Effect on composition of duckweed (Lemna minor) of different levels of biodigester effluent in the growth medium and of transferring nutrient-rich duckweed to nutrient-free water

Dang Thi My Tu, Nguyen Thi Kim Dong* and T R Preston**

Department of Agriculture Science, Mekong University, Vietnam
tudangmk@yahoo.com
* Faculty of Animal Science, College of Agriculture and Applied Biology, Can Tho University
** TOSOLY, AA#48, Socorro, Santander, Colombia

Abstract

Two experiments were conducted on a private farm in Binh Thuy District, Cantho City to study: (i) the yield and composition of duckweed cultivated with different levels of biodigester effluent; and (ii) the effect on duckweed composition of a “shock” treatment of transferring high quality duckweed to plain water containing no nutrients. In experiment 1, the treatments were 6 levels (0, 4, 8, 12, 16 and 20%) of biodigester effluent added to fresh water in plastic containers containing duckweed. The surface of water in each container was 0.4 m² with 20 cm depth giving a volume of 60 liters. Duckweed was inoculated at a rate of 400 g/m². The yield of duckweed was measured over a period of 14 days by removing and weighing one third of the biomass every 48 h.

There was a curvilinear response in yield, and in crude protein content of the duckweed, to level of effluent with maximum values for both at a ratio of 12% effluent and 88% water (72 mg N/liter).  Duckweed from this treatment was then transferred to fresh water and the composition studied over 5 days. The content of starch increased and that of crude protein decreased with increasing time in the fresh water.

It was concluded that yield and crude protein content of duckweed was optimized when the culture medium contained 12% biodigester effluent and 88% water.  The “shock” treatment of transferring the best quality duckweed to nutrient-free fresh water led to decreases in crude protein and corresponding increases in starch. However, the effects were relatively small and unlikely to have significance from the points of view of duckweed as a feed for ducks or as a substrate for ethanol production.

Key words: ash, NDF, fertilizer, protein, shock treatment, starch

Introduction

Duckweed (Lemna minor) is a simple tiny water plant that grows very well on pond surfaces. It can tolerate high nutrient stress and appears to be more resistant to pests and diseases than other aquatic plants. Moreover, it has high protein and carotene contents (Bui Xuan Men et al 1995). The protein content of duckweed responds quickly to the availability of nutrients in a water environment (Leng et al 1995). Duckweed has been used as a main protein supplement for pigs (Bui Hong Van et al 1997) and ducks (Bui Xuan Men et al 1995; Nguyen Duc Anh et al 1997b). Duckweed has received research attention because of its high nutritive value, especially the high protein content and also because of its capacity to grow rapidly on nutrient-rich waste water and produce biomass rich in protein (Leng et al 1995).

The use of tubular plastic biodigesters for anaerobic digestion to convert organic matter to biogas and effluent (Botero and Preston 1995) is a very simple and practical system that is flexible and uses low-cost materials (Preston and Rodríguez 2002; Mette 1998; Bui Xuan An et al 1997) when compared to other types of biodigester (Mikkle et al 1996; Timothy and Gohl 1996). The effluent has been shown to be a good fertilizer for duckweed (Rodríguez and Preston 1996; Le Ha Chau 1998; Lampheuy 2003). Biomass yield increased with level of organic fertilizer and was higher for the effluent from a biodigester than for the raw manure (Lampheuy 2003).  This agrees with the findings of Le Ha Chau (1998) who also compared biodigester effluent with manure but at a fixed N level of 150 kg/ha. There was no interaction level*fertilizer for biomass production.

The results from these studies showed that the growth of duckweeds is similar to that of any other plant. Under experimental conditions the annual production reached 183 tonnes/ha of DM, however, under practical conditions a yield of up to 30 tonnes of DM/ha is more feasible (Leng et al 1995). Moderate conditions of temperature and light and liquid medium with the necessary nutrients are essential for good growth. Also, duckweeds adapt well to a wide range of conditions and are easy to grow (Cross 2001). It is considered that the use of effluent from biodigester for growing duckweeds could be a way of increasing feed availability for animals and at the same time reducing problems of pollution to the environment.

It appears there are possibilities to modify the composition of certain strains of duckweed (Cheng and Stomp 2009).  According to these authors, a starch content of 45% was achieved in Spiridela polyrrhiza through simple transfer of the fresh duckweed fronds from a nutrient-rich solution to tap water for 5 days.  The mechanism of the formation of starch was described by Armstrong (2003).  He identified the lateral dark bodies at the base of the mother plant of Lemna turionifera called turions and indicated that they formed when the plant was subjected to nutrient or environment stress (eg: low temperature).  Because the specific gravity of starch is about 1.5, the turions sink to the bottom of the pond or container in which the duckweed is growing. It is not known if other species of Lemna (eg: Lemna minor) respond in a similar manner when subjected to environmental stress.

Hypothesis

The hypotheses to be tested were:

• When duckweed is grown on high nutrient density substrate (eg: with biodigester effluent) and then is suddenly transferred to zero-nutrient water, the metabolism changes to convert some of the protein and other nutrients into starch.
  
  
• Duckweed exposed to environment “shock” (by transfer from nutrient-rich pond to plain water pond) will have a higher content of starch and thus have a higher nutritive value for growing ducks.

Materials and methods

Location and duration

The experiment was conducted on a farm in Binh Thuy District, Cantho City and in the laboratory of the Department of Agriculture and Applied Biology, Cantho University, Vietnam, from April to June 2011. 

Treatment and experimental design

The experiment was a completely randomized design with 6 treatments and three replications. The treatments were 6 levels of biodigester effluent (BE) added to duckweeds growing in plastic containers, used as experimental ponds.  The percentage of biodigester effluent was 0, 4, 8, 12, 16 and 20% (Table 1).  For different concentrations the quantities of biodigester effluent and water were adjusted accordingly.

The surface of water in each basket was 0.4 m² with 20 cm depth giving a volume of 60 liters. Duckweed fronds obtained from natural ponds around the University were inoculated at a rate of 400 g/ m² (160 g/ basket) in each treatment. The different proportions of biodigester effluent and water were added to plastic containers to produce different concentrations of N (Table 2). The biodigester effluent was stored in a container (160 liters) and a sample analyzed at the beginning of the experiment.

The “shock” treatment was carried out by selecting the best treatment (based on biomass yield and N content of the duckweed) to measure the duckweed response to the transfer from nutrient-rich to plain water.  After the transfer, samples of the duckweed were harvested daily for 6 days and analyzed for DM, N, starch, NDF and ash.

Water

Water used in the experiment was taken from only source water at the farm and applied for all plastic containers.

Biodigester effluent

The biodigester effluent was obtained from a plug-flow tubular plastic biodigester charged with cattle manure (Photo 2).

Measurements and chemical analyses

The duckweed was allowed to grow for a period of 14 days at which time all the biomass was harvested and weighed and samples taken for analysis of DM, CF, Ash, NDF, N and starch. The root length of the duckweed was measured with a graduated ruler (Rodriguez and Preston 1996). The contents of DM, CF, Ash, NDF, N and starch were determined by procedures of AOAC (1990).

Photo 3. Harvesting of duckweed (one third of the surface is harvested every 48h)	Photo 4. Measurement of the root length of duckweed

Statistical analysis

The data were analyzed by the General Linear Model procedure of the ANOVA program in the Minitab (2000) software. Sources of variation were: Biodigester effluent level and error. When the F test showed significant differences at P<0.05, Tukey’s test for treatment comparisons was used (Minitab 2000).

Results and discussion

Chemical composition of duckweed

The DM content of the duckweed did not vary with level of effluent that was applied (Table 3). The values (4.99 to 5.04%) were similar to those (4.99 to 5.85%) reported in the study of Lampheuy (2003). The content of ash tended to increase with level of effluent.

There was a close negative relationship between the NDF content of the duckweed and the level of biodigester effluent that was applied (Figure 2). However, the relationship between crude protein and biodigester effluent was curvilinear with the maximum protein content at the 12% level of effluent (Figure 1), equivalent to 108 kg N/ha. By contrast, Lampheuy (2003) reported linear increases in crude protein content of duckweed (from 16.7 to 34.5% in DM) with levels of biodigester effluent N up to 200 kg/ha.  The maximum level of crude protein reached in the present experiment (30.6% in DM with 108 kg N/ha) was similar to the level reported by Lampheuy (2003) for the 100 kg/ha level of N.

Figure 1. Relationship between effluent concentration and crude protein content of duckweed	Figure 2. Relationship between effluent concentration and NDF content of duckweed

Biomass yield and root length of duckweed

The relationship between biomass yield and effluent level showed a similar curvilinear relationship as was observed for the crude protein content (Table 4, Figure 3), with maximum yield being obtained with the 12% level, equivalent to 108 kg N/ha. In terms of annual yield of DM, this was equivalent to 19 tonnes/ha. This agrees with the findings of Leng et al (1998) who reported yields of duckweed from 10 to 20 tonnes of DM/ha/year.

The root length of the duckweed was negatively related with crude protein content (Table 5; Figure 4), although the relationship (R²=0.32) was not as strong as was reported by Rodriguez and Preston (1996a) (R²=0.86) and Lampheuy et al (2003) (R²=0.82). There were closer relationships between root length and biomass yield (Figure 5) and between the N content of the water in the ponds and root length (Figure 6). There are many experimental observations (Nguyen Duc Anh et al 1997; Le Ha Chau 1998; Rodriguez and Preston 1996) that have shown that over short growth periods there is a close negative relationship between root length and  the N content of the pond water.

Therefore the root length of duckweed is a good indicator of the crude protein of the duckweed, biomass yield and the N content of the pond water.

Figure 4. Relationship between root length and crude protein content of duckweed	Figure 5. Relationship between root length and biomass yield of duckweed

Figure 6. Relationship between N content of the pond water and the root length of the duckweed

Effect of the “shock” treatment on the composition of the duckweed

There were changes in the composition of the duckweed after transfer from nutrient-rich to plain water (Table 6). The crude protein and the ash content decreased and the starch content increased with increasing exposure to the nutrient-free water (Figures 7-9).

Figure 7. Relationship between time exposed to nutrient-free water and starch content of duckweed	Figure 8. Relationship between time exposed to nutrient-free water and crude protein content of duckweed

The above trends agree with the findings of Cheng and Stomp (2009) who reported that the starch content of duckweed increased after transfer for 5 days from nutrient-rich to plain water. However, the degree of change of starch concentration in our experiment (from 2.05 to 2.63%) was much less than that in the report of Cheng and Stomp (2009). According to these authors, a starch content of 45% was achieved in Spiridela polyrrhiza through a simple transfer of the fresh duckweed fronds from a nutrient-rich solution to tap water for 5 days. The reason for this difference is perhaps because of the different kind of duckweed used (Lemna minor in my experiment compared with Spiridela polyrrhiza in the report of Cheng and Stomp 2009) (Photos 5 and 6).

Photo 5. Lemna minor (Wikipedia No date)	Photo 6. Spiridela polyrrhiza (Wikipedia, No date

Conclusions

• Yield and crude protein content of duckweed was optimized when the culture medium contained 12% biodigester effluent and 88% water, equivalent to 72 mg N/liter
  
  
• The “shock” treatment of transferring the best quality duckweed to nutrient-free fresh water led to decreases in crude protein and corresponding increases in starch;  however, the effects were relatively small and unlikely to have significance from the points of view of duckweed as a feed for ducks or as a substrate for ethanol  production.

Acknowledgments

This paper forms part of the thesis submitted to Cantho University, Vietnam as part of the requirement of the Senior Author for the Master of Science degree “ Specialized in Response to climate changes and Depletion of Non-renewable resources”. We would like to sincerely thank the MEKARN Program for financial support.  We also want to express gratitude to all the people who helped to carry out this study. We thank the Department of Agriculture and Applied Biology, Cantho University, Vietnam, for providing facilities for the research..

References

AOAC 1990 Official Methods of Analysis.13th edn.Association of Official Analytical Chemists. Washington, DC

Armstrong 2003 No title http://waynesword.palomar.edu/1wayindx.htm#Disclaimer

Botero R and Preston T R 1995 Low-cost biodigester for production of fuel and fertilizer from manure (Spanish). Manuscrito ineditado CIPAV, Cali, Colombia, pp 1-20.

Bui Hong Van, Le Thi Men, Vo Van Son and Preston T R 1997 Duckweed (Lemnaspp) as protein supplement in an ensiled cassava root diet for fattening pigs. Livestock Research for Rural Development 9(1) http://www.lrrd.org/lrrd9/1/lemen912.htm

Bui Xuan An, Rodriguez L, Sarwatt S, Preston T R and Dolberg F 1997 Installation and performance of low-cost polyethylene tube biodigesters on small-scale farms.  World Animal Review 88: 38-47. FAO

Cheng and Stomp 2009 Growing High-Starch Duckweed on Wastewater for Ethanol Production http://insciences.org/article.php?article_id=4150

Cross J W 2001 Cham of duckweed  http://www.usra.edu/~jwcross/duckweed.html (1 Apr 2001).

Lampheuy K 2003 Manure or biodigester effluent as fertilizer for duckweed.  Retrieved, from MEKARN Mini-projects http://www.mekarn.org/MSc 2003-05/miniprojects/pheu.htm

Le Ha Chau 1998 Biodigester effluent versus manure, from pigs or cattle, as fertilizer for duckweed (Lemna spp.). Livestock Research for Rural Development (10) 3 http://www.cipav.org.co/lrrd/lrrd10/3/chau2.htm

Leng R A, Stamboli J H and Bell R 1995 Duckweed-A potential high protein feed resource for domestic animals and fish. Livestock Research for Rural Development 7(1) http://www.lrrd.org/lrrd7/1/3.htm

Men Bui Xuan, Ogle B and Preston T R 1995 Use of duckweed (lemnaspp) as replacement for soya bean meal in a basal diet of broken rice for fattening ducks. Livestock Research for Rural Development 7(3) http://www.lrrd.org/lrrd7/3/2.htm

Mikkle S L, Sandie S A and Torry- Smith M 1996 Building of a flexible bag biogas digester in Tanzania, Student Report of Center for developing countries Technical University of Denmark, 8809 Environmental field studies in developing countries.

Mette I L 1998 Evaluation of the impact on women's lives of the introduction of low cost polyethylene biodigesters on farms in villages around Ho Chi Minh City, Vietnam. Livestock research for rural development, vol. 3 No 3 http://www/lrrd10/3/met1031.htm

Mikkle S L, Sandie S A and Torry- Smith M 1996 Building of a flexible bag biogas digester in Tanzania, Student Report of Center for developing countries Technical University of Denmark, 8809 Environmental field studies in developing countries.

Minitab 2000 Minitab reference manual release 13.31. User’s guide to statistics. Minitab Inc. USA

Nguyen DucAnh and Preston T R 1997b Evaluation of protein quality in duckweed (Lemna spp.) using a duckling growth assay. Livestock Research for Rural Development 9(2) http://www.lrrd.org/lrrd9/2/anh92.htm

Rodriguez L and PrestonT R 1996 Use of effluent from low cost plastic biodigesters as fertilizer for duck weed ponds. Livestock Research for Rural Development. 8(2) http://www.lrrd.org/lrrd8/2/lylian2.htm

Preston T R and Rodríguez L 2002 Low-cost biodigester as the epicenter of a ecological farming systems. Proceedings biodigester workshop March 2002 http://www.mekarn.org/probiod/pres.htm

Timothy E S and Gohl B 1996 Tubular plastic bio-digester design, installation and management. Farm level Applied by Research Methods in East and Southern Africa.

Received 4 March 2012; Accepted 25 March 2012; Published 2 April 2012

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