Utilization of biodigester effluent for production of Chlorella

The aim of the research was to study the utilization of biodigester effluent as fertilizer to raise biomass of Chlorella. The design was completely randomized (CRD) with 5 treatments and 3 replicates. Biodigester effluent (880 mg N/liter) was diluted 40 times to yield the diluted effluent (22 mg N/liter). The individual treatments were: diluted biodigester effluent (22 mg N/liter) replacing tap water at 100, 75, 50, 25 and 0%. The control treatment (0% effluent) was fertilized with Wanle additive solution. The experiment was carried out using colloidal glass containers of 15 liter capacity which were put in a building with a transparent roof and 24h light from a 40W bulb. The trial lasted for 7 days. The density of algae as well as environmental factors (pH, temperature) were monitored daily.

The treatment using 25% of diluted biodigester effluent (5.5 mg N/liter in the growth medium) supported the highest density of Chlorella, with stable growth, which was comparable with the control treatment which used a standard growth medium for Chlorella (Wanle additive).

Introduction

Aquaculture is a key economic sector in Vietnam, bringing in much foreign currency for the country with exports of frozen products such as black tiger shrimp and catfish. The growth of aquaculture production has led to the production of a wide range of species with high economic value such as black tiger shrimp, giant fresh water prawn, sea bass and grouper. These species require during the larval stage natural food of a size corresponding to the size of their mouths (eg: microalgae, rotifers, Moina and Artemia). Chlorella is one of the most important microalgae due to its high nutritive value (contains in DM: 65-68% protein, 17% sugar (glucan), 6% fat (fatty acids) and several vitamins (Pham Thanh Ho 2008).

Today, most areas allocated to development must be coupled with environmental protection. So the study of waste treatment and reuse of waste for other purpose is of great interest. Our response to the requirements of environmental protection led to research about using excreta from livestock after processing in a biodigester system.

Biodigester systems not only reduce pollution, but also create additional resources as biogas which can be used for cooking, which partially reduces the cost of living for farmers. Especially, biodigester effluent can be used in aquatic systems to promote production of natural food. Waste water from biodigesters may contain up to 3.59% dry matter and 967 mg/liter of N (Bui Phan Thu Hang 2003). For these reasons we conducted the project on utilization of biodigester effluent at different concentrations to feed Chlorella algae which is a natural food for aquaculture.

Materials and methods

The study was conducted at the aquaculture experimental farm of An Giang University. The study was arranged in a completely randomized design (CRD) with 5 treatments and 3 replicates, including four combinations of tap water and biodigester effluent and a control treatment. The tap water was aerated to remove the chlorine. The experimental system was a series of colloidal glass containers (15 liters) continuously aerated during the experiment and situated in a closed building with transparent roof. Light from a 40W bulb was provided during the whole experiment. The volume of water at the beginning was 8 liters/container. The nitrogen level of the pure biodigester effluent was 876 mg/liter. This was diluted 40 times to reduce the N content to 22 mg/liter, and added to tap water according to the prescribed treatments which were:

The initial density of Chlorella in the water in all treatments was 100,000 cells/ml. The experiment lasted for 7 days. Each day one liter of the culture solution in the containers was removed by siphon and replaced with one liter of experimental solution for treatments BE100 to BE25. In the control treatment, Walne solution was added daily at one ml for each liter of culture solution.

Measurements were made of temperature and pH (2 times/day). Chlorella density and specific growth rate were determined daily by the method of Coutteu (1996). Density of algae was counted in a Sedgewick Rafter counting chamber according to the formula :

Specific growth rate (SGR) of Chlorella was calculated according to the formula:

In which:
N_t: Final density of algae (cells)
N_o: Initial density of algae (cells)
T: Number of experimental days (day)

Statistics

The data for amount of algae density, specific growth rate of Chlorella were analyzed using the General Linear Model (GLM) of the ANOVA program with the Tukey pair-wise comparison in Minitab software (Minitab 2010). Sources of variation were: Level of biodigester effluent and error.

Results and discussion

The growth of Chlorella

In all treatments the density of Chlorella increased linearly over the first 4 days reaching maximum values at day 5 and then declining (Table 1; Figure 1). From day 1 to day 4, the highest values were in the control treatment, followed by BE25. On day 5 the control and BE25 treatments had the highest values which did not differ from each other while on days 6 and 7 the BE25 treatment was higher than all the others. Over the whole 7 day period the Chorella densities were inversely related to the concentration of biodigester effluent in the growth medium with best results being obtained with the BE25 treatment containing 5.5 mg N/liter.

Table 1. Mean values for density of Chlorella (thousand cells/ml) during the experiment
	Treatments
Day	Control	BE100	BE75	BE50	BE25	SEM	P - value
0	100.00	100.00	100.00	100.00	100.00
1	192.6^c	102.8^a	111.3^ab	123.3^ab	144.8^b	7.45	<0.0001
2	420.3^c	238.0^a	312.3^b	269.5^ab	253.7^ab	13.21	<0.0001
3	739.7^c	309.7^a	343.0^a	444.2^b	489.0^b	12.79	<0.0001
4	1,011^d	494.7^a	599.0^b	500.3^a	667.3^c	5.15	<0.0001
5	1,029^c	325.8^a	411.0^a	776.3^b	1,079^c	19.52	<0.0001
6	551.3^b	266.3^a	288.5^a	375.3^a	1,107^c	25.11	<0.0001
7	350.5^c	158.4^a	171.5^a	206.5^b	729.3^d	5.48	<0.0001
^a,b,c,dMeans with different superscripts within species and within rows are different at P<0.05

Weed algae began to appear on days 3 and 4 (Table 2) with lowest values on treatments BE25 and the control (Table 2; Figure 2). Most of the weed algae belonged to green algae, such as Scenedesmus, Protococcus viridis and Botrydiopsis.

Table 2. Mean values for density of weed algae (cells/ml) on days 3 to 7 of the experiment
	Treatments					SEM	P - value
Day	Control	BE100	BE75	BE50	BE25	SEM	P - value
3	7711^a	9117^b	11489^c	11027^c	8617^ab	274.4	<0.0001
4	17000^a	21667^b	25917^c	28583^c	18250^ab	885.1	<0.0001
5	54500^a	64250^c	75250^d	60500^bc	57500^ab	1054.8	<0.0001
6	85333^a	93417^b	96250^b	94500^b	88917^a	983.2	<0.0001
7	36833^a	43583^b	48917^b	44167^b	34333^a	1251.1	<0.0001
^a,b,c,dMeans with different superscripts within species and within rows are different at P<0.05

Density of Chlorella in treatments BE100 to 50 were low probably because the high concentration of organic matter in these treatments led to the water having a dark color (Photo 1) which reduced photosynthesis activities of the Chlorella. By contrast the colour of the water in the BE25 treatment was green (Photo 2). In general, the BE25 treatment supported similar increases in growth of Chlorella (Table 2) as in the control treatment, and during the last few days had a higher density of Chlorella than in the control.

The specific growth rate of the Chlorella was highest on the control treatment during the first 3 days after which it declined. The treatments with biodigester effluent showed a similar pattern but with lower values. Treatment BE25 had the most stable growth rate that was relatively higher than the others (Table 3).

Table 3. Specific growth rate of Chlorella in the experiment (%/day)

Day

Treatments

SEM

P - value

Control

BE100

BE75

BE50

BE25

1

64.90 ^c

2.78^a

10.49^a

20.89^ab

36.99^b

4.24

<0.0001

2

71.60^c

43.31^a

56.91^b

49.56^ab

46.52^a

1.75

<0.0001

3

66.70^c

37.67^a

41.08^a

49.61^b

52.88^b

0.932

<0.0001

4

57.84^d

39.97^a

44.75^b

40.25^a

47.45^c

0.197

<0.0001

5

46.61^d

23.62^a

28.27^b

40.98^c

47.55^d

0.387

<0.0001

6

28.40^c

16.31^a

17.66^a

22.04^b

40.04^d

0.549

<0.0001

7

17.92^d

6.57^a

7.70^b

10.36 ^c

28.38^e

0.197

<0.0001

^{a, b, c, d, e} Means with different superscripts within rows are different at P<0.05

The environmental factors

The values for temperature and pH in the culture media (Table 4) were within the normal range recommended for Chlorella (Liao 1983).

Table 4. Average values of environmental factors in the treatments
	Control	BE100	BE75	BE50	BE25
Temperarure in morning, ⁰C	28.6	29.1	29.1	29.6	29.5
Temperature in afternoon, ⁰C	32.5	33	32.67	32.67	32.5
pH	7.5	7.67	8.33	8	8.5

Conclusions

References

Coutteau P 1996 Micro-algae. In: Manual on the production and use of live food for aquaculture. Patrick Lavens and Patrick Sorgeloos (Eds). Published by Food and Agriculture Organization of the United Nations: 9-59.

Bui Phan Thu Hang 2003 Effect of dimensions of plastic biodigester (width:length ratio) on gas production and composition of effluent [on-line], mekarn, vailable from: http://mekarn.org/msc2003-05/miniprojects/webpage/hangctu.htm Accessed: 20.07.2011).

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

Minitab 2010 Minitab User's guide. Data analysis and quality tools. Release 16.1 for Windows. Minitab Inc., Pennsylvania, USA.

Pham Thanh Ho 2008 Introduction to Biotechnology, Education Publishing House. Chlorella algae. Bachelor thesis of Can Tho University.

Appendix 1.The composition of the solution Walne
	Amount
Solution A (1 – 2 ml for 1 liter of culturing algae).
FeCl₃.6H₂O	1.30g
MnCl₂.4H₂O	0.36g
H₃BO₃	33.60g
EDTA	45.00g
NaH₂PO₄.2H₂O	20.00g
NaNO₃	100.00g
Solution B	1.0ml
Distilled water to.	1000ml
Solution B
ZnCl₂	2.1g
CoCl₂.6H₂O	2.0g
(NH₄)₆.Mo₇O₂₄.4H₂O	0.9g
CuSO₄.5H₂O	2.0g
HCL condense	10.0ml
Distilled water to	1000ml
Solution C (0.1ml for 1 liter of culturing algae)
Vitamin B12	10mg
Vitamin B1	200mg
Distilled water to	1000ml
Solution D (2ml for 1 liter of culturing algae)
Na₂SiO₃.5H₂O	40.0g
Distilled water to	1000ml

Utilization of biodigester effluent for production of Chlorella

Trinh Thi Lan, Nguyen Huu Yen Nhi and Le Tran Minh Khoa

An Giang University, Vietnam
ttlan@agu.edu.vn

Abstract

Introduction

Materials and methods

Statistics

Results and discussion

The growth of Chlorella

The environmental factors

Conclusions

References


Figure 1. Fluctuation of Chlorella density among treatments during the experiment	Figure 2. Fluctuation of weed algae density among treatments during the experiment


Photo 1. Water color in treatments BE100, BE75 and BE50	Photo 2. Water color in treatment BE25

Utilization of biodigester effluent for production of Chlorella

Trinh Thi Lan, Nguyen Huu Yen Nhi and Le Tran Minh Khoa

An Giang University, Vietnam ttlan@agu.edu.vn

Abstract

Introduction

Materials and methods

Statistics

Results and discussion

The growth of Chlorella

The environmental factors

Conclusions

References

An Giang University, Vietnam
ttlan@agu.edu.vn