Synergic effect of cassava (manihot esculenta crantz) foliage, brewer’s grains, and biochar on methane production and performance of ruminants

HUE UNIVERSITY 
UNIVERSITY OF AGRICULTURE AND FORESTRY
LE THUY BINH PHUONG 
SYNERGIC EFFECT OF CASSAVA (MANIHOT ESCULENTA CRANTZ) FOLIAGE, BREWER’S GRAINS, AND BIOCHAR ON METHANE PRODUCTION AND PERFORMANCE OF RUMINANTS 
DOCTOR OF PHILOSOPHY IN ANIMAL SCIENCES
HUE, 2020
HUE UNIVERSITY
UNIVERSITY OF AGRICULTURE AND FORESTRY
LE THUY BINH PHUONG
SYNERGIC EFFECT OF CASSAVA (MANIHOT ESCULENTA CRANTZ) FOLIAGE, BREWER’S GRAINS, AND BIOCHAR ON METHANE PRODUCTION AND PERFORMANCE OF RUMINANTS 
SPECIALIZATION: ANIMAL SCIENCES
CODE: 9620105
DOCTOR OF PHILOSOPHY IN ANIMAL SCIENCES
SUPERVISOR 1: ASSOC. PROF. NGUYEN HUU VAN 
SUPERVISOR 2: DR. DINH VAN DUNG
HUE, 2020
Declaration
I declare that this dissertation is the result of my work and that it has not been presented previously as a dissertation at this university or elsewhere. To the best of my knowledge, it does not breach copyright law, and has not been taken from other sources except where such work has been cited and acknowledged within the text. All results have been published at Journal of Livestock Research for Rural Development (LRRD) 
 Hue University, 2020
Le Thuy Binh Phuong
Dedication 
To my parent who spends their immense loves to me.
To my husband, Than Van Dang, and my two daughters, Than Ngoc Kim Nguyen and Than Ngoc Hai An, who encouraged me to pursue my dreams.
Acknowledgements
	My Ph.D. has been an amazing experience with Professor Thomas Reginal Preston. He has been teaching me how good experiment is done and how to be real researcher. I have been grown up like that, thus, I would like to thank with all my heart to his guidance. 
	I am thankful to Professor Ron A. Leng who gave me the background knowledge in biochemistry for stimulating the ideas in research. 
	I would like to thanks to Assoc. Prof. Nguyen Huu Van and Dr. Dinh Van Dung who gave me the most helpful advice and instructed me to complete the dissertation.
	I gratefully acknowledge financial support from the SIDA-financed project, MEKARN II for 3 years that made my Ph.D. work possible.
	My classmate in Ph.D. course, the group is source of friendship as well as good collaboration.
	Lastly, I would like to thank my family for all their love and encouragement. For my parents who take care of my children during course time and support me to participate in learning and research activities. Most of all for my loving, supportive, encouraging, and patient husband Dang who faithful support during the final stages of this Ph.D. is so appreciated. Thank you.
Abstract
	This dissertation was aimed to develop a greater understanding of both the constraints in the presence of cyanide toxin and benefits of using cassava foliage as bypass protein in order to improve its utilization in ruminant feeding systems.
	The study comprised two in vitro rumen incubations, one feeding trial on cattle and a digestibility/N retention experiment on goats, in each case involving comparisons of varieties of cassava known to be rich (KM94) or poor (Gon) in cyanogenic glucosides. 	In the first experiment (Chapter 2), cassava foliage varieties (Japan, KM94, KM140 and Gon) with different level of cyanide concentration were considered their effect on methane production in ruminal in vitro incubation. The second experiment (Chapter 3) examined the relative responses of cattle fed cassava root pulp and urea as basal diet with foliage from “sweet” (Gon) or “bitter” (KM140) cassava foliage as protein source. The third experiment (Chapter 4) determined methane production in an in vitro rumen incubation of cassava pulp - urea with additives of brewers’ grain, rice wine yearst culture, yeast-fermented cassava pulp and leaves of sweet or bitter cassava variety. The fourth experiment (Chapter 5) measured effect of additives (brewer’s grain and biochar) on the nitrogen retention and rumen methane production when goats had access to mixed sweet and bitter varieties of cassava foliage compared with the sweet variety alone.
	The results of these experiments indicated that bitter cassava foliage containing high levels of cyanogenic glucosides greatly reduces methane production, compared with sweet varieties, in the rumen in vitro incubations. However, the toxicity of cyanide in vivo in ruminants (cattle and goats) can be reduced by “prebiotic” properties provided by either brewers’ grains or biochar. In the presence of these “prebiotics”, HCN-linked challenges from feeding bitter cassava leaves at up to 50% of the diet of goats did not negatively impact to feed intake, growth and animal health. On the contrary, the HCN precursors present in bitter cassava leaves may lead to a partial shift in digestion of nutrients from the rumen to the lower parts of the ruminant digestive tract leading to improvement in productivity.
Key words: Prebiotic, cyanide, bitter cassava, rumen fermentation, in vitro. 
Table of Contents
List of Figures
Figure 1.1 Pathway of VFA in metabolism	7
Figure 1.2 The reaction of methane generation	10
Figure 1.3 The pathway of hexose conversation to end-products	11
Figure 1.4 A sulfurtransferase reaction catalyzed by rhodanese	27
Figure 1.5 The porous structure of biochar invites microbial colonization- pine saw dust-derived biochar	37
Figure 2.1 Relationship between methane in the gas and HCN content in treatments	69
Figure 2.2 Effect of HCN content in treatment on ammonia production is expressed as all data in each treatment during the in vitro fermentation	69
Figure 2.3 Effect of HCN content in treatment on ammonia production is expressed as average in each treament.	69
Figure 3.1 The negligible growth rate of Laisind cattle fed bitter cassava foliage as protein source in Period 1 was dramatically increased by adding 4% of brewers’ grains to the diet in Period 2.	82
Figure 3.2 Growth curves of Laisind cattle showing the change in live weight gain after introduction of 4% brewers’ grains (as % of diet DM) to those fed bitter cassava foliage	83
Figure 3.3 Mean values for VFA proportions in rumen fluid from cattle in Period 2	85
Figure 4.1 Effect of additives, and source of cassava leaf (bitter or sweet variety) on gas production after 24h fermentation	100
Figure 4.2 Effect of stage of the fermentation on the methane content of the gas	100
Figure 4.3 Effect of additives, and source of cassava leaf on methane content of the gas after 24h fermentation	101
Figure 4.4 Interaction between source of cassava leaf and additive with brewers’ grains on methane content in the gas	103
Figure 4.5 Viable Saccharomyces cells in brewers’ grain and in cassava pulp fermented only with yeast (YFCP) or with yeast and urea (YFCP-U-DAP) after 07 days of fermentation	104
Figure 4.6 Lactobacilli in brewers’ grain and in cassava pulp fermented only with yeast (YFCP) or with yeast and urea (YFCP-U-DAP) after 07 days of fermentation	104
Figure 5.1 Condensed tannin in petiole and leaf from cassava foliage	116
Figure 5.2 HCN equivalent in petiole and leaf from cassava foliage	116
Figure 5.3 DM intake of leaf and petiole of sweet and bitter cassava varieties when the goats had free access to both (sweet+ bitter foliage) in Square 2.	117
Figure 5.4 Individual and combined effects of additives and source of cassava foliage on N retention; (SW as Sweet; SW-BIT as Sweet and Bitter)	119
Figure 5.5 Bach Thao goat from Latin Square 2	120
Figure 5.6 Methane: carbon dioxide ratios in mixed eructed gas and air in goats	120
Figure 5.7a Relationship between methane: carbon dioxide ratio in mixed eructed gas and air and nitrogen retention (includes all 8 goats)	121
Figure 5.7b Relationship between methane: carbon dioxide ratio and nitrogen retention (excluding the outlier result)	121
List of Tables
Table 1.1 Nutrient composition of fresh cassava leaf	20
Table 1.2 Essential amino acid profile of cassava leaf	21
Table 1.3 Chemical composition of brewers’ grain	31
Table 2.1 Composition of the substrates	65
Table 2.2 Ingredients in buffer solution	66
Table 2.3 Chemical composition of the ingredients in the substrate	67
Table 2.4 Mean values for gas production in 24 hours, methane in the gas and per unit DM mineralized in an in vitro rumen fermentation.	68
Table 2.5 Mean values for content of condensed tannin and HCN in the leaves of sweet and bitter varieties of cassava leaves, ammonia concentration and methane production per DM mineralized after 24h incubation	68
Table 3.1 The chemical composition of ingredients	78
Table 3.2a Mean values for feed intake, change in live weight and feed conversion of Laisind cattle in Period 1	81
Table 3.2b. Mean values for feed intake, change in live weight and feed conversion of Laisind cattle (in Period 2)	82
Table 3.3 Mean values for thiocyanate in the urine of cattle	84
Table 3.4 Mean values for VFA proportions in rumen fluid of cattle (in Period 2)	84
Table 3.5 Mean values of methane: carbon dioxide ratios in mixed eructed gas/air of cattle	85
Table 4.1 Chemical composition of substrates	99
Table 4.2a Effect of source of cassava variety on gas production and methane percentage in the gas	101
Table 4.2b Effect of additive on gas production and methane percentage in the gas	102
Table 5.1 Layout of each Latin Square	112
Table 5.2 Dry matter (DM) and crude protein (CP) of ingredients	112
Table 5.3 Mean values for effects of cassava foliage (sweet or bitter) on % DM, tannin and HCN equivalent in leaves and petioles	115
Table 5.4 Mean intakes of leaf and petiole for the goats in Square 2 that had free access to foliage of both sweet and bitter varieties	116
Table 5.5 Mean values (g/d) for effects of cassava foliage (bitter or sweet) and of additives on DM intake (DMI), apparent digestibility of DM and crude protein (CP) and N balance	118
Table 5.6 Mean values for effects of additives on N retention	118
Table 5.7 Mean values for VFA proportions (mol %), acetic: propionic ratio, rumen ammonia, daily urine volume, daily excretion of thiocyanate (SCN) in urine and CH4:CO2 ratio in mixed eructed gas and air	119
Abbreviation
ADG
Average daily gain
ATP
Adenosine tri-phosphate
ADF
Acid detergent fiber
BG
Brewers’ grain
CP
Crude protein
CF
Crude fiber
CFU
Colony-forming unit
DM
Dry matter
DMI
Dry matter intake
EPS
Extracellular polymeric substances
EE
Ether extract
HCN
Hydrocyanic acid
GE
Gross energy
LW
Live weight
MOS
Manna-oligosaccharide
N
Nitrogen
NADH
Nicotinamide adenine dinucleotide hydride
NDF
Neutral detergent fiber
NPN
Non-protein nitrogen
RDP
Rumen degradable protein
PEP
Phosphoenolpyruvate
SEM
Standard error mean
UDP
Un-degradable protein
VFA
Volatile fatty acid
INTRODUCTION
1. Problem statement
	Cassava is perspective plant to climate change adaptation; its pests and its diseases resistance and greater drought tolerance is a major factor in ranking cassava in the food security of the world (Jarvis et al. 2012). In Vietnam, cassava is second crop, is grown mainly in both at the household and small-scale processor level (Hoang Kim et al. 2000). From the successful experiment in utilization of cassava foliage (sweet variety) as protein source on cattle which was originally reported by Ffoulkes and Preston (1978), and then have been successfully fed as fresh state to goat and cattle in Cambodia (See report of Preston and Rodríguez Lylian, 2004), that make cassava foliage become important plant protein source in ruminant diet. Nevertheless, cyanide toxin in fresh cassava foliage, especially bitter cassava foliage, is the main obstacle for animal such as restricting the consumption intake of ruminant or causing poisoning when they consume rapidly. Currently, as the quantity of bitter cassava (high cyanide content) develop more predominant than sweet cassava (lower cyanide content) on the field, utilization of bitter cassava foliage in diet will match reality more; however, looking for feeding method of minimizing negative effect of cyanide toxin, from that can be utilized bitter cassava foliage in diet will more match reality but will a challenge.
	Many studies are beginning to be interested in cyanide toxic that has certain effect on methanogenic bacteria population by inhibited methanogenesis activity lead to diminish methane production (Ch Olga Rojas et al. 1999; Phuong et al. 2012; Phanthavong et al. 2015). However, whether cyanide may affect overall microbial activity and impact the rate of rumen fermentation indirectly, it is still not fully understood. Previously, the knowledge of ruminant nutritionists focused on rumen, but the impact of cyanide on rumen fermentation may profoundly influence lower digestive physiology of ruminant and must be considered to fully understand when utilizing bitter cassava foliage in diet. Even so, the challenge of bitter cassava foliage diet (high cyanide content) is a new approach but must require the safety for animal's health. Therefore, building appropriate feeding method for fresh cassava foliage diet, particularly bitter cassava foliage, without cyanide poisoning is needed to utilize cassava foliage more effective in the ruminant feeding system.
2. Aim and objective of the study
2.1 Aims of the study
	The aim of this thesis was to develop a greater understanding of both the constraints and benefits of using cassava foliage in ruminant feeding systems. From these things can improve the utilization of cassava foliage in ruminant feeding by enhancing its properties as a source of bypass protein and verify the role of HCN toxin in cassava foliage on the reduction of methane production that was built on earlier findings.
2.2 Objective of the study
	The following objectives are required to accomplish the aim of this research:
Determining the trend influences of HCN concentration in cassava foliage on the characteristic of in vitro rumen fermentation such as gas and methane production, ammonia concentration.
Considering the benefit of brewers’ grain to “bitter” cassava foliage (KM94) diet by examining Saccharomyces and acid lactic bacteria in fresh brewers’ grain and compare it with potential fermented cassava pulp on gas and methane production of ruminal in vitro incubation.
Building feeding method of “bitter” cassava foliage (KM 94 variety; moderate HCN content) diet by added 4% brewers’ grain (of DM) and/or 1% biochar (as DM), then evaluating the effects of this feeding method on growth, digestibility/N retention, excretion of thiocyanate in urine and methane production of cattle and goat.
3. Research hypotheses
	The research hypothesizes following: 
Higher HCN content in bitter cassava foliage would be more effective in reducing methane production in both rumen in vitro incubations and in vivo experiment rather than foliage from a sweet variety (low HCN content).
By added 4% brewers’ grain and/or 1% biochar in bitter cassava foliage (KM94 variety) diet would lead to: (1) improving the growth rate of cattle fed a basal diet of cassava pulp-urea; and (2) i ... ducing methane production and excretion of thiocyanate. The effect of adding both brewers’ grains and biochar in combination with mixed sweet and bitter cassava foliage was a 58% higher N retention than on the control treatment of only sweet cassava and no additives. An important finding was the negative relationship between methane production and N retention, confirming the original comments of Johnson et al. (1993) (cited by Johnson and Johnson 1995) regarding the potential gain in dietary net energy by reducing enteric methane production.
6.2 Conclusions
	In summary, the body of work presented in this dissertation has shown that supplementing ruminant diets with cassava foliage reduces rumen methane production, and the effect is more pronounced with varieties containing higher levels of cyanogenic glucosides, which give rise to HCN in the rumen. However, the risk of toxicity of cyanide in ruminants (cattle and goats) can be reduced by adding to the diet either brewer’s grains or biochar or both as a “prebiotic” source. In the presence of “prebiotic”, challenges of bitter cassava leave in the feeding system of goat and cattle did not negatively impact to feed intake and animal’s health. In more detail, adding restricted (4% of DM) brewers’ grain into “bitter” cassava foliage as a main protein source appeared reduction of excreted thiocyanate in urine, lead to significant improvement of growth rate of cattle compare to only “bitter” cassava foliage. Moreover, synergistic of brewers’ grain (4% of DM) and biochar (1% of DM) as additives could show substantially same effectiveness even when goat was fed “bitter” cassava foliage at up to 50% in the diet. On the contrary, the feeding of the bitter cassava foliage appeared to modify the rumen fermentation leading to an improved balance of nutrients at the whole animal level, as manifested by the 20% increase in nitrogen retention associated with decreased production of methane. 
 6.3 Implication and further research
	The dissertation is the process with step-by-step experiments has provided evidence for the benefits of restricted amounts (4 %) of brewers’ grains and/or biochar (1%) as a means of avoiding risks of HCN toxicity in ruminant feeding systems using bitter cassava foliage as the source of bypass protein. However, the implications of the proposed partial shift in sites of digestion (from rumen to small intestine and the cecal-colon region) must be substantiated in long-term feeding trials. Research is also needed to monitor how modifications in the rumen fermentation (eg: increased escape of digestible organic matter) affect fermentation in the cecum. 
References
 Delaney, B., Nicolosi, R. J., Wilson, T. A., Carlson, T., Frazer, S., Zheng, G. H., Hess, R., Ostergren, K., Haworth, J., Knutson, N., 2003. Beta-glucan fractions from barley and oats are similarly antiatherogenic in hypercholesterolemic Syrian golden hamsters. Journal of Nutrition 133(2): 468-475.
Ffoulkes, D. and Preston, T. R., 1978. Cassava or sweet potato forage as combined sources of protein and roughage in molasses based diets: effect of supplementation with soybean meal. 
Gerlach, K., Pries, M., Südekum, K-H., 2018. Effect of condensed tannin supplementation on in vivo nutrient digestibilities and energy values of concentrates in sheep. Small Ruminant Research 161: 57-62.
Hojjatollah Shokri, Farzad Asadi and Ali Reza Khosravi, 2008. Isolation of β -glucan from the cell wall of Saccharomyces cerevisiae. Natural Product Research: Formerly Natural Product Letters, 22:5, 414-421. 
Inthapanya, S., Preston, T. R. and Leng, R. A., 2016. Ensiled brewers’ grains increased feed intake, digestibility and N retention in cattle fed ensiled cassava root, urea and rice straw with fresh cassava foliage or water spinach as main source of protein. Livestock Research for Rural Development. Volume 28, Article #20.  
Jarvis, A., Ramirez-Villegas, J., Herrera Campo, B.V., Navarro-Racines, C., 2012. Tropical Plant Biology Volume 5, Issue 1, Page 9-29. https://doi.org/10.1007/s12042-012-9096-7
Johnson, K. A. and Johnson, D. E., 1995. Methane emissions from cattle. Journal of Animal Science 73:2483-2492. 
Kuma, R., Singh, M., 1984. Tannins: their adverse role in ruminant nutrition. Journal of Agriculture and Food Chemistry 32, pages 447-458. 
Leng, R. A., 2017. Biofilm compartmentalisation of the rumen microbiome: modification of fermentation and degradation of dietary toxins . Animal Production Science 57(11) 2188-2203 https://doi.org/10.1071/AN17382
Leng, R. A., 2018. Unravelling methanogenesis in ruminants, horses and kangaroos: the links between gut anatomy, microbial biofilms and host immunity. Animal Production Science, 58, 1175–1191. Perspectives on Animal Biosciences. https://doi.org/10.1071/AN15710
Majak, W., 1991. Metabolism and absorption of toxic glycosides by ruminants. Journal of Range Management, 45(1):67-71 
Phanthavong, V., Preston, T. R., Vorlaphim, T., Dung, D. V. and Ba, N. X., 2018. Fattening “Yellow” cattle on cassava root pulp, urea and rice straw: completely mixed ration system with cassava foliage as protein supplement compared with feeds not mixed and brewers’ grains as protein source. Livestock Research for Rural Development. Volume 30, Article #169. Retrieved January 4, 2019, from  
Phanthavong, V., Preston, T. R., Viengsakoun, N. and Pattaya, N., 2016. Brewers' grain and cassava foliage (Manihot esculenta Cranz) as protein sources for local “Yellow” cattle fed cassava pulp-urea as basal diet. Livestock Research for Rural Development. Volume 28, Article #196. 
Phonethep, P., Preston, T. R. and Leng, R. A., 2016. Effect on feed intake, digestibility, N retention and methane emissions in goats of supplementing foliages of cassava (Manihot esculenta Crantz) and Tithonia diversifolia with water spinach (Ipomoea aquatica). Livestock Research for Rural Development. Volume 28, Article #72. 
Outhen, P., Preston, T. R. and Leng, R. A., 2011. Effect of supplementation with urea or calcium nitrate and cassava leaf meal or fresh cassava leaf in an in vitro incubation using a basal substrate of sugar cane stalk. Livestock Research for Rural Development. Volume 23, Article #23. Retrieved July 25, 2019, from 
Phuong, L. T. B., Preston, T. R. and Leng, R. A., 2012. Effect of foliage from “sweet” and “bitter” cassava varieties on methane production in in vitro incubation with molasses supplemented with potassium nitrate or urea. Livestock Research for Rural Development. Volume 24, Article #189. Retrieved August 19, 2018, from  
Promkot, C. and Wanapat, M., 2003. Ruminal degradation and intestinal digestion of crude protein of tropical protein resources using nylon bag technique and three-step in vitro procedure in dairy cattle. Livestock Research for Rural Development. Volume 15, Article #81. 
Ravindran, V., 1993. Cassava leaves as animal feed: Potential and limitations. Journal of the Science of Food and Agriculture 61(2): 141-150. 
Sath, K., Borin, K. and Preston, T. R., 2008. Effect of levels of sun-dried cassava foliage on growth performance of cattle fed rice straw. Livestock Research for Rural Development. Volume 20, from  
Sengsouly, P. and Preston, T. R., 2016. Effect of rice-wine distillers’ byproduct and biochar on growth performance and methane emissions in local “Yellow” cattle fed ensiled cassava root, urea, cassava foliage and rice straw. Livestock Research for Rural Development. Volume 28, Article #178. Retrieved January 2, 2019, from 
Sivilai, B., Preston, T. R., Leng, R. A., Hang, D. T., and Linh, N. Q., 2018. Rice distillers’ byproduct and biochar as additives to a forage-based diet for growing Moo Lath pigs; effects on growth and feed conversion. Livestock Research for Rural Development. Volume 30, Article #111. 
Sina, V., Preston, T. R. and Tham, T. H., 2017. Brewers’ grains have a synergistic effect on growth rate of goats fed fresh cassava foliage (Manihot esculenta Crantz) as basal diet. Livestock Research for Rural Development. Volume 29, Article #137. Retrieved August 16, 2018, from 
Thuy Hang, L. T., Preston T. R., Ba, N. X., and Dung, D. V., 2018. Digestibility, nitrogen balance and methane emissions in goats fed cassava foliage and restricted levels of brewers’ grains. Livestock Research for Rural Development. Volume 30, Article #68. Retrieved January 3, 2019, from  
Wanapat, M., Pimpa, O., Petlum, A. and Boontao. U., 1997. Cassava hay: A new strategic feed for ruminants during the dry season. Livestock Research for Rural Development. Volume 9, Article #18.
Wanapat, M., 1995. The use of local feed resources for livestock production in Thailand. Proceedings of the International Conference on Increasing Animal Production with Local Resources (Editor: Guo Tingshuang). China Forestry Publishing House, Ministry of Agriculture, China.
PUBLICATION LIST
Paper 1. Phuong L T B, Khang D N and Preston T R 2015: Methane production in an in vitro fermentation of cassava pulp with urea was reduced by supplementation with leaves from bitter, as opposed to sweet, varieties of cassava. Livestock Research for Rural Development. Volume 27, Article #162.  
 Paper 2. Binh P L T, Preston T R, Duong K N and Leng R A 2017: A low concentration (4% in diet dry matter) of brewers’ grains improves the growth rate and reduces thiocyanate excretion of cattle fed cassava pulp-urea and “bitter” cassava foliage. Livestock Research for Rural Development. Volume 29, Article #104.  
 Paper 3. Binh P L T, Preston T R, Van H N and Dinh V D 2018: Methane production in an in vitro rumen incubation of cassava pulp-urea with additives of brewers’ grain, rice wine yeast culture, yeast-fermented cassava pulp and leaves of sweet or bitter cassava variety. Livestock Research for Rural Development. Volume 30, Article #77.  
 Paper 4. Phuong L T B, Preston T R, Van N H and Dung D V 2019: Effect of additives (brewer’s grains and biochar) and cassava variety (sweet versus bitter) on nitrogen retention, thiocyanate excretion and methane production by Bach Thao goats. Livestock Research for Rural Development. Volume 31, Article #1. 
APPENDIX. PROTOCOL D1- DETERMINATION OF THIOCYANATE IN URINE
(Source: 
Components 
A. Protocol D1 instructions for thiocyanate analysis of urine. 
B. Thirty (30) flat-bottomed plastic bottles with screw capped lids. 
C. Three (3) graduated 1 ml plastic pipettes. 
D. Ten (10) white standard papers with thiocyanate equal to 10 ppm. 
E. One hundred (100) yellow indicator papers glued to strips of clear plastic. STORE IN FREEZER. Stable for one month only at room temperature. 
F. Colour chart with ten (10) shades of colour which correspond to 0-100 ppm thiocyanate. G. Bottle containing 0.5 g potassium permanganate. 
Method 
(Complete steps 4 to 7 quickly as the enzyme acts rapidly to release HCN) 
1. Urine samples should either be fresh or stored in the freezer for up to 6 months. 
2. Prepare a sulphuric acid solution, add 5.5 ml of concentrated (96%) sulphuric acid to 100 ml water in a beaker slowly with stirring. TAKE CARE! heat is produced do not add water to acid! 
3. Prepare a permanganate solution by dissolving 100 mg potassium permanganate in 5 ml water. This is stable for 2 months if stored at room temperature and away from direct sunlight. 
4. Follow sketch 1. Use plastic pipette to add 1.0 ml urine to a flat-bottomed plastic bottle followed by three (3) drops of sulphuric acid solution from a second plastic pipette and mix. 5. Add three (3) drops of permanganate solution from a third plastic pipette and mix gently. 6. IMMEDIATELY add a yellow indicator paper attached to a plastic strip so that the paper does not touch the liquid in the bottle. When not in use STORE INDICATOR PAPERS IN FREEZER. 
7. IMMEDIATELY close the bottle with a screw capped lid. The magenta colour should disappear. 
8. A positive and negative control should be run for each set of experiments. a. For a negative control, prepare another sample as shown in sketch 1 but with 1.0 ml water instead of urine. b. For a positive control follow sketch 2. Place a white standard paper disc in the bottle. Add 1.0 ml water, three drops of sulphuric acid solution, mix and add three drops of permanganate solution. IMMEDIATELY add the yellow indicator paper and IMMEDIATELY close the bottle with a screw capped lid and carefully mix to avoid wetting the paper. The magenta colour may remain for some time. (wash the pipettes thoroughly to remove urine, sulphuric acid and permanganate). 
9. Allow the bottles to stand for 16-24 hr at room temperature. 
10. Open the bottles and match the colour of the indicator papers against the shades of colour on the colour chart supplied. 
11. Read from the colour chart the thiocyanate content in ppm in the urine sample (see 16b). Check that the negative control is zero and that the positive control gives a colour of about 10 ppm. THIS SECTION TO BE FOLLOWED IF YOU HAVE A SPECTROPHOTOMETER 
12. For each sample, carefully remove the plastic backing sheet from the indicator paper. 
13. Place the paper in a test tube and add 5.0 ml of water measured accurately. 
14. Leave the test tube at room temperature for about 30 min with occasional gentle stirring. 15. Measure the absorbance at 510 nm of the solution, subtract the value of the negative control. 
16. The thiocyanate content in ppm is calculated by the equation1 a. thiocyanate content (ppm) = 78 x absorbance b. thiocyanate content in µmol/Lt = thiocyanate content (ppm) x 17.2 
17. The thiocyanate content obtained for the same sample of urine, from both measurements 11 and 16 should be about the same. Also check the standard value agrees using both methods. 
Troubleshooting 
The thiocyanate content of the white standard paper should be about 10 ppm. Possible problems could be:
 • If the indicator paper is left at room temperature it gradually becomes darker and after one month its colour will be around 1-2 ppm on the colour chart. 
• If the indicator paper has been left in bright sunlight it becomes bleached on one side and is no good. 
• Permanganate solution has decomposed which would give a low result. Make up a new solution. 
• Sulphuric acid solution was not made up properly or may be too dilute. Make up a new solution. 
• If you use a bottle which is not gas tight (e.g. the screw cap is cracked) then gas could escape and this would give a low result. 
• As stated in steps 6 & 7, it is important to add the indicator paper immediately after the permanganate and then immediately close the lid otherwise there will be a loss of gas which would give a low result. 
Reference 
Haque, R. and Bradbury, J. H., 1999. Simple kit method for determination of thiocyanate in urine. Clinical Chemistry, 45, 1459-1464. 
Correspondence 
Konzo Prevention Group, Research School of Biology, Australian National University, 46 Sullivans Creek Rd, Acton, ACT, 2601, Australia. Email: konzo@anu.edu.au. Web: