedical press, New York, pp: 389-399.

Pratt R 1948 Studies on Chlorella vulgaris: XI. Relation between surface tension and accumulation of Chlorellin. Am. J. Bot. 35: 634-637.

Sunda W G 2006 Trace Metals and Harmful Algal Blooms. In: Granéli E, Turner J, editors. Ecology of Harmful Algae. Springer; Berlin/Heidelberg, Germany: 2006. pp. 203–214.

Tran Chan Bac, Le Thi Quyen Em, Pham Hong Nga, Nguyen Xuan Loc and Nguyen Minh Chon 2015 Using wastewater from catfish ponds to raise biomass of Chlorella sp. Scientific Journal of Can Tho University. 39 (2015): 90-96.

Tran Thi Thuy 2008 Effects of temperature, pH, and nutrition on the growth of Chlorella. Bachelor thesis, Can Tho University.

Truong Sy Ky 2004 Techniques for raising some species as food for aquatic larvae. Ho Chi Minh City Agricultural Publishing House.

Xiong W, Li X, Xiang J and Wu Q 2008 High density fermentation of microalgae Chlorella protothecoides in bioreactor for microbio-diesel production. Applied Microbiology and Biotechnology 78: 29-36.

Xu H, Miao X X and Wu Q 2006 High quality biodiesel production from a microalga Chlorella protothecoides by heterotrophic growth in fermenters. Journal of Biotechnology 126: 499-507.