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Effect of harvesting rate and harvesting frequency on biomass yield of microalgae Chlorella vulgaris cultured with waste mineral water

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

The study to evaluate the effect of harvesting rate and harvesting frequency on biomass yield of microalgae Chlorella vulgaris was carried out at a bottled mineral water factory in An Hao commune, Tinh Bien district, An Giang province with the aim was to find out the frequency and harvesting rate of Chlorella vulgaris during the rearing process for the best biomass. The experiment was arranged in a completely randomized design with 3 replicates and 5 treatments including treatment 1: harvest 100% of the algae volume when the algae density was maximum; treatment 2: harvest 30% of the volume and collect once a day; treatment 3: harvest 60% and collect once a day; treatment 4: harvest 30% volume and collect 2 days/time; treatment 5: harvest 60% and collect 2 days /time. The environmental factors during the culture process such as temperature, pH, NH3, PO43- and NO2 were all in the appropriate range for the normal growth and development of Chlorella vulgaris. The experimental results showed that the highest algal biomass was 597.5 g/day in treatment 5 (harvested 60% volume, 2 days/time) and has a statistically significant different compared with the remaining treatments. The lowest algae biomass was 304.1 g/day in the treatment 3 (harvested 60% volume, once a day). Thus, the most appropriate harvesting rate and frequency for the growth and development of Chlorella vulgaris is harvest at a rate of 60% by volume and harvest every 2 days.

Keywords: algae biomass, Chlorella vulgaris, harvest rate, harvest frequency


Introduction

In aquaculture, Chlorella is an ideal food for rotifers, because algae has the ability to increase biomass of rotifers quickly in rearing conditions as well as ensure adequate nutrition in rotifers for larvae of various fish, crab species. According to Aranda et al (1994), when studying the catching ability and digestive rate of Strombus gigas larvae (molluscs) for 8 species of algae, the larvae's ability to catch prey was detected with Chlorella was higher than that of other algae such as Isochrysis aff galbana, Dunaliella tertiolecta, Chlamydomonas coccoides, Tetraselmis fluviatilis and Tetraselmis suecica . Digestibility begins one hour after ingestion with the digestibility ofChlorella being faster than that of D. tertiolecta and T. fluviatilis. In addition, Chlorella is also used in green water systems when rearing giant freshwater prawn larvae, sea crab larvae, and marine fish larvae such as milkfish (Chanos chanos). The addition of algae to the nursery tank of giant freshwater shrimp is provide some water-soluble trace elements that are essential nutrients that are not found in the feed. On the other hand, the ability to cause diseases from bacteria is limited thanks to the growth of algae in the nursery tank. Chlorella with high nutritional value can be used as an alternative protein source for conventional protein sources in animal feed. In addition, Chlorella algae also have a role in wastewater treatment, algae was remove nitrogen and phosphorus from the water environment. Several experiments have been carried out to test the conversion of nitrogen (TN) and phosphorus (TP) out of wastewater by Chlorella algae such as the experiment of Gozalez (1997) discovered that Chlorella vulgaris and Scenedesmus dimorphus absorbed 95% NH4+ and 50% TP in wastewater. In addition, Lan et al (2022) used 100% mineral wastewater from RO filter column to grow Chlorella vulgaris algae with very positive results. Thereby showing that there is a prospect in reusing the water source and making use of this waste mineral water to create biomass for the other objects.


Materials and methods

The experiment was arranged at SM bottled mineral water factory in An Hao commune, Tinh Bien district, An Giang province.

Prepare research materials

The source of algae farming water is waste mineral water taken directly from the waste RO filter column at SM company (An Hao commune, Tinh Bien, An Giang). Waste mineral water before using in the experiment was treated with Chlorine at a concentration of 50ppm to remove harmful bacteria for algae growth, in order to create quality algal biomass.

Chlorella vulgaris samples were collected and isolated from the catfish ponds. The medium for isolation used is the Walne environment.

Experimental layout

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

NT1: Harvest 100% of the algae volume when the algae density was maximum (Trinh Thi Lan et al, 2022)

NT2: Harvest 30% of the volume and collect once a day.

NT3: Harvest 60% of the volume and collect once a day.

NT4: Harvest 30% volume and collect 2 days/time.

NT5: Harvest 60% volume and collect 2 days/time.

Algae were grown in plastic-wrapped cylinders, the volume of each cylinder algal culture was 300 liters. The volume of seed algae inoculated into each cylinder was 10 liters and the volume of Walne medium (nutrient medium) was 2 ml for each liter of algae culture water.

All algal culture cylinders were aerated throughout the experiment, the cylinders were located outdoors to get direct sunlight and were covered to avoid the infection of other algae.

The experiment lasted 30 days

In treatment 1, the algae were harvested when the density was maximum (Trinh Thi Lan et al, 2022). All the algae in the plastic-wrapped cylinders was be collected after kept amount of enough algae seed for the next batch. The plastic-wrapped cylinders was be clean and renewed the new batches with the algae seed were taken and kept from the old batches.

Photo 1. Prepare to disinfect water Photo 2. Algae arrangement in treatments




Photo 3. Algae after placement and harvesting 15 days Photo 4. Algae was concentrated after harvest
Measurements

Algae biomass was determined by pumping the amount of algae to be harvested into the container, and the pH was adjusted to 11.5 with 0.1N NaOH. Wait for the algae to settle to the bottom. Remove the water from the top. Centrifuge the algae deposited at the bottom at 3000 rpm/15 min. Then, add a little distilled water to wash the algae and continue to centrifuge to remove the remaining water. Weigh the algae biomass obtained after centrifugation.

Water quality including NO2-, NH4 +, PO43- were collected and analyzed according to APHA (1995).

Dry matter was determined by drying in an oven at 105 oC until constant weight. Ash (mineral) content was determined by incineration of the sample at 550 oC for 4 h. Crude protein was calculated as 6.25 % N analysed by the Kjeldahl method, ether extract (EE) was measured using the Soxhlet method and crude fibre (CF) content was analysed using standard methods (AOAC, 2000).

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

Environmental parameters during the experiment

Table 1. Water quality parameters of treatments during the experiment

Parameters

NT 1

NT 2

NT 3

NT 4

NT 5

p value

PO43- (mg/L)

0.37

0.50

0.43

0.55

0.38

0.64

NH4+ (mg/L)

0.27

0.16

0.26

0.33

0.15

0.49

NO2 - (mg/L)

0.33

0.13

0.33

0.22

0.36

0.37

pH

8.93

8.88

8.8

8.78

8.69

0.44

DO (mg/L)

5.4

6.9

6.8

5.9

6.2

0.39

Light intensity (lux)

15000

15000

15000

15000

15000

0.49

Temperature (oC)

28.5

28.5

27.9

28.0

28.5

0.45

Table 1 showed that the water quality parameters were always maintained at a stable level for the growth of Chlorella vulgaris and this difference was not statistically significant between treatments (p>0.05).

Harvested algae biomass

After one week of harvesting, the algae had a noticeable difference in color. Treatments 2 and 3 were lighter in color than the other treatments because of continuous harvesting, so the growth of algae in these 2 treatments was slower. In treatment 4, there was a phenomenon of algae settling to the bottom due to the superior growth in biomass, leading to the phenomenon of algal decay. The growth of algae in treatment 5 was more stable and had a higher biomass than that of the other treatments. Treatment 1, 100% harvested when the biomass reached the maximum, after collection, it was necessary to change the container, treat the water with chlorine and replant the algae as before, so the collection time and the number of harvests in the treatment 1 were not a lot of.

Table 2. Algae biomass obtained in different treatments

Treatments

Algae biomass (g/day)

1

364.72bc

2

358.60bc

3

304.05c

4

408.28b

5

597.46a

p value

<0.001

abcd Means with different superscript letters within column are significantly different (P<0.05)

Table 2 showed that the difference in weight of algae harvested between different treatments was statistically significant. In the treatments, harvesting every 2 days was higher in biomass than that of the other treatments. The highest in treatment 5 (60% harvest, 2 days/time) and lowest in treatment 3 (60% harvest, 1 day/time). Compared with the results of Truong Vinh (2018) in the experiment of cultivating Chlorella sp., the highest harvest volume reached 684 g/day, during the collection period every 2 days with the collection rate of 50% of the total volume of culture and cultured in Bold Basal medium compared to the experimental results were similar.

Algae quality

After weighing, concentrating and analyzing the nutritional content of the algae, the chemical composition of algae as below (Table 3)

Table 3. Chemical composition of algae after harvest (%)

Chemical composition

NT1

NT2

NT3

NT4

NT5

p value

Dry matter

9.23

9.10

9.28

9.65

9.10

0.59

% in dry matter

Protein

50.34

48.64

52.7

48.40

48.11

0.88

Lipid

16.07

16.3

17.12

16.41

15.96

0.65

Minerals

6.69

5.67

5.75

5.73

5.47

0.10

Means without different superscript letters within rows are no significantly different (P>0.05)

In general, algae harvesting time every 2 days with 60% volume had the best results and better biomass growth compared with the continuous harvesting and 100% collection treatments.


Acknowledgement

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


References

AOAC 2000 Official methods of analysis of AOAC International (17th ed.) . Retrieved from AOAC International, Gaithersburg, MD, USA.

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

Aranda D A, Suares V P and Brule T 1994 Ingestion and digestion of eight algae by Strombus gigas larvae (Mollusca, Gastropoda) studied by epifluorescence imcroscopy. Aquaculture, Vol.126, issues 1-2.

Gonzales L E, Canizares R O, Beana S 1997 Efficiency of ammonia and phosphorus removed from a comlombian argroindustrial waste water by microalgae Chlorella vulgaris and Scenedesmus dimorphus. Bioresource Technology. Volumne 60, Issue 3, Pages 259-262

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.

Minitab 2010 Minitab 16 Statistical Software 16.2.0.

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 2022 Using waste water mineral from RO column to culture Chlorella vulgaris. Livestock Research for Rural Development. Volume 34, Article #71. Retrieved January 13, 2023, from http://www.lrrd.org/lrrd34/8/3471lan.html

Truong V, Truong V T, Ho T T C, Nguyen D Q, Nguyen T T T 2018 Harvesting of Chlorella vulgaris grown in closed-photobioreactor with chitosan for use in food. The Journal of Agriculture and Development 17(4), 102-111. www.jad.hcmuaf.edu.vn