Effect of method of processing of cassava leaves on protein solubility and methane production in an in vitro incubation using cassava root as source of energy

Sangkhom Inthapanya, T R Preston*, Duong Nguyen Khang** and R A Leng***

Faculty of Agriculture and Forest Resource, Souphanouvong University, Lao PDR
inthapanyasangkhom@yahoo.com
* Finca Ecologica, TOSOLY, AA#48, Socorro, Santander, Colombia
** Department of Animal Physiology and Biochemistry, Nong Lam University, Ho Chi Minh City, Vietnam
*** University of New England, Armidale NSW, Australia

Abstract

Two experiments were carried out to study effects of processing of cassava leaves on the solubility of the protein and on methane production when they were incubated with cassava root meal in an in vitro incubation with urea or potassium nitrate as source of NPN.

Experiment 1: The treatments in a 2*4 factorial arrangement in a randomized block design were:   leaves or petioles of cassava, and form of processing (fresh, ensiled, sun-dried and oven-dried). Protein solubility was decreased by ensiling and with the severity of drying.  

Experiment 2: The treatments in a 2*4 factorial arrangement in a randomized block design were: form of processing cassava leaves (fresh, ensiled, sun-dried and oven-dried) and source of NPN (urea or potassium nitrate).  An in vitro incubation system was used to determine the effects of the treatments on methane production and substrate fermentation. The quantity of substrate was 12g DM to which was added 240 ml rumen fluid (from slaughtered buffalo) and 960 ml of buffer solution. The incubation was for 12 and 24h with measurements of gas production, percent methane, substrate fermented and methane produced per unit substrate fermented. 

The gas production and methane percentage in the gas were increased with incubation time. The percentage of methane in the gas, and methane per unit substrate fermented were reduced when nitrate replaced urea as the NPN source and were lower for fresh and ensiled cassava leaves than for dried leaves at both 12 and 24h of incubation. Methane produced per unit of fermented DM was inversely related to protein solubility. 

Key words: Climate change, gas production, greenhouse gases, hydrogen cyanide

Introduction

Cassava is currently the third most important crop in Laos, after rice and maize. It is widely grown throughout the country by upland farmers but in small areas using local varieties and with very few inputs (CIAT 2001). The potential use of cassava foliage as an animal feed has been studied and described by several authors. Phuc et al (2001) investigated the use of cassava foliage as a feed for pigs and poultry. Ffoulkes and Preston (1978) reported the feeding of fresh cassava foliage as the only source of roughage and protein in diets for cattle based on molasses-urea. Van et al (2001) and Khang (2004) described the use of foliage of cassava as a protein source for small ruminants and cattle. The potential of cassava foliage as a protein source in ruminant feeds has not been fully exploited, probably because of the risk of toxicity resulting from the content of precursors of hydrogen cyanide (Wanapat 2001). However, it is known that the capacity to liberate HCN from cassava foliage is reduced by processing such as sun drying or ensiling (Khieu Borin et al 2005; Phengvichith and Ledin 2007).The role of cyanide as inhibitor of methanogenesis in sludge fermentation has been discussed by Gijzen et al (2000); Eikmanns and Thauer (1984); Smith et al (1985) and Cuzin and Labat (1992).

Cassava leaves are known to contain variable levels of condensed tannins; about 3% in DM according to Netpana et al (2001) and Bui Phan Thu Hang and Ledin (2005).  Condensed tannins at moderate levels are known to have positive effects on the nutritive value of the feed by forming insoluble complexes with dietary protein, resulting in "escape" of the protein from the rumen fermentation (Barry and McNabb 1999). Numerous studies have also shown the potential of the tannin content in cassava leaves to play an anthelminthic role for the control of nematode parasites in ruminants (Seng Sokerya and Preston 2003; Neptpana et al, 2001; Khoung and Khang 2005). Condensed tannins (CT) are also reported to decrease methane production and increase the efficiency of microbial protein synthesis (Makkar et al 1995; Grainger et al 2009). Reductions of CH₄ production by 13 to 16% were reported by Carulla et al (2005), Waghorn et al (2002), Grainger et al (2009) and Woodward et al (2004), apparently through a direct toxic effect on methanogens.

In a recent study, Ho Quang Do et al (2010, unpublished data) showed major effects of protein solubility on methane production in an in vitro incubation. Methane production was reduced by almost 50% when the protein source was fish meal (protein solubility 16.6%) compared with groundnut (protein solubility 76.2%) when sodium nitrate was the source of NPN and was 25% less when urea was the source of NPN.

The purpose of the present study was to determine the possible relationship between the solubility of the protein in cassava foliage and how this might affect methane production in an in vitro incubation, in which the NPN source was either potassium nitrate or urea.

Hypothesis

It was hypothesized that the solubility of the protein in cassava leaves would be reduced by drying and that this would result in a reduced production of methane in an in vitro incubation with cassava root meal as energy source and potassium nitrate or urea as source of NPN.

Materials and methods

Location and duration

The experiment was conducted in the laboratory of the Department of Animal Science, Faculty of Agriculture and Forest Resources, Souphanouvong University, Luang Prabang province, Lao PDR, from September to October 2011. 

Experiment 1: Protein solubility of leaves and stems of cassava foliage 

Treatments and experimental design 

The experimental design was arranged as a random block design with a 2*4 factorial arrangement of the following treatments:

Source of material:

·         Leaves

·         Petioles

Processing:

·         Fresh

·         Ensiled

·         Sun-dried

·         Oven-dried

Experimental procedure

Cassava foliage was collected from the Souphanouvong University campus. The samples were collected in the morning and immediately put into sealed plastic bags to avoid moisture loss.  

Leaves and petioles were separated and chopped into small pieces around 0.5-1.0cm of the length and then ground through a 1mm sieve. Part of the material was sun-dried for 24h. Another part was dried in the oven for 24h at 80°C. For the ensiling treatment, the material was put in sealed plastic bags, and the air removed prior to storage at room temperature for 14 days.

Protein solubility was measured by weighing 3 g of samples (DM basis), followed by liquidizing and shaking in 100 ml of M NaCl  for 3h. The suspension was then filtered through Whatman No. 4 filter paper and washed 3 times with distilled water. All the filtrate was then transferred to a kjeldahl flask for digestion, distillation and titration according to AOAC (1990). Protein solubility was calculated as the N content of the filtrate as a percentage of the N in the original sample.

Photo 1. The shaking	Photo 2. The filtering	Photo 3. The digestion	Photo 4. The distillation

Statistical analysis

Data were analyzed by the General Linear Model option in the ANOVA program of the Minitab software (Minitab 2000). Sources of variation in the model were: plant part, processing and interaction plant part*process and error.

Experiment 2: Effect of processing of cassava leaves on methane production in an in vitro incubation with cassava root meal as energy substrate and potassium nitrate or urea as source of NPN

Treatments and experimental design 

The experiment was arranged as a 2*4 factorial of the following treatments in a random block design:

Source of NPN:

·         Urea

·         Potassium nitrate

Processing of cassava leaves: 

·         Fresh

·         Ensiled

·         Sun-dried

·         Oven-dried

The basal substrate was cassava root meal.   

The in vitro system

The equipment and procedure was that used by Sangkhom Inthapanya et al (2011) (Photos 5 and 6). 

Photo 5. The in vitro system	Photo 6. Gas production after fermentation

Experimental procedure  

The leaves of cassava were chopped into small pieces of around 0.5-1.0 cm of length), then ground (1mm sieve). They were mixed with cassava root meal and either potassium nitrate or urea (Table 1) prior to adding 0.96 liters of buffer solution (Table 2) and 240 ml of rumen fluid (obtained from a newly slaughtered buffalo in the village abattoir), and put in the incubation bottle which was then gassed with carbon dioxide. The bottles were incubated at 38 ⁰C in a water bath for 12 and 24 h.  

Data collection and measurements

The gas volume and the percentage of methane in the gas (Crowcon infra-red analyser; Crowcon Instruments Ltd, UK) were recorded for the separate incubations after 12 and 24h.  At the end of each incubation the residual DM in the incubation bottle was determined by filtering through cloth and drying (100°C for 24h) the residue.

Statistical analysis

The data were analyzed by the General Linear Model (GLM) option in the ANOVA program of the Minitab (2000) Software. Sources of variation in the model were: NPN source, leaves of cassava, interaction NPN*leaves and error.

Results and discussion

Chemical composition  

On all processing treatments, the petioles had lower content of DM and of crude protein in the DM (Table 3).

Protein solubility

The protein solubility decreased in the order: fresh, ensiled, sun-dried and oven-dried processing (Table 4; Figure 1).  Petioles had lower values than leaves.

Figure 1.  Protein solubility in leaves and petioles of cassava

Methane production

Gas production, per cent methane in the gas, substrate fermented and methane produced per unit substrate fermented were higher for 24 than 12h incubations and lower for nitrate than for urea (Figures 2-9).   

Gas production was lower for fresh leaves than for ensiled or dried leaves when urea was the NPN source, but with no differences for processing with nitrate as NPN. Per cent methane in the gas was lower for fresh and ensiled leaves compared with dried leaves at 12h with a similar but less marked tendency for the 24 h incubation. The proportion of substrate fermented was higher for fresh and ensiled leaves compared with dried leaves for both nitrate and urea supplements and for both times of incubation. Methane produced per unit substrate fermented was lower for fresh and ensiled leaves than for dried leaves at both12 and 24h incubation, the differences being more marked for the shorter incubation time. 

Figure 2. Effect of NPN source on gas production for 12h incubation  time with fresh, ensiled,  sun-dried and oven-dried cassava leaves	Figure 3. Effect of NPN source on gas production for 24h incubation  time with fresh, ensiled,  sun-dried and oven-dried cassava leaves

Figure 4. Effect of NPN source on methane per cent  in the gas for 12h incubation  time with fresh, ensiled,  sun-dried and oven-dried cassava leaves	Figure 5. Effect of NPN source on methane per cent  in the gas for 24h incubation  time with fresh, ensiled,  sun-dried and oven-dried cassava leaves

Figure 6. Effect of NPN source on substrate fermented  for 12h incubation  time with fresh, ensiled,  sun-dried and oven-dried cassava leaves	Figure 7. Effect of NPN source on substrate fermented  for 24h  incubation  time with fresh, ensiled,  sun-dried and oven-dried cassava leaves

Figure 8. Effect of NPN source on methane per unit substrate fermented for 12h incubation  time with fresh, ensiled,  sun-dried and oven-dried cassava leaves	Figure 9. Effect of NPN source on methane per unit substrate fermented for 24h incubation  time with fresh, ensiled,  sun-dried and oven-dried cassava leaves

The higher methane production per unit of dry matter digested in dried cassava leaves compared with fresh and ensiled indicates that treatments that reduce cyanide concentrations (eg: sun or oven-drying) increase the amount of methane produced, which may be explained if cyanide inhibits the metabolism of acetate to methane and carbon dioxide as happens in sludge type fermentations (Gijzen et al 2000; Eikmanns and Thauer 1984; Smith et al 1985 and Cuzin and Labat 1992).

The original hypothesis that decreasing protein solubility would reduce methane production was disproved as the contrary effect was observed (Figure 10).  It would appear that the effects of drying on cyanide release potential were more important than the slight differences in protein solubility brought about by drying.

Figure 10. Apparent relationship between protein solubility in cassava leaves and production of methane per unit substrate fermented

Conclusions

• The percentage of methane in the gas and quantity of methane produced per unit of fermented substrate was lower with fresh cassava leaves when compared with dried cassava leaves in an in vitro incubation with cassava root meal as energy source.

•  The gas production, percentage of methane in the gas and methane produced per unit substrate fermented, were reduced when nitrate replaced urea as the NPN source in the in vitro incubation

Acknowledgements

The authors expresses gratitude to the MEKARN project, supported by Sida, for financial support for this research. Special thanks are given to Mr Sengsouly Phongphanith who provided valuable help in the laboratory. We also acknowledge the Souphanouvong University, Faculty of Agriculture and Forest Resources, Department of Animal Science, for providing infrastructure support.

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Received 2 January 2012; Accepted 26 January 2012; Published 7 February 2012

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