Effect of foliage from “sweet” and “bitter” cassava varieties on methane production in in vitro incubation with molasses supplemented with potassium nitrate or urea

Le Thuy Binh Phuong, T R Preston* and R A Leng**

Nong Lam University, Viet Nam
binhphuongty27@yahoo.com
* Finca Ecologica, TOSOLY, UTA (Colombia)
AA#48, Socorro, Santander, Colombia
** University of New England, Armidale NSW, Australia

Abstract

This study was on the effect of variety (sweet or bitter) and processing (fresh or sun-dried) of cassava foliage on protein solubility and on methane production in an in vitro rumen fermentation in which the NPN sources were nitrate or urea.

The sun-drying of cassava foliage reduced protein solubility and potential  HCN release. The bitter varieties  contained more HCN precursors and the protein was less soluble than in the sweet variety. Bitter varieties had lower crude protein solubility and higher potential release of HCN than the sweet variety. Sun-dried processing decreased N solubility and potential HCN release compared with fresh leaves. Nitrate compared with urea, and bitter compared with the sweet cassava, independently reduced methane production resulting in an overall reduction of 43% for the  combination of the bitter varieties and supplementation with nitrate compared with the sweet variety and urea.

Key words: HCN, protein solubility, rumen fermentation

Introduction

Cassava (Manihot esculenta, Crantz) is a crop of major importance in the tropics. Fresh cassava foliage has been used as a source of by-pass protein for cattle fed basal diets of molasses (Ffoulkes and Preston 1978) and rice straw (Sath et al 2010; Tham et al 2010). Recent research reports also indicate that methane emissions were less when fresh cassava leaf rather than dried leaf meal was the protein source in in vitro incubations with sugar cane (Phommasack  et al 2011) and cassava root meal (Sangkhom et al 2012). Heating of intact cassava leaves causes liberation of hydrogen cyanide. This phenomenon appears to be caused by β-glucosidase-catalysed decomposition of the cyanogenic glycosides linamarin and lotaustralin. It is known that the capacity to liberate HCN from cassava foliage is higher in “bitter “ cassava leaves than in “sweet” cassava leaves (Chhay Ty et al 2007) and is reduced by processing such as sun drying or ensiling (Khieu Borin et al 2005; Phengvichith and Ledin 2007). Hydrogen cyanide is a potent  inhibitor of acetoclastic methanogens which are slow growing and  mainly present in such ecosystems as  biodigestors but it also inhibitory to hydrogenotrophic Archae (Cuzin and Labal 1992; Annachhatra and Amonkuew 2001),  which are  the main producers of methane  in the rumen, although the  toxicity to this group of methanogens  is low (Smith et al 1985).

According to Ho Quang Do et al (2012) a further factor in feeds that affects methane production in in vitro rumen incubations is the solubility of the protein. These authors reported that methane production was less when fish meal of low solubility was the protein source compared with groundnut meal having highly soluble protein. The groundnut diet would be expected to be more rumen-fermentable, than the fish meal diet,  hence producing more methane, even though  protein meals fermented in the rumen produce approximately half the methane that results from the same weight of carbohydrate (Van Nevel  and Demeyer1996).

The apparent conversion of glycosides in cassava to hydrogen cyanide and  loss of hydogen cyanide in storage or after heating allowed us to assess the effects of high or low levels of hydrogen cyanide on rumen fermentation.

Leng (2008) has emphasized that nitrate as a feed component replacing urea has a dual role as an electronic sink for hydrogen produced by rumen fermentation, and as a source of ammonia for rumen microbial protein synthesis. The effects of cyanide on other terminal electron processes is unknown and as nitrate  is now regarded as a potential alternative electron sink (see Leng 2008) it is essential to understand whether cyanide is toxic to nitrate reducing bacteria in the rumen. A further consideration in these studies was the potential benefits of sulphur availability from S-amino acids  which would be considerably greater where soluble vs insoluble  protein meals are part of the substrate.

Hypothesis

•      The solubility of the protein in cassava foliage and the potential to produce HCN will be greater in bitter than in sweet varieties; and that both characteristics will be reduced by sun-drying.

•      There will be an interaction  between cassava foliage variety and supplementary nitrate on the enhancement of the reduction of methane production.

Materials and methods

Location and duration

The experiment was conducted in the laboratory of Nong Lam University, Ho Chi Minh city, Viet Nam, in May, 2012.

Experiment  1:  

Experimental design

The experiment was designed as a 3*2 factorial arrangement of the following treatments:

Variety:

• Bitter 1 (KM95 variety)

• Bitter 2 (KM94 variety)

• Sweet

Processing  

• Fresh

• Sun-dried

Material preparation

Cassava leaves of the three varieties were collected in the morning from fresh stands in a nearby farm, and immediately put into plastic bags to avoid moisture loss. One portion was ground immediately through a 1mm sieve  and the other dried in the sun for 2 days prior to grinding.  

Measurements

The content of DM and N in the leaves was determined according to AOAC (1990) methods. Solubility of the protein was determined by shaking a 3g sample (DM basis) with 100ml 1M NaCl  for 3 hours, filtering through Whatman No.4 filter paper and then washed 3 times with distilled water. The filtrate was  analysed for N  according to AOAC (1990).  N solubility was calculated as N content of the filtrate as a percentage of the N  in the original sample.

Hydrogen cyanide (HCN) concentration was determined by putting the  sample (20 g fresh leaf; or 5 g dry leaf)  into a kjeldalh flask, and adding 250 - 300 ml distilled water and  8 ml of chloroform, then boiling until the solution changed to green colour. The contents of the kjeldahl flask were then transferred to an Erlenmeyer 250 ml flask containing 8 ml of 0.1N KOH and titrated with 0.1N Silver nitrate (1 ml 0.1 N Silver nitrate =  0.005204 g HCN).

The content of crude protein in the cassava samples was adjusted to deduct the N present as HCN

Statistical analysis

The data were analyzed by the General Linear Model (GLM) option in the ANOVA program of the Minitab Softwar (Minitab 2000). Sources of variation in the model were: cassava varieties, processing of cassava foliage, cassava varieties * processing and error.

Experiment 2

Experimental design

The experiment was arranged as a 3*2*2 factorial with 12 treatments and 4 repetitions .

The first factor was cassava leaf variety

• Sweet

• Bitter 1 (KM95)

• Bitter 2 (KM94)

The second factor was processing of the leaves

• Fresh

• Sun-drying

The third factor was the source of fermentable nitrogen:

• Potassium nitrate [6% of substrate DM]

• Urea [1.8% of substrate DM)

The carbohydrate source was molasses.  The in vitro procedure was that described by Sangkhom Inthapanya et al (2011).

Material preparation and implementation of the method

Molasses was purchased in the market. For the cassava leaves, the collection and processing were the same as described in Experiment 1. Representative samples (Table 1) of the ingredients (molasses, cassava leaves and NPN source)  were put in the incubation bottle to which was added 960 ml of buffer solution (Tilly and Terry 1963) and 240 ml of cattle rumen fluid. The rumen fluid was taken immediately from a cow that was slaughtered at the local abattoir, and held in a thermos flask for about 1 h until placed in the incubation flask which was then gassed with carbon dioxide. The incubation was for 24 h in a water-bath held at 37°C. 

Measurements

The gas volume and percentage methane were determined after 6 and 24h. Gas volume was measured by water displacement; methane percentage in the gas was determined with an infra-red meter (Crowcom Instruments Ltd, UK). The substrate solubilised was the residue after 24h incubation, which was filtered through 2 layers of cloth then dried at 120°C for 72 h. The DM and N in the molasses and cassava leaves were determined according to AOAC (1990) methods.

Statistical analysis

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

Results and discussion

Chemical composition

The crude protein content of the KM94 (bitter 2) variety was less than in the bitter 1 (KM93) and sweet varieties  (Table 3). The crude protein equivalent from the HCN was only a small fraction (about 1%) of the total crude protein (Table 3).

Crude protein solubility and HCN concentration

The bitter 1 variety had lowest crude protein solubility and highest HCN content ; sun-drying decreased crude protein solubility and HCN content as compared with the fresh leaves (Table 4; Figures 1-3).

Figure 1. Effect of variety on CP solubility of cassava leaves	Figure 2. Effect of processing on CP solubility of cassava leaves	Figure 3. Effect of variety and processing on HCN potential release in cassava leaves

 Methane production

After 24h incubation, gas production was lower from substrates with  nitrate than with urea but there were  no differences among varieties nor for effects of processing (Table 5; Figure.4-5). The methane percentage in the gas was always lower for nitrate than urea as NPN source (Figures 6 and 7). The proportion of the substrate solubilized was not affected by any of the treatments. There was a tendency for methane production per unit substrate solubilized to be less for the bitter compared with the sweet variety (P=0.17) and for fresh compared with sun-dried cassava leaves (P=0.21) (Figures 8 and 9). When the data for the two bitter varieties were combined and compared with the sweet variety (Figure 10) the effect  of the bitter varieties in reducing methane production was accentuated (P=0.051). There was no interaction on methane production between NPN source and cassava variety, thus it can be concluded that both nitrate and variety (ie: potential HCN release) independently reduced methane production resulting in an overall reduction of 43% for the combination of the bitter varieties and supplementation with nitrate compared with the sweet variety and urea.

Ammonia after 24 hours incubation

The ammonia concentration in the media  after 24h fermentation was not affected by any of the treatments (Table 6).

Figure 4. Effect of NPN source on gas production for 24h incubation with cassava leaf varieties	Figure 5. Effect of NPN source on gas production for 24h incubation with fresh and sun-dried cassava leaves
Figure 6. Effect of NPN and cassava varieties on methane percentage in gas for 24h incubation w	Figure 7. Effect of NPN source on methane percentage in gas for 24h incubation with fresh and sun-dried cassava leaves
Figure 8. Effect of NPN source on methane produced per unit substrate solubilized for 24h incubation with cassava leaves from sweet and bitter varieties	Figure 9. Effect of NPN source on methane produced per unit substrate solubilized for 24h incubation with fresh and sun-dried cassava leaves

The tendency for methane production to be higher (P=0.21) for dried compared with fresh cassava leaves as the protein source is in line with recent reports by Phommasack  et al (2011) and Sangkhom et al (2012) where methane production was reduced when fresh cassava leaves replaced dried leaves in in vitro rumen fermentations. Phommasack et al (2011) cited the research of Eikmanns and Thauer (1984) and Smith et al (1985) which supports the concept that cyanide is somewhat toxic to methanogens  or reduces their potential growth by lowering the availability of sulphur by formation of thiocyanates (Majak and Cheng 1984). Additions of 5, 10, and 25 mg 1itre^-l cyanide (from KCN or linamarin) temporarily inhibited methanogenesis in biodigesters charged with cassava root waste, but when the concentration of cyanide returned to lower levels (as it was before KCN or linamarin addition), methane production recovered (Cuzin and Labat 1992)  The biodigester methanogenic microflora were sensitive to the added cyanide,

There appears to be no effect of presence of cyanide on the  nitrate reducing bacteria and indirectly on the sulphur reducing bacteria in the rumen. In fact the combined effects of  cyanide levels and nitrate as a fermentable N source appeared to be additive in lowering methane production

It is likely that cyanide does have an effect on methanogens directly but the rapid deactivation of cyanide to thiocyanate (Majak and Cheng 1984) may relieve this inhibition.  It is assumed that cattle accustomed to cyanide in the diet develop very effective processes to remove it. Thus it is unlikely that there is additional benefit to methane mitigation by feeding cyanide containing plants. However, in a recent experiment in our laboratory with cattle adapted to cassava foliage in a molasses-based diet, methane production was reduced by 13% when fresh cassava foliage was fed compared with cassava leaf meal (Phuong et al 2012). More research with animal growth trials is needed to clarify this issue.

Protein solubility appears to play some role in methanogenesis but it is small and possibly entirely due to the  change in the site of protein digestion -- either being fermented in the rumen or digested in the intestines. From the known pathways of degradation of the amino acid complement of proteins, hydrogen release is about half that from a similar weight of  carbohydrate (Van Nevel and Demeyer 1996), but  not all the protein in highly soluble protein entering the rumen is fermented to ammonia and depending on many factors, amino acids and peptides from these sources are incorporated into microbial protein. The concept that protein solubility may be a factor contributing to methane generation is probably not of major significance.  However, bypass (escape) protein can have major effects  feed intake and productivity in ruminants (Prestn and Leng 1987)  particularly on diets of agro industrial byproducts where a major reduction in methane production per unit of animal production would be a most important outcome.  

Conclusions

• Bitter varieties had lower crude protein solubility and higher potential release of HCN content than the sweet variety.

  
  

• Sun-dried processing decreased N solubility and potential HCN release compared with fresh leaves.

  
  

• Nitrate compared with urea, and bitter compared with the sweet cassava, independently reduced methane production resulting in an overall reduction of 43% for the  combination of the bitter varieties and supplementation with nitrate compared with the sweet variety and urea.

  
  

• The fresh cassava foliage tended to support lower methane  production than sun-dried cassava foliage, independent of the variety.

Acknowledgements

The authors acknowledge support for this research from the MEKARN project financed by Sida. Thanks are due to the Department of Animal Nutrition laboratory, Faculty of Animal Science and Veterinary Medicine, Agriculture Forestry University for providing the facilities to carry out this research. 

References

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Received 14 August 2012; Accepted 24 September 2012; Published 1 October 2012

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