Utilization of biodigester effluent for production of chlorella

Trinh Thi Lan and Le Tran Minh Khoa

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

Abstract 

The research was about utilization of biodigester effluent to raise biomass of chlorella and was conducted in the experimental farm of AnGiang University. This study aimed to find the amount of  biodigester effluent which is suitable for culturing Chlorella. The design was  completely randomized (CRD) with 5 treatments and 3 replicates. Individual treatments were: 100% diluted biodigester effluent, 75% diluted biodigester effluent + 25% tap water, 50% diluted biodigester effluent + 50% tap water, 25% diluted biodigester effluent + 75% tap water, and control treatment (tap water with Wanle additive).   Nitrogen level of the biodigester effluent was 22 mg/liter (this concentration resulted from diluting 40 times the pure biodigester effluent). The experiment was carried out in colloidal glass containers of 15 liters which were put in a building with tonle roof and were followed during 8 days. The density of algae as well as environmental factors (pH, temperature) were monitored daily, while other factors such as TAN, NO₃^- were measured every 2 days.

The treatment using 25% of diluted biodigester effluent had the highest density of algae and stable growth, comparable with the control treatment which used a standard growth medium for chlorella (Wanle additive). 

Key words: Algae, growth, nitrogen, Wanle

Introduction 

Aquaculture is a key economic sector in Vietnam, bringing in more foreign currency for the country with the strength of frozen products such as black tiger shrimp, tra and basa fish. The growth of aquaculture production has led to growth of aquatic breeds. The production of aquatic products with high economic value such as black tiger shrimp, giant fresh water prawn, sea bass, grouper ... requires natural food size corresponding to the size of the larval mouth, such as microalgae, rotifers, Moina, Artemia ... in which, we have to say Chlorella because this kind of natural food has more nutritious containing 65-68% protein, 17% sugar (glucan), 6% fat (fatty acids), and some 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 for biogas systems.

Biodigester systems not only reduce pollution sources, but also create additional sources of gas which partially reduces the cost of living for farmers. Especially, biodigester effluent can be used in polyculture to promote production of natural food. Wastewater from biodigesters may contain up to 3.59% dry matter and 967 mg/liter of N (Bui Phan Thu Hang 2003) which is favorable for natural food development. For these reasons we conducted the project "Utilization of biodigester effluent at difference concentrations to raise biomass of chlorella" in order to utilize the nutrients in biodigester effluent 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  in colloidal glass containers (15 liters)  continuously aerated during the 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 and added to tap water according to the prescribed treatments which were:

• Control treatment (DC): Walne solution (Appendix 1)) ...

• BE100: 100% biodigester effluent (equivalent N content was 22 mg/litre)

• BE75: 75% biodigester effluent + 25% tap water (N content was 16.5 mg/litre)

• BE50: 50% biodigester effluent + 50% tap water (N content was 11mg/liter)

• BT25: 25% biodigester effluent + 75% tap water (N content was 5.5mg/liter)

The initial density of chlorella in the water in all treatments was 100,000 cells/ml. The experiment lasted for 7 days. During the experiment, each day one liter of the culture solution in the containers was removed by syphon 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, pH (2 times/day). TAN and NO₃^- were measured every 2 days. Chlorella density and specific growth rate were determined daily by the method of Coutteu (1996). Density of algae was counted in Sedgewick Rafter counting chamber according to the formula :           

M (cells/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  

Specific growth rate (SGR) of chlorella was calculated according to the formula:
SGR (%/day) = (lnN_t – lnN_o)*100/T
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 were processed statistically using SPSS software.

Results and discussion

The environmental factors

Water temperature during the experimental period fluctuated slightly.  The highest average temperature in the morning at 8 o'clock was 29.2⁰C and evening at 14 hours was 32.7 ⁰C. This was the right temperature for Chlorella development (Liao 1983). The average pH over the experimental day was 8. In particular, the highest was 8.5 and the lowest was 7.5.  The pH in the treatments was in the range suitable for the growth of Chlorella.

pH in the treatments using biodigester effluent were higher than in the control treatment using Walne solution. Typically, treatment BE25 (use 25% of biodigester effluent) on day 5 reached pH 8.50 while the control treatment pH was 8.33. Due to density of chlorella increased rapidly led to absorb more CO2 so make the pH rise. At the end of the experiment due to death of chlorella populations so algae density decreased as the pH decreased. In addition, the decay of dead algae and organic matter in biodigester effluent increased the amount of CO₂ that would lead also to pH decrease. 

 According to Oh-Hama (1986), Chlorella always prefer using ammonia-N in the water although there were other forms of nitrogen such as nitrate and urea that were used. The average amount of TAN in the treatments using biodigester effluent were more than in the control treatment using Walne solution. The concentration of TAN in BE100, 75 and 50 were always high because at the beginning the levels of biodigester effluent provided for these treatment were high, and the density of algae in these treatments was which led to nutrients from biodigester effluent not being absorbed. In treatment BE25, biodigester effluent offered was moderate at the beginning (25% of biodigester effluent) and density of algae in this treatment was high, so greater amount of TAN was absorbed. With the death of chlorella, the decomposition of dead algae led to higher level of TAN at the end of the experiment.  

The lowest concentration of NO3- was 0.02mg/liter on day 1 of the control treatment and the highest was 1.00 mg/liter in treatments BE100 and BE75. At the end of the experiment. NO3- concentration in treatment BE100 and BE75 were the highest since also high levels of TAN and algal density declining so nutrients were not absorbed. In the remaining treatments, at the beginning, thee were low levels of NO3- because algae used NO3- to increase algal biomass. When nearing the end of the experiment, the concentration of NO3- increased due to death of algae and algae density reduction which resulted in decreased absorption of nutrients and made levels of NO3- higher. 

 The growth of algae

On the first day after the experiment, the density of algae in the control treatment was highest 192.6 thousand cells/ml) and lowest in the BE treatments (102.83 thousand cells/ml). Highest algal density on day 5 in treatment 4 was 1079 thousand cells/ml, next to the control treatment was 1028.67 thousand cells/ml. On day 6, the density of algae in treatment 4 still highest, but in the remaining treatments had clearly reduced in the density of algae. At the end of the experiment, the density of al treatments were reduced but treatment 4 still had higher density than the other treatments. The difference between treatments in the experiment was statistically significant (p <0.05).

Figure 1. Fluctuation of chlorella density among treatments

The densities of algae in the treatments using biodigester effluent were higher than control treatment using Wanle solution, typically in NBE25 (using 25% biodigester effluent) had the highest density of algae. Figure 1 shows that in the early days of the experiment the density of algae in control treatment was always the highest. The density of algae in treatments using biodigester effluent increased slowly in the early days and increase faster on days 4, 5, 6 as by now algae were familiar with living environment of biodigester effluent. Algae grew fast in the first 5 days and then the rate  gradually decreased. Density of algae in treatments BE100 to 50 were not high and can be explained that biodigester effluent provided so much organic matter in these treatments that led to the water having a dark color (Photo 1) which  reduced photosynthesis ability of the algae.  By contrast the colour of the water in the BE25 treatment was green (Photo 2). Because the micro-algae are autotrophic species, they use solar energy, nutrients and trace minerals to synthesize organic matters for their body.  

In general, the BE25 treatment supported similar increases in growth of chlorella (Table 2) as in the control treatment, and in fact during the last few days had a higher density of chlorella than in the control.

Weed algae began to appear on days 3, 4 (Table 4) and were highest in treatments BE100, 75 and 50. On days 6 and 7  the proportions of weed algae were lowest in treatment BE25. 

The ratio between weed algae and chlorella was highest on the 6th day in most treatments. In particular, treatment BE100 on day 6 had the highest weed algae rate which was 26.0%. Most of the weed algae belong to green algae, such as scenedesmus, protococcus viridis and botrydiopsis

Conclusions

• The concentration of biodigester effluent suitable for the growth of Chlorella was 0.6%, equivalent to 5.5 mg N/liter.

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. 

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

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.

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