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Simultaneous colorimetric determination of thiosulphate and thiocyanate in the culture medium of Rhizopus oryzae
Journal o f Microbiological Methods 12 (1990) 189-195 Elsevier
189
MIMET 00401
Simultaneous colorimetric determination of thiosulphate and thiocyanate in the culture medium of Rhizopus oryzae R a m e s h C. Ray, B a l a g o p a l a n a n d G. P a d m a j a Central Tuber Crops Research Institute, Trivandrum, India (Received 12 February 1990; revision received 20 June 1990; accepted 13 August 1990)
Summary Thiosulphate and thiocyanate are two intermediate forms of S produced during oxidation of elemental S to sulphate in the culture medium of Rhizopus oryzae but no simple and specific method is available for determination of microgram quantities of these S forms in culture filtrates. A simple, rapid and precise colorimetric method is described for the simultaneous determination of thiocyanate and thiosulphate. The method is based on the cyanolytic reduction of thiosulphate in presence of Cu 2÷ to tbiocyanate under alkaline conditions and determining the thiocyanate formed and/or present in the medium by reacting with ferric nitrate under acidic (HNO3) conditions which gives a reddish-brown colour. The colour intensity is measured spectrophotometrically at 460 nm. The method is rapid, sensitive and accurate and it permits determination of microgram quantities of thiocyanate or thiosulphate or both. The method was applied for the determination of thiosulphate and thiocyanate in the culture medium of R. oryzae amended with elemental S. Accumulation of thiosulphate and thiocyanate progressed for up to 6 days of incubation and thereafter declined, possibly due to conversion into sulphate and/or utilization in cell metabolism.
Key words: Rhizopus oryzae; S oxidation; Thiocyanate; Thiosulphate
Introduction There is a growing interest in the biochemistry of S transformations by microorganisms [1] but a lack of sensitive and accurate methods for simultaneous determination of various intermediate S compounds have hampered the progress in this field. In an earlier work, we have reported that a mucoraceous fungus, Rhizopus oryzae isolated from rotten cassava tubers possesses rhodanese (thiosulphate:cyanide sulphurtransferase, EC 2.8.1.1) [2 - 3], an enzyme implicated in S oxidation in soil [4 - 5] and microorganisms [6]. Some preliminary studies with this fungus have shown that it is capable Correspondence to: R.C. Ray, Scientist S-2 (Microbiology), Regional Centre of Central Tuber Crops Research Institute, Indian Council of Agricultural Research, N-1/224, Nayapalli, Bhubaneswar-751012, India.
0167-7012/90/$ 3.50 © 1990 Elsevier Science Publishers BN. (Biomedical Division)
190 of oxidizing elemental S and produces thiosulphate (82 O2- ) and thiocyanate (SCN - ) as intermediate S compounds. These studies presumed that the fungus could possibly be utilized as a microbial inoculant in rectifying S-deficient soils where the process of S oxidation is either slow or non-existent. Several methods are available for the individual determination of $2O2- [ 7 - 9] and S C N - [10] but no simple and accurate method is available for determining microgram quantities of these S forms when present in combination. The methylene blue method of Johnson and Nishita [8] is sensitive but the reagents used for the reduction of $2O2- or S C N - also reduce the higher valence states of S such as SO~- and polythionates. Likewise, the iodimetric method has its inherent problem, for example, some organic S compounds and other reduced S forms (tetrathionate, hydrosulphite, etc.) will also decompose during acidification leading to erratic results in determining $20 ~- or S C N - [9]. The method described by Nor and Tabatabai [9] is somewhat sensitive for determination of $2O2- in microgram quantities but it does not account for the microgram amount of S C N - present in the medium as is the case with R. oryzae and other biological systems [11]. This paper describes a chemical method for simultaneous determination of $20 ~- and S C N - in the culture medium of R. oryzae. The method is based on colorimetric determination of: (1) $2O2- - by alkaline cyanolysis of $2O2- in the presence of CU2+: Cu2+ $20 ~- + C N - catalyst ) $20~ + S C N - . (2) S C N - - by directly reacting S C N - with Fe (NO3) 3 under acidic (HNO3) conditions, thus forming F e - S C N complex: S C N - + Fe 3+ ~ F e - SCN complex. Materials and Methods
R. oryzae and growth conditions The fungus R. oryzae isolated from rotten cassava tubers was used in this study. Spore suspensions of R. oryzae were prepared from 6 days-old cultures grown at room temperature (30+ 2 °C) on potato dextrose ( P D ) - a g a r slants. Spores were karvested in sterile distilled water, washed and diluted to a concentration of 6 x 107 spores, m l - ~. The same concentration was used throughout the study. R. oryzae and S oxidation To study S oxidation by R. oryzae, a spore suspension of the fungus was inoculated into 250-ml Erlenmeyer flasks containing 100 ml PD broth and 50 mg elemental S. Controls were maintained with 100 ml PD broth in 250-ml Erlenmeyer flasks inoculated with spores but without elemental S. Likewise, blanks were maintained with PD broth with elemental S but without spores being inoculated. After 3, 6 and 9 days of incubation at room temperature (30+2 °C), triplicate flasks were assayed for $2O2- , S C N - and SO42-.
191
Determination of S C N - and S2&3Reagents The following reagents were used: (1) Na2S203 stock solution (1 mg $2O 2 - - S •ml-1); (2) KSCN stock solution (1 ml S C N - - S . ml-1); (3) KCN solution (0.125 M); (4) CuC12 solution (0.05 M); (5) Fe (NO3) 3 - H N O 3 solution (0.25 M Fe(NO3) 3.9 H20 : 3.1 M HNO3); (6) deionized distilled water. Procedure Fungal cell mass was determined by filtering cultures through Whatman No. 42 filter-paper and drying overnight at 80 °C. The solutions were filtered twice and the clear filtrate was used for assay of S C N - , $20 ]- and SO 2- . Triplicate cultures were maintained for each treatment and the values reported were an average of the assays. S C N - - S. To determine the S C N - content, an 1- 20-ml aliquot of the culture filtrate containing 2 5 - 2 0 0 #g S as S C N - was taken to 25-ml flasks and treated with 1 ml Fe(NO3) 3 - H N O 3 reagent. In a separate experiment, it was observed that the highest S C N - - S concentration acceptable to keep the linear response of the test should not exceed that of 200/zg S C N - - S standard and samples with higher S C N - concentrations were further diluted. The contents were mixed and the volume was made to 25 ml with water. After thorough mixing, the absorbance of the reddish-brown colour of the F e - SCN complex was measured at 460 nm. A blank was run by taking water and 1 ml Fe(NO3)2 - H N O 3 reagent and the volume was adjusted to 25 ml. The S C N - content was calculated by reference to a standard thiocyanate curve. $20~3- - S . To determine the $2032- content, an 1-20-ml aliquot of the culture filtrate containing 2 5 - 2 5 0 #g S as $2O2- was taken to 25-ml flasks and was added with 1 ml 0.125 M KCN. After mixing and incubation for 20 min, 2.5 ml 0.05 M CuC12 solution was added and the volume was made to 25 ml with water and then proceeded as described for analysis of S C N - in culture filtrate. To quantify the $2032- content, either of the following two procedures could be adopted. (1) The blanks could be run by taking the same amount of the aliquots (as the test samples) in 25-ml flasks and were added with 1 ml Fe(NO3) 3 - HNO3 reagent. This would delete the S C N - content, if any, already present in the mixture and give values only for $2032- - S which can be calculated by reference to a standard thiosulphate curve• This procedure is suitable when only small number of samples are to be analysed because for each sample 100°70 transmission of light must be adjusted• (2) Alternately, the blanks could be run by taking water and 1 ml Fe(NO3)3 - HNO3 reagent. The reddish-brown colour of the F e - SCN complex formed from the test samples is quantified using the standard curve for S C N - - S , then subtract the F e - S C N formed only due to S C N - , if any, present in the medium. The residual S C N - would account for the cyanolytic reduction of $202- to S C N - . Theoretically, when 1 tool thiosulphate undergoes cyanolysis in the presence of Cu 2+ , 1 mol thiocyanate is produced that contains 50o7o of the amount of S present in the thiosulphate (Reaction 1) [ 7 - 9 ] . Therefore, a factor of 2 is used for the residual
192 S C N - values in order to calculate the quantities of thiosulphate present in samples containing thiocyanate. However, in practice this is never achieved when analysing thiosulphate by this method and a factor for calculating the quantities of thiosulphate is 1.75. This procedure is appropriate when large numbers of samples are to be analysed.
Standard curves SCN- -S. The S C N - content of the aliquot was determined by reference to a standard curve prepared with standards containing 0, 50, 100, 150 and 200 t~g S C N - S .
$20~3- -S. The $2O2- - S content of the aliquot was determined by reference to a standard curve prepared with standards containing 0, 50, 100, 150, 200 and 250/~g $2O2- -- S.
S0~4- determination SO 2- - S in the culture filtrate was determined by turbidimetry [12]. Results and Discussion
The methods developed for the determination of S C N - and $2O2- are based on the measurement of colour intensity of the F e - SCN complex formed from the reaction of Fe 3+ with S C N - under aci~lic (HNO3) conditions. The methods are based on systematic studies of factors affecting F e - S C N complex at room temperature, i.e., amount and concentration of KCN, CuCI 2 and Fe(NO3) 3 - H N O 3 reagent. The absorption spectra of the F e - SCN complex formed when standard S C N - or $2O2- solutions were analysed by the method described were studied. The optimum absorption peak was found at 460 nm which is similar to that reported by Aldrige [10] for determination of S C N - and by Nor and Tabatabai [9] for determination of $2O2 . The reddish-brown colour of F e - S C N complex was found to be stable up to 15 min in laboratory light and 1 h in the dark. The fading of F e - SCN colour has been long recognized as one of the limitations of the colorimetric SCN method for determining Fe [13] which is believed to be due to the reaction between H N O 3 and S C N - a n d is promoted by the presence of Fe 3+ ions. The results reported in this paper were obtained by measuring the colour intensity within 10 min after final adjustment of the volume. Figs. 1 and 2 show typical standard curves obtained by the methods for S C N - and $2O2- , respectively. There are linear relationships between absorbance and the concentration of S C N - (up to 200 #g S) and $2O2- (up to 250/~g S) which are in accordance with Beer's law. The standard curves are reproducible. The method described has been tested to determine the SzO2- content in a mixture of $2O2- and S C N - (100 #g S) and the results are shown in Table 1. It shows that nearly 98°70 of the initial $202- - S or S C N - - S content could be estimated by the method described in all the cases. Thus, the method appears to be precise and accurate. The data reported here were obtained by determining these S forms by reference to the standard curves (Figs. 1, 2).
193
08
06
0.4
0.2
o
~o
4o SCN
Fig. I.
-s
~o
~oo
(pg)
Standard curve for S C N - - S by method described.
Because this method was developed for analyses of organic/synthetic microbial culture media, we studied the effects of various cations (Ca 2+, Mg 2+, Na +, K + and Zn 2+ as sulphate salts at 0.1o70 level) and anions ( N O r , N O r and C1- as K salt at 0.1°70 level) as well as some organic compounds, viz., peptone, yeast extract and amino acids (cysteine and methionine) individually at 1°70 level on the determination of S C N - and $2 O2- . The solutions which were to be analysed by the method described
0.4
E
c ©
c
03
02
o
<~ 01
50
Fig. 2.
100 2$203 - S
150
200
250
(..ug)
Standard curve for $2 O2- - S by method described.
194 TABLE 1 PRECISION OF METHOD DEVELOPED Forms of S
Incubation condition
S added 0zg .ml 1)
S recovered* (#g.ml l) Range
~
SD
SCN- - S
_+100 tzg-ml i $2O2 S
50 100 200
49.0- 50.6 98.0- 101.2 198.0- 199.6
50.2 99.5 199.0
0.75 0.57 0.83
$20 ~ - S
_+100 #g.ml 1 SCN- - S
50 100 200
48.5- 49.5 96.2 99.5 194.4- 198.5
49.0 98.2 198.0
0.52 0.77 0.78
* Six analyses. were m a d e to c o n t a i n these salts or organic c o m p o u n d s in a d d i t i o n to 100 tzg S as S C N - or $2 O2 - . T h e tests indicated that these substances have little effect as the estim a t i o n o f 100 #g S C N - - S or $2 O2- - S was q u a n t i t a t i v e (<98°70).
Cell m a s s a n d S oxidation by R. o r y z a e E arl i er studies have s h o w n that the fungus R. oryzae p r o d u c e s extracellular r h o d a n e s e [ 2 - 3] a n d its role in d e t o x i f i c a t i o n o f cyanide to S C N - has been explained [2]. Since r h o d a n e s e has m u l t i p l e roles in b i o s p h e r e [14], we have u n d e r t a k e n prelimin a r y studies to find o u t o t h e r possible roles o f r h o d a n e s e activity by R. oryzae, especially in relation to S o x i d a t i o n . Table 2 shows the fungus g r o w t h (cell mass) a nd different f o r m s o f S a c c u m u l a t e d in P D b r o t h as a result o f S o x i d a t i o n by R. oryzae. Th er e was an increase in g r o w t h (cell mass) up to 6 days o f i n c u b a t i o n after w h i c h g r o w t h r e m a i n e d static an d sporulat i o n h ad initiated. O u r earlier studies with the fungus also exhibited a similar trend w h e n g ro wn in P D b r o t h [3]. However, sulphate a c c u m u l a t e d in m e d i u m linearly with i n c u b a t i o n t i m e whereas the c o n c e n t r a t i o n s o f $203z- an d S C N - showed g r ad u al inTABLE 2 CELL MASS AND AMOUNT OF $20 ~- - S, SCN- - S and SO~- - S PRODUCED DURING OXIDATION OF ELEMENTAL S BY R. OR YZAE IN PD BROTH Variable measured
Cell mass (mg + SD) Forms of S (tzg.ml - l + SD) $202--S SCN--S SO2 - S * Results of four replicate analyses.
Incubation 3 days
6 days
9 days
158 _+12
306 _+14
285 + 12
16_+ 1.3 100_+ 2.6 30_+ 2.6
32_+ 1.5 253_+ 4.0 62_+ 4.0
18_+ 1.6 78_+ 1.8 143_+ 8.5
195 crease up to 6 days, thereafter, a decline in the concentration was observed suggesting their conversion in part to sulphate and/or utilization in cell metabolism.
Acknowledgements We thank G.G. Nayar, Director of the Central Tuber Crops Research Institute, for providing facilities and encouragement.
References 1 Wainwright, M. (1984) Sulphur oxidation in Soils. Adv. Agron. 37, 350-396. 2 Padmaja, G. and Balagopalan, C. (1985) Cyanide degradation by Rhizopus oryzae. Can. J. Microbiol. 31, 663-669. 3 Ray, R.C., Padmaja, G. and Balagopalan, C. (1990) ExtraceUular rhodanese production by Rhizopus oryzae. Zentralbl. Mikrobiol. 145, 259-268. 4 Tabatabai, M. A. and Singh, B. B. (1976) Rhodanese activity of soils. Soil Sci. Soc. Am. J. 40, 381 - 385. 5 Ray, R. C., Behera, N. and Sethunathan, N. (1985) Rhodanese activity of flooded and non-flooded soils. Soil Biol. Biochem. 17, 159-162. 6 Lettl, A. (1983) Occurrence of thiosulphate sulphurtransferase producers in the population of mesophillic heterotrophic bacteria and microfungi of spruce humus. Folia Microbiol. (Prague) 28, 106-111. 7 Sorbo, B. (1957) A colorimetric method for the determination of thiosulphate. Biochim. Biophys. Acta 23, 412-416. 8 Johnson, C. M. and Nishita, H. (1952) Microestimation of sulphur in plant materials, soils and irrigation waters. Anal. Chem. 24, 736-742. 9 Nor, Y. M. and Tabatabai, M.A. (1976) Extraction and colorimetric determination of thiosulphate and tetrathionate in soils. Soil Sci. 122, 171-178. 10 Aldrige, W. N. (1944) A new method for the estimation of microquantities of cyanide and thiocyanate. Analyst 69, 262-265. 11 Solomonson, L.P. (1981) Cyanide as a metabolic inhibitor. In: Cyanide in Biology (Vennesland, B., Conn, E.E., Knowles, C.J., Westley, J. and Wissing, E, eds.), pp. 11-28, Academic Press, London. 12 Massoumi, A. and Cornfield, A.H. (1963) A rapid method for determining sulphate in water extracts of soils. Analyst 88, 321-322. 13 Sandell, E.B. (1950) Colorimetric Determination of Traces of Metals, Second Edition, p. 367, Interscience, New York. 14 Volini, M. and Alexander, K. (1981) Multiple forms and multiple functions of the rhodaneses. In: Cyanide in Biology (Vennesland, B., Corm, E.E., Knowles, C.J., Westley, J. and Wissing, E, eds.), pp. 77-92, Academic Press, London.
189
MIMET 00401
Simultaneous colorimetric determination of thiosulphate and thiocyanate in the culture medium of Rhizopus oryzae R a m e s h C. Ray, B a l a g o p a l a n a n d G. P a d m a j a Central Tuber Crops Research Institute, Trivandrum, India (Received 12 February 1990; revision received 20 June 1990; accepted 13 August 1990)
Summary Thiosulphate and thiocyanate are two intermediate forms of S produced during oxidation of elemental S to sulphate in the culture medium of Rhizopus oryzae but no simple and specific method is available for determination of microgram quantities of these S forms in culture filtrates. A simple, rapid and precise colorimetric method is described for the simultaneous determination of thiocyanate and thiosulphate. The method is based on the cyanolytic reduction of thiosulphate in presence of Cu 2÷ to tbiocyanate under alkaline conditions and determining the thiocyanate formed and/or present in the medium by reacting with ferric nitrate under acidic (HNO3) conditions which gives a reddish-brown colour. The colour intensity is measured spectrophotometrically at 460 nm. The method is rapid, sensitive and accurate and it permits determination of microgram quantities of thiocyanate or thiosulphate or both. The method was applied for the determination of thiosulphate and thiocyanate in the culture medium of R. oryzae amended with elemental S. Accumulation of thiosulphate and thiocyanate progressed for up to 6 days of incubation and thereafter declined, possibly due to conversion into sulphate and/or utilization in cell metabolism.
Key words: Rhizopus oryzae; S oxidation; Thiocyanate; Thiosulphate
Introduction There is a growing interest in the biochemistry of S transformations by microorganisms [1] but a lack of sensitive and accurate methods for simultaneous determination of various intermediate S compounds have hampered the progress in this field. In an earlier work, we have reported that a mucoraceous fungus, Rhizopus oryzae isolated from rotten cassava tubers possesses rhodanese (thiosulphate:cyanide sulphurtransferase, EC 2.8.1.1) [2 - 3], an enzyme implicated in S oxidation in soil [4 - 5] and microorganisms [6]. Some preliminary studies with this fungus have shown that it is capable Correspondence to: R.C. Ray, Scientist S-2 (Microbiology), Regional Centre of Central Tuber Crops Research Institute, Indian Council of Agricultural Research, N-1/224, Nayapalli, Bhubaneswar-751012, India.
0167-7012/90/$ 3.50 © 1990 Elsevier Science Publishers BN. (Biomedical Division)
190 of oxidizing elemental S and produces thiosulphate (82 O2- ) and thiocyanate (SCN - ) as intermediate S compounds. These studies presumed that the fungus could possibly be utilized as a microbial inoculant in rectifying S-deficient soils where the process of S oxidation is either slow or non-existent. Several methods are available for the individual determination of $2O2- [ 7 - 9] and S C N - [10] but no simple and accurate method is available for determining microgram quantities of these S forms when present in combination. The methylene blue method of Johnson and Nishita [8] is sensitive but the reagents used for the reduction of $2O2- or S C N - also reduce the higher valence states of S such as SO~- and polythionates. Likewise, the iodimetric method has its inherent problem, for example, some organic S compounds and other reduced S forms (tetrathionate, hydrosulphite, etc.) will also decompose during acidification leading to erratic results in determining $20 ~- or S C N - [9]. The method described by Nor and Tabatabai [9] is somewhat sensitive for determination of $2O2- in microgram quantities but it does not account for the microgram amount of S C N - present in the medium as is the case with R. oryzae and other biological systems [11]. This paper describes a chemical method for simultaneous determination of $20 ~- and S C N - in the culture medium of R. oryzae. The method is based on colorimetric determination of: (1) $2O2- - by alkaline cyanolysis of $2O2- in the presence of CU2+: Cu2+ $20 ~- + C N - catalyst ) $20~ + S C N - . (2) S C N - - by directly reacting S C N - with Fe (NO3) 3 under acidic (HNO3) conditions, thus forming F e - S C N complex: S C N - + Fe 3+ ~ F e - SCN complex. Materials and Methods
R. oryzae and growth conditions The fungus R. oryzae isolated from rotten cassava tubers was used in this study. Spore suspensions of R. oryzae were prepared from 6 days-old cultures grown at room temperature (30+ 2 °C) on potato dextrose ( P D ) - a g a r slants. Spores were karvested in sterile distilled water, washed and diluted to a concentration of 6 x 107 spores, m l - ~. The same concentration was used throughout the study. R. oryzae and S oxidation To study S oxidation by R. oryzae, a spore suspension of the fungus was inoculated into 250-ml Erlenmeyer flasks containing 100 ml PD broth and 50 mg elemental S. Controls were maintained with 100 ml PD broth in 250-ml Erlenmeyer flasks inoculated with spores but without elemental S. Likewise, blanks were maintained with PD broth with elemental S but without spores being inoculated. After 3, 6 and 9 days of incubation at room temperature (30+2 °C), triplicate flasks were assayed for $2O2- , S C N - and SO42-.
191
Determination of S C N - and S2&3Reagents The following reagents were used: (1) Na2S203 stock solution (1 mg $2O 2 - - S •ml-1); (2) KSCN stock solution (1 ml S C N - - S . ml-1); (3) KCN solution (0.125 M); (4) CuC12 solution (0.05 M); (5) Fe (NO3) 3 - H N O 3 solution (0.25 M Fe(NO3) 3.9 H20 : 3.1 M HNO3); (6) deionized distilled water. Procedure Fungal cell mass was determined by filtering cultures through Whatman No. 42 filter-paper and drying overnight at 80 °C. The solutions were filtered twice and the clear filtrate was used for assay of S C N - , $20 ]- and SO 2- . Triplicate cultures were maintained for each treatment and the values reported were an average of the assays. S C N - - S. To determine the S C N - content, an 1- 20-ml aliquot of the culture filtrate containing 2 5 - 2 0 0 #g S as S C N - was taken to 25-ml flasks and treated with 1 ml Fe(NO3) 3 - H N O 3 reagent. In a separate experiment, it was observed that the highest S C N - - S concentration acceptable to keep the linear response of the test should not exceed that of 200/zg S C N - - S standard and samples with higher S C N - concentrations were further diluted. The contents were mixed and the volume was made to 25 ml with water. After thorough mixing, the absorbance of the reddish-brown colour of the F e - SCN complex was measured at 460 nm. A blank was run by taking water and 1 ml Fe(NO3)2 - H N O 3 reagent and the volume was adjusted to 25 ml. The S C N - content was calculated by reference to a standard thiocyanate curve. $20~3- - S . To determine the $2032- content, an 1-20-ml aliquot of the culture filtrate containing 2 5 - 2 5 0 #g S as $2O2- was taken to 25-ml flasks and was added with 1 ml 0.125 M KCN. After mixing and incubation for 20 min, 2.5 ml 0.05 M CuC12 solution was added and the volume was made to 25 ml with water and then proceeded as described for analysis of S C N - in culture filtrate. To quantify the $2032- content, either of the following two procedures could be adopted. (1) The blanks could be run by taking the same amount of the aliquots (as the test samples) in 25-ml flasks and were added with 1 ml Fe(NO3) 3 - HNO3 reagent. This would delete the S C N - content, if any, already present in the mixture and give values only for $2032- - S which can be calculated by reference to a standard thiosulphate curve• This procedure is suitable when only small number of samples are to be analysed because for each sample 100°70 transmission of light must be adjusted• (2) Alternately, the blanks could be run by taking water and 1 ml Fe(NO3)3 - HNO3 reagent. The reddish-brown colour of the F e - SCN complex formed from the test samples is quantified using the standard curve for S C N - - S , then subtract the F e - S C N formed only due to S C N - , if any, present in the medium. The residual S C N - would account for the cyanolytic reduction of $202- to S C N - . Theoretically, when 1 tool thiosulphate undergoes cyanolysis in the presence of Cu 2+ , 1 mol thiocyanate is produced that contains 50o7o of the amount of S present in the thiosulphate (Reaction 1) [ 7 - 9 ] . Therefore, a factor of 2 is used for the residual
192 S C N - values in order to calculate the quantities of thiosulphate present in samples containing thiocyanate. However, in practice this is never achieved when analysing thiosulphate by this method and a factor for calculating the quantities of thiosulphate is 1.75. This procedure is appropriate when large numbers of samples are to be analysed.
Standard curves SCN- -S. The S C N - content of the aliquot was determined by reference to a standard curve prepared with standards containing 0, 50, 100, 150 and 200 t~g S C N - S .
$20~3- -S. The $2O2- - S content of the aliquot was determined by reference to a standard curve prepared with standards containing 0, 50, 100, 150, 200 and 250/~g $2O2- -- S.
S0~4- determination SO 2- - S in the culture filtrate was determined by turbidimetry [12]. Results and Discussion
The methods developed for the determination of S C N - and $2O2- are based on the measurement of colour intensity of the F e - SCN complex formed from the reaction of Fe 3+ with S C N - under aci~lic (HNO3) conditions. The methods are based on systematic studies of factors affecting F e - S C N complex at room temperature, i.e., amount and concentration of KCN, CuCI 2 and Fe(NO3) 3 - H N O 3 reagent. The absorption spectra of the F e - SCN complex formed when standard S C N - or $2O2- solutions were analysed by the method described were studied. The optimum absorption peak was found at 460 nm which is similar to that reported by Aldrige [10] for determination of S C N - and by Nor and Tabatabai [9] for determination of $2O2 . The reddish-brown colour of F e - S C N complex was found to be stable up to 15 min in laboratory light and 1 h in the dark. The fading of F e - SCN colour has been long recognized as one of the limitations of the colorimetric SCN method for determining Fe [13] which is believed to be due to the reaction between H N O 3 and S C N - a n d is promoted by the presence of Fe 3+ ions. The results reported in this paper were obtained by measuring the colour intensity within 10 min after final adjustment of the volume. Figs. 1 and 2 show typical standard curves obtained by the methods for S C N - and $2O2- , respectively. There are linear relationships between absorbance and the concentration of S C N - (up to 200 #g S) and $2O2- (up to 250/~g S) which are in accordance with Beer's law. The standard curves are reproducible. The method described has been tested to determine the SzO2- content in a mixture of $2O2- and S C N - (100 #g S) and the results are shown in Table 1. It shows that nearly 98°70 of the initial $202- - S or S C N - - S content could be estimated by the method described in all the cases. Thus, the method appears to be precise and accurate. The data reported here were obtained by determining these S forms by reference to the standard curves (Figs. 1, 2).
193
08
06
0.4
0.2
o
~o
4o SCN
Fig. I.
-s
~o
~oo
(pg)
Standard curve for S C N - - S by method described.
Because this method was developed for analyses of organic/synthetic microbial culture media, we studied the effects of various cations (Ca 2+, Mg 2+, Na +, K + and Zn 2+ as sulphate salts at 0.1o70 level) and anions ( N O r , N O r and C1- as K salt at 0.1°70 level) as well as some organic compounds, viz., peptone, yeast extract and amino acids (cysteine and methionine) individually at 1°70 level on the determination of S C N - and $2 O2- . The solutions which were to be analysed by the method described
0.4
E
c ©
c
03
02
o
<~ 01
50
Fig. 2.
100 2$203 - S
150
200
250
(..ug)
Standard curve for $2 O2- - S by method described.
194 TABLE 1 PRECISION OF METHOD DEVELOPED Forms of S
Incubation condition
S added 0zg .ml 1)
S recovered* (#g.ml l) Range
~
SD
SCN- - S
_+100 tzg-ml i $2O2 S
50 100 200
49.0- 50.6 98.0- 101.2 198.0- 199.6
50.2 99.5 199.0
0.75 0.57 0.83
$20 ~ - S
_+100 #g.ml 1 SCN- - S
50 100 200
48.5- 49.5 96.2 99.5 194.4- 198.5
49.0 98.2 198.0
0.52 0.77 0.78
* Six analyses. were m a d e to c o n t a i n these salts or organic c o m p o u n d s in a d d i t i o n to 100 tzg S as S C N - or $2 O2 - . T h e tests indicated that these substances have little effect as the estim a t i o n o f 100 #g S C N - - S or $2 O2- - S was q u a n t i t a t i v e (<98°70).
Cell m a s s a n d S oxidation by R. o r y z a e E arl i er studies have s h o w n that the fungus R. oryzae p r o d u c e s extracellular r h o d a n e s e [ 2 - 3] a n d its role in d e t o x i f i c a t i o n o f cyanide to S C N - has been explained [2]. Since r h o d a n e s e has m u l t i p l e roles in b i o s p h e r e [14], we have u n d e r t a k e n prelimin a r y studies to find o u t o t h e r possible roles o f r h o d a n e s e activity by R. oryzae, especially in relation to S o x i d a t i o n . Table 2 shows the fungus g r o w t h (cell mass) a nd different f o r m s o f S a c c u m u l a t e d in P D b r o t h as a result o f S o x i d a t i o n by R. oryzae. Th er e was an increase in g r o w t h (cell mass) up to 6 days o f i n c u b a t i o n after w h i c h g r o w t h r e m a i n e d static an d sporulat i o n h ad initiated. O u r earlier studies with the fungus also exhibited a similar trend w h e n g ro wn in P D b r o t h [3]. However, sulphate a c c u m u l a t e d in m e d i u m linearly with i n c u b a t i o n t i m e whereas the c o n c e n t r a t i o n s o f $203z- an d S C N - showed g r ad u al inTABLE 2 CELL MASS AND AMOUNT OF $20 ~- - S, SCN- - S and SO~- - S PRODUCED DURING OXIDATION OF ELEMENTAL S BY R. OR YZAE IN PD BROTH Variable measured
Cell mass (mg + SD) Forms of S (tzg.ml - l + SD) $202--S SCN--S SO2 - S * Results of four replicate analyses.
Incubation 3 days
6 days
9 days
158 _+12
306 _+14
285 + 12
16_+ 1.3 100_+ 2.6 30_+ 2.6
32_+ 1.5 253_+ 4.0 62_+ 4.0
18_+ 1.6 78_+ 1.8 143_+ 8.5
195 crease up to 6 days, thereafter, a decline in the concentration was observed suggesting their conversion in part to sulphate and/or utilization in cell metabolism.
Acknowledgements We thank G.G. Nayar, Director of the Central Tuber Crops Research Institute, for providing facilities and encouragement.
References 1 Wainwright, M. (1984) Sulphur oxidation in Soils. Adv. Agron. 37, 350-396. 2 Padmaja, G. and Balagopalan, C. (1985) Cyanide degradation by Rhizopus oryzae. Can. J. Microbiol. 31, 663-669. 3 Ray, R.C., Padmaja, G. and Balagopalan, C. (1990) ExtraceUular rhodanese production by Rhizopus oryzae. Zentralbl. Mikrobiol. 145, 259-268. 4 Tabatabai, M. A. and Singh, B. B. (1976) Rhodanese activity of soils. Soil Sci. Soc. Am. J. 40, 381 - 385. 5 Ray, R. C., Behera, N. and Sethunathan, N. (1985) Rhodanese activity of flooded and non-flooded soils. Soil Biol. Biochem. 17, 159-162. 6 Lettl, A. (1983) Occurrence of thiosulphate sulphurtransferase producers in the population of mesophillic heterotrophic bacteria and microfungi of spruce humus. Folia Microbiol. (Prague) 28, 106-111. 7 Sorbo, B. (1957) A colorimetric method for the determination of thiosulphate. Biochim. Biophys. Acta 23, 412-416. 8 Johnson, C. M. and Nishita, H. (1952) Microestimation of sulphur in plant materials, soils and irrigation waters. Anal. Chem. 24, 736-742. 9 Nor, Y. M. and Tabatabai, M.A. (1976) Extraction and colorimetric determination of thiosulphate and tetrathionate in soils. Soil Sci. 122, 171-178. 10 Aldrige, W. N. (1944) A new method for the estimation of microquantities of cyanide and thiocyanate. Analyst 69, 262-265. 11 Solomonson, L.P. (1981) Cyanide as a metabolic inhibitor. In: Cyanide in Biology (Vennesland, B., Conn, E.E., Knowles, C.J., Westley, J. and Wissing, E, eds.), pp. 11-28, Academic Press, London. 12 Massoumi, A. and Cornfield, A.H. (1963) A rapid method for determining sulphate in water extracts of soils. Analyst 88, 321-322. 13 Sandell, E.B. (1950) Colorimetric Determination of Traces of Metals, Second Edition, p. 367, Interscience, New York. 14 Volini, M. and Alexander, K. (1981) Multiple forms and multiple functions of the rhodaneses. In: Cyanide in Biology (Vennesland, B., Corm, E.E., Knowles, C.J., Westley, J. and Wissing, E, eds.), pp. 77-92, Academic Press, London.