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Mineralization of soil sulfur and its relation to soil carbon : nitrogen and phosphorus Kowalenko, Charles Grant 1973

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MINERALIZATION OF SOIL SULFUR AND ITS RELATION TO SOIL CARBON, NITROGEN AND PHOSPHORUS by CHARLES GRANT KOWALENKO B.S.A., University of Saskatchewan (Saskatoon), 196? M.Sc, University of Saskatchewan (Saskatoon), 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of S o i l Science We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July 19 7 3 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f S o i l Science  The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, C a n a d a Abstract The mineralization of s o i l s u l f u r was studied p a r t i c u l a r l y i n r e l a t i o n to s o i l carbon, nitrogen and phosphorus. The investigation involved two aspects; an examination of pertinent methods and a consideration of the processes involved i n s o i l s u l f u r mineralization using an incubation procedure. The examination of pertinent methods involved both a n a l y t i c a l and incubation techniques. The bismuth s u l f i d e colorimetric f i n i s h was shown to be a suitable alternative to the methylene blue f i n i s h i n the hydriodic acid reduction method of quantifying s u l f a t e . A chromotropic acid n i t r a t e analysis method was shown to provide several advantages over the phenoldisulfonic acid method. The aerobic status of a p a r t i c u l a r closed incubation system was evaluated by comparing mineralization r e s u l t s with an open incubation system. The nature of the reagent, the s o i l to solution r a t i o and a i r drying were evaluated for t h e i r influence on using extractable sulfate as a measure of s o i l s u l f u r mineralization during an incubation study. U t i l i z a t i o n of data not considering these influences were c r i t i c a l l y discussed and several recommendations were made. The established techniques were used to examine the mineralization of su l f u r p a r t i c u l a r l y i n r e l a t i o n to other s o i l parameters. Relatively close correlations were found among calcium chloride extractable sulfate values and carbon dioxide evolved, nitrogen mineralized, and s o i l pH through an incubation over 14 weeks at 3 0°C and 10 0 cm water tension. Poorer correlations of calcium chloride extract-able sulfate with both sodium bicarbonate extractable phos-phate and ary l s u l f a t a s e a c t i v i t y were found. Relationships between t o t a l phospholipid phosphorus and su l f u r mineralized were r e l a t i v e l y c l o s e , however, the reason for t h i s remained unclear. It appeared that phospholipids may be an important phosphorus or carbon source for microorganisms. Relationships among carbon, nitrogen and sulfur mineralization were discussed. The form and d i s t r i b u t i o n of s u l f u r com-pounds , whether Hl-reducible S or C-bonded S was indicated as being an important consideration i n mineralization studies. Attempts at separating organically bound sulfate from inorganic sulfate i n phosphate buffer and bicarbonate extracts were la r g e l y unsatisfactory, however, s i g n i f i c a n t amounts of the organic form were shown to be present. It was suggested that the in c l u s i o n of organic sulfate i n - i v -phosphate b u f f e r , bicarbonate and probably acetate extract analyses contributed to poor correlations with microbial a c t i v i t y . An incubation experiment using two s o i l samples, treated with f i v e nitrogen sources at four rates was conducted to study the influence of nitrogen additions on s u l f u r mineralization. The analyses were done after eight weeks incubation using the previously established methods. Simple, p a r t i a l and multiple correlations showed that the sulfate mineralized was influenced by nitrogen applications through the l a t t e r ' s e f f e c t on microbial a c t i v i t y and the r e s u l t i n g s o i l pH. An analysis of variance using a f a c t o r i a l design showed the following very highly s i g n i f i c a n t i n t e r a c t i o n s : nitrogen source with r a t e ; nitrogen source with s o i l ; rate with s o i l ; and s o i l , source and rate a l l together. Hence, the nitrogen source had an influence on microbial a c t i v i t y and microbial environment which i n turn influenced the s o i l s u l f u r mineralized. This e f f e c t varied with the s o i l sample, and nitrogen source and r a t e . The possible implications that nitrogen additions may have on plant-available s o i l sulfur and areas for further research were b r i e f l y discussed. - V -TABLE OF CONTENTS Page INTRODUCTION 1 CHAPTER 1 OBSERVATIONS ON THE BISMUTH SULFIDE COLORIMETRIC PROCEDURE FOR SULFATE ANALYSIS IN SOIL 5 INTRODUCTION 5 METHODS AND MATERIALS . . . . 6 RESULTS AND DISCUSSION 7 Effect of Nitrogen Gas Flow Rate . . 7 Time of D i s t i l l a t i o n 8 E f f e c t of Predrying Samples 10 E f f e c t of Sample Nitrate Content . . 11 Comparison of the Bismuth and Methylene Blue Results on S o i l Materials . . . 11 Microdeterminations Using the Bismuth Method r - 7 ~ 7 ~ 7 ~ 7 ~ 7 _ r ~ r - 7 ~ r - 7 ~ r . . . 12 REFERENCES 14 CHAPTER 2 DETERMINATION OF NITRATES IN SOIL EXTRACTS 15 REFERENCES 19 CHAPTER 3 EVALUATION OF VARIOUS SULFATE EXTRACTANTS AND A CLOSED INCUBATION METHOD FOR STUDYING SOIL SULFUR MINERALIZATION . . . 20 INTRODUCTION 20 METHODS AND MATERIALS 21 Soils 21 A n a l y t i c a l and Extraction Methods . . 23 Incubation Methods 24 - v i -Page RESULTS AND DISCUSSION 25 CONCLUSION 34 REFERENCES 36 CHAPTER 4 MINERALIZATION OF SOIL SULFUR AND ITS RELATION TO SOIL CARBON, NITROGEN AND PHOSPHORUS 3 9 INTRODUCTION 39 METHODS AND MATERIALS 40 RESULTS AND DISCUSSION 41 CONCLUSION 62 REFERENCES 64 CHAPTER 5 THE EFFECT OF NITROGEN ADDITIONS ON MINERALIZATION OF SOIL SULFUR DURING INCUBATION 6 6 INTRODUCTION 66 METHODS AND MATERIALS 68 RESULTS AND DISCUSSION 69 CONCLUSION 75 REFERENCES 78 GENERAL SUMMARY AND CONCLUSIONS 81 APPENDIX 1 EVALUATION OF METHODS FOR DIRECT DETERMINATION OF CARBON-BONDED SULFUR IN SOIL 88 INTRODUCTION 88 METHODS AND MATERIALS 91 RESULTS AND DISCUSSION 93 CONCLUSION 96 REFERENCES 97 - v i i -Page APPENDIX 2 OBSERVATIONS ON AN ALKALINE OXIDATION METHOD FOR DETERMINATION OF TOTAL SULFUR IN SOILS 98 INTRODUCTION 98 METHODS AND MATERIALS 99 RESULTS AND DISCUSSION 101 CONCLUSION 106 REFERENCES 107 APPENDIX 3 CHEMICAL ANALYSES OF FOUR SOIL SAMPLES DURING AN INCUBATION EXPERIMENT 108 APPENDIX 4 EXAMINATION OF VARIOUS METHODS FOR SEPARATION OF ORGANIC FROM INORGANIC SULFATE IN SOIL EXTRACTS 115 INTRODUCTION 115 METHODS AND MATERIALS 116 RESULTS AND DISCUSSION 118 CONCLUSION 123 REFERENCES 125 APPENDIX 5 CARBON DIOXIDE AND pH ANALYSES OF TWO SOIL SAMPLES TREATED WITH VARIOUS NITROGEN SOURCES AND RATES AFTER EIGHT WEEKS INCUBATION IN A CLOSED SYSTEM . . 127 - v i i i -LIST OF TABLES Page CHAPTER 1 OBSERVATIONS ON THE BISMUTH SULFIDE COLORIMETRIC PROCEDURE FOR SULFATE ANALYSIS IN SOIL TABLE 1. Effect of nitrogen flow rate on sulfate-S determinations using the bismuth sulfide finish 9 CHAPTER 3 EVALUATION OF VARIOUS SULFATE EXTRACTANTS AND A CLOSED INCUBATION METHOD FOR STUDYING SOIL SULFUR MINERALIZATION TABLE 1. Some properties of soil samples TABLE 2. Mineralized sulfur by four extraatants at 1, 2, 4, 8 and 14 weeks incubation compared with carbon dioxide evolved for fresh and dried samples for four soils . . . TABLE 3. Linear correlation coefficients (r) of sulfate extracts and inorganic nitrogen at 0} 1, 2, 4, 8 and 14 week intervals during incubation at 30°C and 100 cm water CHAPTER 4 MINERALIZATION OF SOIL SULFUR AND ITS RELATION TO SOIL CARBON, NITROGEN AND PHOSPHORUS TABLE 1. Some chemical analyses of soil samples TABLE 2. Mineralization of carbon and nitrogen from four soils during 14 week incubation TABLE 3. Net sulfur mineralized in 14 weeks expressed as percentage of various sulfur fractions 27 42 43 59 - ix -TABLE 4. Comparison of mineralizable sulfur (14 weeks) with two soil to solution ratios for extraction of soluble sulfate (ppm S)3 C:N and C:S ratios Page 61 CHAPTER 5 EFFECT OF NITROGEN ADDITIONS ON THE MINERALIZATION OF SOIL SULFUR DURING INCUBATION TABLE 1. Calcium chloride (0.15%) extractable sulfate of soil samples treated with various sources at several rates after eight weeks incubation at 100 cm moisture tension at 30°C 71 TABLE 2. Correlation coefficients (r and R) of extractable sulfate (Y) with carbon dioxide evolved (Xj) and pH (X%) of results after an eight week incubation of two soils treated with nitrogen 73 APPENDIX 1 EVALUATION OF METHODS FOR DIRECT DETERMINATION OF CARBON-BONDED SULFUR IN SOIL TABLE 1. Carbon-bonded sulfur (ppm S) of soils determined by two direct methods 94 TABLE 2. Carbon-bonded sulfur (ppm S) in several soil extracts measured by two direct determination methods , . 95 APPENDIX 2 OBSERVATIONS ON AN ALKALINE OXIDATION METHOD FOR DETERMINATION OF TOTAL SULFUR IN SOILS TABLE 1. Soil samples used in total sulfur content studies , 100 - X -Page APPENDIX 3 TABLE 2. Comparison of total sulfur (ppm) of soil samples by sodium hypobromite and sodium hypochlorite oxidations • TABLE 3. Comparison of soil sulfur values (ppm S) by two direct determinations and a summation method for a few of the samples . . CHEMICAL ANALYSES OF FOUR SOIL SAMPLES DURING AN INCUBATION EXPERIMENT 104 105 APPENDIX 4 TABLE 1. Soil analysis results of four samples during an incubation experiment using a closed system . . 108 TABLE 2. Cumulative carbon dioxide carbon (mg/g/soil) evolved from four soils during a fourteen week incubation . 1 112 TABLE 3. Soil analysis results of soil samples determined on fresh material at various intervals during an incubation experiment using an open system with daily water additions . 113 EXAMINATION OF VARIOUS METHODS FOR SEPARATION OF ORGANIC FROM INORGANIC SULFATE IN SOIL EXTRACTS APPENDIX 5 TABLE 1. Percentage organic sulfate sulfur in soil extracts using an ion retardation resin separation . . 122 CARBON DIOXIDE AND pH ANALYSES OF TWO SOIL SAMPLES TREATED WITH VARIOUS NITROGEN SOURCES AND RATES AFTER EIGHT WEEKS INCUBATION IN A CLOSED SYSTEM . • 127 - x i -LIST OF FIGURES Page CHAPTER 3 EVALUATION OF VARIOUS SULFATE EXTRACTANTS AND A CLOSED INCUBATION METHOD FOR STUDYING SOIL SULFUR MINERALIZATION FIGURE 1. Sulfate s u l f u r extracted from four s o i l s with 0.15% calcium chloride at various s o i l : s o l u t i o n r a t i o s 30 CHAPTER 4 MINERALIZATION OF SOIL AND ITS RELATION TO SOIL CARBON, NITROGEN AND PHOSPHORUS FIGURE 1. Sodium bicarbonate extractable phosphorus of four s o i l samples during 14 week incubation . . 46 FIGURE 2. Arylsulfatase a c t i v i t y of four s o i l samples during 14 week incubation 48 FIGURE 3. Sulfate s u l f u r extracted by four solutions from Prest s o i l sample during 14 week incubation 5 0 FIGURE 4. Sulfate s u l f u r extracted by four extracting solutions from Stevens sample during 14 week incubation 51 FIGURE 5. Calcium chloride extractable sulfate sulfur of four s o i l samples during 14 week incubation 5 4 FIGURE 6. Nitrogen mineralized i n r e l a t i o n to carbon dioxide evolved from four s o i l samples during 14 week incubation 56 FIGURE 7. Sulfur mineralized i n r e l a t i o n to carbon dioxide evolved from s o i l samples during 14 week incubation 5 7 - x i i -Acknowledgements The author wishes to thank Dr. L.E. Lowe for his help f u l supervision throughout t h i s project. Suggestions by Dr. C A . Rowles, Dr. T.M. B a l l a r d , Dr. Mary Barnes and Dr. P.M. Townsley are also g r a t e f u l l y acknowledged. A speci a l thanks to my wife for her patience and encouragement through the entire degree program and p a r t i c u l a r l y for the preliminary typing of t h i s manuscript. The figures were drafted by Beth Loughran and the f i n a l typing was done by Retha Gerstmar. Their work was very much appreciated. Fina n c i a l assistance for t h i s project was supplied by Canada Department of Agriculture and the Leonard S. Klinck Fellowship. INTRODUCTION Recent f e r t i l i z e r developments, agronomic and i n d u s t r i a l practices and an increasing demand for a larger volume of quality food products have increased the need for understanding the role of the s o i l i n supplying s u l f u r to a g r i c u l t u r a l crops. A large portion of the sulfur present i n the surface horizon of s o i l i s of organic nature and since most plants appear to prefer the inorganic sulfate anion, mineralization of the organic f r a c t i o n i s considered an important process. Although i t i s recognized that other forms of s o i l s u l f u r , such as inorganic s u l f u r compounds and adsorbed s u l f a t e , and atmospheric gases must also be considered for the o v e r a l l s u l f u r c y c l e , more knowledge of organic s u l f u r transformations i s important. The primary focus of t h i s i n v e s t i g a t i o n , then, was to consider some aspects of these transformations i n the s o i l . More s p e c i f i c a l l y some of the factors were considered which influence the appearance of extractable s u l f a t e , i n the absence of plants or other photosynthetic a c t i v i t y , during optimum incubation conditions. Because a balance of nutrients i s recognized i n plant growth and microbial a c t i v i t y , other s o i l c h a r a c t e r i s t i c s were also considered to help understand s u l f u r reactions and interactions i n the complex s o i l system. - 2 -This thesis i s i n the form of a series of chapters that would be suitable for publication i n s c i e n t i f i c journals, each chapter being important i n the consideration of the topic o u t l i n e d . A s i g n i f i c a n t proportion of the work was devoted to a study of methods and procedures because there i s a lack of understanding of many methods that are a v a i l a b l e , and that has resulted i n some of the d i f f i c u l t i e s i n i nterpreting r e s u l t s of the past. The f i r s t chapter was a consideration of the bismuth s u l f i d e colorimetric f i n i s h of the hydriodic acid method of quantifying sulfate i n s o i l s and s o i l extracts. The hydriodic acid method of sulfate analysis has advantages of s e n s i t i v i t y and r e l a t i v e freedom from interference by materials common i n s o i l s and the application of the bismuth f i n i s h was found to be important i n increasing the ease of operation and i n reducing the a n a l y t i c a l time of the technique. The analysis of n i t r a t e content i n s o i l extracts was an important consideration i n t h i s study. Many quantitative n i t r a t e a n a l y t i c a l methods are a v a i l a b l e , however each procedure has certai n l i m i t a t i o n s . A procedure that previously had not been reported to have been used on s o i l extracts was discovered. This r e l a t i v e l y simple colorimetric method, which appeared to have suitable s e n s i t i v i t y and - 3 -accuracy for analysis of s o i l e x tracts, applied the use of chronotropic a c i d . The second chapter of t h i s thesis considers the a p p l i c a b i l i t y of t h i s method for n i t r a t e determinations of s o i l s . The t h i r d chapter was a consideration of methods needed for an incubation i n v e s t i g a t i o n . A closed incubation system was examined for aerobic conditions and the analyses of the s o i l samples were also considered. There are numerous extractants available f o r the determination of sulfate i n s o i l s and several were compared for t h e i r a b i l i t y to y i e l d r e s u l t s which could be correlated with microbial a c t i v i t y . The influence of a i r drying samples was studied by comparing res u l t s of analyses of fresh and a i r dried samples. The mineralization of sulfur i n four selected s o i l samples was more closel y examined i n the fourth chapter. This was considered with respect to c e r t a i n s o i l character-i s t i c s such as t o t a l carbon, s u l f u r and nitrogen quantities as well as carbon dioxide evolved, extractable n i t r a t e and ammonium, extractable phosphate, phospholipids, a r y l s u l f a t a s e a c t i v i t y and s o i l pH. Relationships and conclusions were attempted from the numerous analyses that were done, primarily to gain information on c h a r a c t e r i s t i c s which would be most useful for further study i n r e l a t i o n to sulfur mineralization. - 4 -The l a s t chapter describes a study that used information on s o i l s , methods and r e s u l t s from the previous chapters to examine the r e l a t i o n s h i p between the sulfur mineralized from two s o i l samples treated with several nitrogen sources at a series of rates a f t e r an incubation period. This study indicates the effects that nitrogen f e r t i l i z a t i o n practices may have on the potential release of sulfur as sulfate from s o i l s . Because of the large number of analyses involved i n such a study, only two s o i l s were considered with f i v e d i f f e r e n t nitrogen sources applied at four d i f f e r e n t r a t e s . S t a t i s t i c a l tests were computed on the data using a f a c t o r i a l design experiment. Consideration-was also given to the influence of the nitrogen sources and application rates on microbial a c t i v i t y (carbon dioxide evolved) and s o i l pH, and how these might be i n t e r r e l a t e d with sulfur mineralization. - 5 -CHAPTER 1 OBSERVATIONS ON THE BISMUTH SULFIDE COLORIMETRIC PROCEDURE FOR SULFATE ANALYSIS IN SOIL1 INTRODUCTION There are a number of methods for determining sulfate-S i n s o i l and plant m a t e r i a l , but none have proved e n t i r e l y s a t i s f a c t o r y . Perhaps the most widely accepted procedure i s the reduction of sulfate to hydrogen s u l f i d e by hydriodic a c i d , and colorimetric determination of s u l f i d e as methylene blue (Johnson and N i s h i t a , 1952; Johnson and U l r i c h , 1959). This procedure has been investigated i n some d e t a i l (Gustafsson, 1960a, b; Johnson and U l r i c h , 1959). Dean (1966) has proposed an alternative colorimetric f i n i s h to the analysis based on the formation of bismuth s u l f i d e . This procedure appears to o f f e r some advantages, but the significance of v a r i a t i o n i n operating conditions has not been examined i n as much d e t a i l as the methylene blue method. Appears i n Communications i n S o i l Science and Plant Analysis 3(1), 79-86 (1972) with L.E. Lowe as co-author. - 6 -The aims of the present study were to examine the bismuth procedure with respect to nitrogen flow rate and i t s c o n t r o l , n i t r a t e interference, and predrying of aqueous samples; and to further evaluate t h i s procedure as an alternative to the methylene blue procedure. METHODS AND MATERIALS Previously described procedures f o r the reduction of sulfate to s u l f i d e (Johnson and U l r i c h , 1959) and the subsequent colorimetric quantitation by the methylene blue f i n i s h (Johnson and U l r i c h , 1959) and bismuth s u l f i d e (Dean, 1966) were used. D i f f i c u l t i e s were encountered i n c o n t r o l l i n g the r e l a t i v e l y low flow rate used from a compressed nitrogen tank, so a system of c a p i l l a r y tubing was f i n a l l y adopted (Jenkinson, 1968). A buffer tank (500 ml erlenmeyer) was added between the nitrogen p u r i f i c a t i o n bottle and d i s t i l l a t i o n u n i t s . Between the buffer tank and each d i s t i l l a t i o n u n i t , about 30 cm of c a p i l l a r y tubing was i n s e r t e d , which e f f e c t i v e l y reduced and maintained the flow of nitrogen within the desired range. With t h i s simple, inexpensive set up, the nitrogen flowed through one unit of a two unit set up, even when the other unit was disconnected. - 7 -S o i l samples (60 mesh) used i n th i s work were from various areas of B r i t i s h Columbia and included surface horizons of organic s o i l s , podzols, l u v i s o l s and chernozems. They were chosen for t h e i r wide range of s u l f u r content and v a r i a t i o n i n the proportions of sulfur f r a c t i o n s . The s t a t i s t i c a l method for standard error of estimate or the standard deviation of Y for fixed X (a l i n e a r regression) used the following equation (Steele and T o r i e , 1960) : S2y x = Zv2 - (Exy)2/x2 n - 2 The c o e f f i c i e n t of v a r i a t i o n (CV) used CV = 1^_t'ls where x s = average standard deviation and x = average of values. RESULTS AND DISCUSSION Effe c t of Nitrogen Gas Flow Rate Dean (1966) reported that the nitrogen flow rate should be above 500 ml/min, and recovered 82% of a known amount of sulfate at a flow rate of 300 ml/min, using a 10 minute d i g e s t i o n - d i s t i l l a t i o n period. T r i a l s were made using a flow rate of 100 to 200 ml/min, as used with the methylene blue f i n i s h (Gustafsson, 1960b; Johnson and - 8 -N i s h i t a , 1952), but with the time increased to 20 minutes. The r e s u l t s were compared with those obtained with a flow rate of over 500 ml/min (Table 1). Complete recovery was achieved at both flow r a t e s , indicating that a lower flow rate i s not a c r i t i c a l factor i f the time of reaction i s s u f f i c i e n t l y long. At the higher flow r a t e , condensation appeared i n the apparatus above the condenser, which turned reddish-brown (assumed to be i o d i n e ) . This material transferred by the nitrogen at a higher flow rate was assumed to have caused the higher, more v a r i a b l e , reading. For t h i s reason the lower flow rate for a longer time i s recommended. Time of D i s t i l l a t i o n An advantage of the bismuth s u l f i d e over the methylene blue f i n i s h i s that no gas wash i s needed, thus reducing the time required to transfer a l l the hydrogen s u l f i d e to the absorbing s o l u t i o n . A s u l f a t e - r i c h organic s o i l from the Fraser Valley was used to determine the minimum time required for complete d i s t i l l a t i o n of sulfate from a whole s o i l sample by both colorimetric f i n i s h e s . The bismuth s u l f i d e method resulted i n v i r t u a l l y complete (95%) d i s t i l l a t i o n of sulfur i n 10 minutes, but the methylene blue recovered only 80% i n 45 minutes. F i n a l l y , 20 minutes was found to be a s u f f i c i e n t l y - 9 -TABLE 1. Effect of nitrogen flow rate on sulfate-S determinations using the bismuth s u l f i d e f i n i s h Nitrogen flow rate Sulfate-Sa Standard , (ml/min for 20 min) (ppm) deviation 100-200 100 ±1 >500 105 ±4 a100 ppm sulfate standard. ^Four determinations on each. - 10 -long d i s t i l l a t i o n time for the bismuth s u l f i d e and one hour for the methylene blue f i n i s h . Effect of Predrying Samples It has been shown that upon reducing the water content of a sample at the sul f a t e reduction step, there are increased yi e l d s of sulfate determined by the methylene blue procedure (Gustafsson, 1960b). However, analysis of aqueous samples has been commonly reported (Johnson and N i s h i t a , 19 52; Tabatabai and Bremner, 19 70). The effect of predrying 2 ml aliquot standard sulfate solutions was investigated using the bismuth method. I n i t i a l l y , samples i n digestion flasks were oven-dried at 100°C. Subsequently, drying under an infrared lamp was shown to give the same r e s u l t s , but was much more r a p i d . Standard curves for predried samples yielded a steeper slope (m = 176.2) than the aqueous samples (m = 158.1). A standard error estimate for each of the two separate t r i a l s was Syx = ±0.02 f o r predried and Syx = ±0.0 3 for the 2 ml aliquot samples, and combining the r e s u l t s for the two i t was Syx = ±0.2 3 . This shows that the two preparations gave d i s t i n c t l y d i f f e r e n t r e s u l t s , therefore the standard curve must be consistent with the method of sample preparation. Predrying (using an infrared lamp) i s recommended where both s o i l materials - 11 -and extracts are involved. Predrying large aliquots of extracts could be used to increase the s e n s i t i v i t y of the method. Effect of Sample Nitrate Content Johnson and Nishita (195 2) showed that samples containing quantities of n i t r a t e i n excess of 6 mg resulted i n interferences with the methylene blue color development and that the pyrogallol-sodium phosphate gas wash solution incompletely removed the i n t e r f e r i n g compounds. Varying milligram quantities of n i t r a t e (as KNO^) were weighed into reduction flasks containing 100 ppm dried sulfate standard, which were then analyzed for sulfate with the bismuth s u l f i d e f i n i s h . There was a reduction of 75% of sulfate determined with n i t r a t e i n excess of 6 mg. The high n i t r a t e content appeared to a f f e c t the sulfate reduction phase, since quantitative recovery of standard sulfate resulted using NaOH absorber containing n i t r a t e reduction products from a separate run. Comparison of the Bismuth and Methylene Blue Results on  S o i l Materials The average t o t a l sulfate-S (sometimes referred to as Hl-reducible S) content of 17 s o i l s determined i n dupli c a t e , - 12 -using the methylene blue and bismuth methods, were 811 and 804 r e s p e c t i v e l y , indicating that the methods y i e l d e s s e n t i a l l y the same recoveries. The precision of the bismuth method (CV = 7.1%) was somewhat, better than for the methylene blue procedure (CV = 9.6%), and was also free from the occasional very e r r a t i c r e s u l t found with the methylene blue method. The higher precision found for standard material (CV = 1%, Table 1) was expected since more homogeneous and soluble material i s used rather than s o l i d s o i l . When applied to s o i l e x tracts, both aqueous and organic, the bismuth method gave e s s e n t i a l l y the same r e s u l t s as the methylene blue method, with as good or better p r e c i s i o n , however, the standard bismuth procedure of Dean (1966) i s markedly less sensitive than the methylene blue and hence requires modification for use with extracts of low sulfate content. Microdeterminations Using the Bismuth Method A l l of the above information was reported for the bismuth method determined on a range of sulfate between 0 and 200 ppm. By quartering the volumes of NaOH absorber and bismuth reagent, sulfate i n the range of 0-40 ppm was determined s a t i s f a c t o r i l y . The r e s u l t i n g volume of bismuth - 13 -su l f i d e solution (7 . 5 ml) was small enough to require p a r t i c u l a r care i n volume measurement. Measurements of the small quantities i n a Spectronic 2 0 spectrophotometer were done using o p t i c a l l y matched cuvettes. The absorption vessel size was adjusted to ensure that the hydrogen s u l f i d e bubbled through about 2 to 3 cm of solution for complete absorption. Comparison of microdeterminations of the modified procedure of Dean with the methylene blue method was very favorable. The standard error for the methylene blue procedure for standard sulfate was Syx = ±0.7 and for bismuth i t was Syx = ±0 . 5 . With practice and care t h i s error could probably be reduced f u r t h e r . It was concluded that the bismuth s u l f i d e f i n i s h i s suitable for microestimations of sulfate by manipulation of reagent volumes. In conclusion, the bismuth method was shown to be comparable to the methylene blue method i n terms of accuracy and precision but also has the advantage of taking less than one-third the time. The bismuth s u l f i d e f i n i s h to the Johnson-Nishita d i s t i l l a t i o n i s recommended as a suitable a l t e r n a t i v e to the methylene blue method. - 14 -REFERENCES Dean, G.A. 1966. A simple colorimetric f i n i s h for the Johnson-Nishita m i c r o d i s t i l l a t i o n of sulphur. Analyst 91: 530-532. Gustafsson, L. 1960a. Determination of ultramicro amounts of sulphate as methylene blue. I. The color r e a c t i o n . Talanta 4: 227-235. Gustafsson, L. 1960b. Determination of ultramicro amounts of sulphate as methylene blue. I I . The reduction. Talanta 4: 236-245. Jenkinson, D.S. 196 8. T i t r i m e t r i c method for determining t o t a l sulphur i n mineral s o i l s . Analyst 93: 5 35-5 39. Johnson, CM. and H. N i s h i t a . 1952. Microestimation of sulfur i n plant materials, s o i l s and i r r i g a t i o n waters. Anal. Chem. 24: 736-742. Johnson, CM. and A. U l r i c h . 1959. A n a l y t i c a l methods f o r use i n plant a n a l y s i s . C a l i f . Agr. Exp. Sta. B u l l . 766. Steele, R.G.D. and J.H. T o r i e . 1960. Principl e s and procedures of s t a t i s t i c s . McGraw-Hill Book Co., Inc., Toronto. 4 26 pp. Tabatabai, M.A. and J.M. Bremner. 1970. An alk a l i n e oxidation method for determination of t o t a l s u l f u r i n s o i l s . S o i l S c i . Soc. Am. Proc. 34: 62-65. - 15 -CHAPTER 2 DETERMINATION OF NITRATES IN SOIL EXTRACTS The phenoldisulfonic acid method has been used extensively for the determination of nitrates i n s o i l e xtracts, but i s somewhat inconvenient and subject to chloride interference i n some extracts. The publication of a number of recent papers on a l t e r n a t i v e methods of quantifying s o i l n i t r a t e s indicates the need for a rapid and r e l i a b l e procedure to replace the phenoldisulfonic acid method. The p o s s i b i l i t i e s of the n i t r a t e s p e c i f i c ion electrode have been investigated by a number of workers, but t h i s technique appears to be subject to some interference, from inorganic anions such as SO^-, H^PO^-, C l ~ , HCO^- and N02~, organic anions and dispersed c o l l o i d a l material (Mack and Sanderson, 1970; Milham et a l . , 1970; Onkin and Sunderman, 1970). Problems with precision have been encountered (Mack and Sanderson, 1971) p a r t i c u l a r l y with low NO^-N concentrations (0-10 ppm). Recently, colorimetric methods u t i l i z i n g chromotropic acid (CTA) have been applied to s o i l extracts (Basargin and - 16 -Chernova, 19 68; Clarke and Jennings, 196 5; Sims and Jackson, 1971). The method of Sims and Jackson was considered and although i t was found to be rapid and s e n s i t i v e , i t s r e p r o d u c i b i l i t y and the l i n e a r i t y of the standard curve was not s a t i s f a c t o r y . Further, these methods, which include the chloride ion to increase s e n s i t i v i t y of the colored complex, are subject to potential interference from n i t r i t e i o n , i r o n , c e r t a i n organic compounds and high concentrations of Na+ and K+ (Basargin and Chernova, 1968; Clarke and Jennings, 1965; Sims and Jackson, 1971). However, the most recent studies appear to have overlooked the p o s s i b i l i t i e s of a CTA procedure developed by West and Ramachandran (1966) for n i t r a t e analysis of water samples. These authors made a thorough study of the nitrate-CTA reaction and developed a procedure where chloride was excluded and interferences due to n i t r i t e , oxidizing agents and many other cations and anions, common i n s o i l extracts and extracting s o l u t i o n s , were overcome. This procedure of West and Ramachandran was tested on s o i l extracts i n our laboratory, with only minor modifications, and was found to be rapid and reproducible i n the 0-10 ppm range. The minor modifications introduced were as follows: the reaction vessel was a test tube rather than a 10 ml volumetric f l a s k - 17 -and f i n a l volumes were reproduced by making standard volume additions of each solution with a rapid delivery p i p e t t e . S t i r r i n g was done using a glass rod, flattened at the bottom. The reagents were the same as employed by West and Ramachandran, although p u r i f i e d CTA i s now available commercially. The absorption maximum at 410 my was confirmed on a Spectronic 2 0 spectrophotometer and the CTA-NO^ complex was stable from 2 0 minutes to 2 7 hours. Blank material was not as stable and absorbance readings increased a f t e r three hours, p a r t i c u l a r l y i n the presence of daylight. Hence, i t i s recommended that color readings be taken within about three hours of color development. When the modified procedure was applied to extracts of four s o i l s containing a range of 0.4 to 30.0 ppm NO^-N res u l t s were i n good agreement with those obtained with the phenoldisulfonic acid method, and of sa t i s f a c t o r y precision ( c o e f f i c i e n t s of v a r i a t i o n were 2.2% and 2.3% for the phenoldisulfonic acid and CTA methods, r e s p e c t i v e l y ) . It was concluded that the procedure of West and Ramachandran provides a sa t i s f a c t o r y alternative to the phenoldisulfonic acid procedure for the determination of n i t r a t e i n s o i l e x t racts, but with improved s e n s i t i v i t y and speed. It can - 18 -be applied to a variety of extracting agents provided the s o i l extract i s s u f f i c i e n t l y free of colored material. Furthermore, the procedure may well prove suitable for adaptation for automated a n a l y s i s . - D E -REFERENCES Basargin, N.N. and E.A. Chernova. 1968. (Rapid photometric determination of n i t r a t e i n s o i l by means of chronotropic acid.) Zh. ana l t . Khim. 23: 102-108. In S o i l s F e r t . 33: 144 (987). Clarke, A.L. and A.C. Jennings. 196 5. Spectrophotometric estimation of ni t r a t e i n s o i l using chromotropic a c i d . J . Agr. Fd. Chem. 13: 174-176. Mack, A.R. and R.B. Sanderson. 1971. S e n s i t i v i t y of the ni t r a t e - i o n membrane electrode i n various s o i l extracts. Can. J . S o i l S c i . 51: 95-104. Milham, D.J., A.S. Awad, R.E. Paull and J.H. B u l l . 1970. Analysis of plan t s , s o i l s and -waters for n i t r a t e by using an ion-selective electrode. Analyst, Lond. 95: 751-757. Onken, A.B. and H.D. Sunderman. 1970. Use of the ni t r a t e electrode for determination of nit r a t e s i n s o i l s . Commun. S o i l S c i . Plant Analysis 1: 155-161. Sims, J.R. and G.D. Jackson. 19 71. Rapid analysis of s o i l n i t r a t e with chromotropic a c i d . S o i l S c i . Soc. Am. Proc. 35: 603-606. West, P.W. and T.P. Ramachandran. 1966. Spectrophotometric determination of n i t r a t e using chromotropic a c i d . Anal. Chim. Acta 35: 317-324. CHAPTER 3 EVALUATION OF VARIOUS SULFATE EXTRACTANTS AND A CLOSED INCUBATION METHOD FOR STUDYING SOIL SULFUR MINERALIZATION INTRODUCTION Current knowledge about microbial mineralization of s o i l s u l f u r i s limited to rather broad relationships with carbon, nitrogen, temperature, moisture content, pH and plant growth (Freney, 1967). More re c e n t l y , isotope methods have been used to indicate that cert a i n fractions of s o i l organic sulfur may be important i n mineralization-immobilization processes i n s o i l (Freney et al. , 1971), however much more information i s needed. Suitable f u l l y understood methods are l i m i t e d , r e s u l t i n g i n generalizations that are sometimes not f u l l y tested experimentally. Methods used for quantitative determination of sulfate must be evaluated before interpretations are made. Two popular methods u t i l i z e either p r e c i p i t a t i o n with barium or reduction to hydrogen s u l f i d e with hydriodic acid reducing reagent. P r e c i p i t a t i o n with barium i s often considered to be a reaction with inorganic sulfate only, however organic - 21 -c o l l o i d s and cations are known to i n t e r f e r e (Beaton et al.3 1968). Hydriodic acid treatment w i l l r e s u l t i n reduction of inorganic and organic sulfates (Johnson and N i s h i t a , 1952). In a d d i t i o n , methods for sulfate extraction should be examined to determine the r e l a t i v e contributions made by soluble s u l f a t e , adsorbed sulfate and organic-bonded sulfate to the t o t a l sulfate extracted. A l s o , some incubation procedures that have been used with the assumption that aerobic conditions were maintained, have' recently been considered and shown that t h i s was not l i k e l y the case (Bremner and Douglas, 19 71; Douglas and Bremner, 19 71). It i s the purpose of t h i s report to consider several methods for use i n an incubation experiment, namely, extractions of sulfate and aerobic conditions of a p a r t i c u l a r closed incubation system. METHODS AND MATERIALS  Soi l s The s o i l samples represent three B r i t i s h Colubmia s o i l s (Cardinal, Prest and Stevens) and one Saskatchewan s o i l (Oxbow). Some of the c h a r a c t e r i s t i c s of the s o i l samples are given i n Table 1. The dpH values give a r e l a t i v e measure of sulfate adsorption capacity (Chao et al. , 1965). One 22 .TABLE 1. Some properties of soil samples Parent S o i l name Great group material Horizon Texture dpH Cardinal Ferro humic G l a c i a l t i l l B f h SiL 0.59 Podzol Oxbow Black G l a c i a l t i l l A CL 0.31 chernozemic P Prest Humic A l l u v i a l A, SiCL 0.39 T n n gleysol Stevens Black G l a c i a l t i l l A h L 0.10 chernozemic aThe System of S o i l C l a s s i f i c a t i o n for Canada. Canada Department of A g r i c u l t u r e , 19 70. - 23 -sample (Stevens) was included because the s o i l s of the area are known to respond to sulfur f e r t i l i z a t i o n . Each sample was a i r dried at room temperature, crushed, and passed through a 2 mm sieve. A n a l y t i c a l and Extraction Methods S o i l sulfate was extracted by four methods, 0.15% CaCl 2«2H 20 (Williams and Steinbergs, 1959), sodium acetate at pH 4.8 (Jordan and Bardsley, 19 58), 0.5 M sodium phosphate buffer at pH 7.0 (Bart, 1969) and 0.5 M NaHC03 at pH 8.5 (Kilmer and Nearpass, 1960). Aliquots of these extracts were dried and examined for sulfate using hydriodic acid reduction using the bismuth colorimetric method (Kowalenko and Lowe, 1972 - Chapter 1). Inorganic nitrogen was extracted with 1% KAKSO^^ (I:1 4 s o i l to solution) with ammonium nitrogen determined by a microdiffusion-Nesslerization procedure ( Z o t t l , 1960) and n i t r a t e by a chromotropic acid method (Chapter 2). Analyses were determined on both fresh s o i l samples and on samples a i r dried at about 20°C. Fresh samples were stored at 4°C to arrest microbial a c t i v i t y u n t i l analyses were completed. A l l r e s u l t s were corrected to oven dry weight. Examination of the s o i l : s o l u t i o n r a t i o e f f e c t was done with the CaCl- extractant, with one-half hour shaking time. - 24 -Weighed air-dry samples, i n t r i p l i c a t e , were shaken for 30 minutes at r a t i o s of 1:1, 1:2, 1:4, 1:5, 1:8, 1:10, 1:20, centrifuged, f i l t e r e d and sulfate measured i n dried a l i q u o t s . Incubation Methods Two incubation methods were employed i n th i s study, one open and one closed. The closed system involved weighing 200 g of sample (oven dry basis) into glass jars (9 00 ml capacity with a 7.5 cm diameter opening) and s u f f i c i e n t d i s t i l l e d water to take the moisture content to 100 cm tension. The s o i l was mixed, r e s u l t i n g i n a loose, well aerated sample. One hundred f i f t y ml beakers containing 4 0 ml of 1 N NaOH were suspended i n the jars above the s o i l with tape and the mouth of the jars were sealed with polyethylene (Barrow, 1960c) and incubated at 30°C i n darkness. The moisture and temperature conditions used were considered optimum for sulfur mineralization (Williams, 1967). Carbon dioxide evolved was determined by t i t r a t i o n with normal acid at 1, 2, 4, 8 and 14 week int e r v a l s on duplicate jars and enough in d i v i d u a l jars were included to allow duplicate samplings for the s o i l analyses at each i n t e r v a l . Blank jars not containing s o i l were included to determine the carbon dioxide contribution of the atmosphere - 25 -under the experimental conditions used. One extra jar of each s o i l was included with test papers for hydrogen s u l f i d e and s u l f u r dioxide evolution using lead acetate and nickel hydroxide, respectively ( F e i g l , 19 56). The open incubation u t i l i z e d the same jars and s o i l conditions except that the jars were l e f t open (Williams, 1967). The moisture content was maintained by d a i l y weighings and addition of water. No estimates were made of carbon dioxide evolution, but sulfate (0.15% CaC^ extractable) and inorganic nitrogen analyses on fresh samples were the same as previously outlined. RESULTS AND DISCUSSION Although Williams (1967) had established that air-drying samples resulted i n a release of sulfate for both calcium chloride and potassium phosphate extractions, for convenience a i r drying the samples p r i o r to analysis was used. It appeared i n his study that the amount of sulfate released during incubation at 30°C over in t e r v a l s up to nine weeks was not affected when samples were predried p r i o r to a n a l y s i s . With the closed incubation, the four s o i l s i n t h i s experiment were examined for changes i n extractable sulfate at various i n t e r v a l s on fresh and then a i r dried samples. Paired t tests - 26 -on the re s u l t s showed s i g n i f i c a n t differences (95% level) for extractable sulfate with calcium c h l o r i d e , sodium acetate and phosphate buffer as well as for extractable n i t r a t e and ammonium nitrogen. Sodium bicarbonate extractable sulfate values were s i g n i f i c a n t l y d i f f e r e n t at the 90% l e v e l . It appears then, that d i f f e r e n t r e s u l t s for these samples were obtained when analysing fresh samples as compared to drying at 20°C. A less s i g n i f i c a n t r e s u l t with sodium bicarbonate extract may be because th i s extractant more completely extracts sulfate fractions (soluble, adsorbed, organic) that may influence the res u l t s for the other three extractions. However, i f the differences of sulfate extracted from fresh and dry samples at each incubation i n t e r v a l were constantly s i m i l a r , r e s u l t s from both the fresh and dry analyses should indicate s i m i l a r r e s u l t s for microbial release of s u l f a t e . Comparison of the mineralized sulfur (sulfate extractable at a given time minus sulfate at beginning) at each i n t e r v a l (1, 2, 4, 8 and 14 weeks) with microbial a c t i v i t y r e f l e c t e d by carbon dioxide evolution revealed that the two res u l t s of dried and fresh samples are not the same (Table 2). It was concluded from these re s u l t s that the sulfate during incubation extracted by four extractants on - 27 -TABLE 2. Mineralized sulfur by four extraotants at 1, 2} 4, 8 and 14 weeks incubation compared with carbon dioxide evolved for fresh and dried samples for four soils Extractant Correlation c o e f f i c i e n t s (r) Fresh sample A i r dried sample Calcium chloride 0 . 86' 0.41 Sodium acetate 0 . 82: -0 .01 Phosphate buffer 0.55* -0 .17 Sodium bicarbonate 0.60** 0 .46 -''Significant (9 5%). — Highly s i g n i f i c a n t (99%). ***Very highly s i g n i f i c a n t (99.9%). - 28 -these four s o i l s was greatly influenced by a i r drying and that the fresh analyses were more closely related to microbial a c t i v i t y . In a further consideration of the method of comparison of fresh and dry s o i l extractions, i t was r e a l i z e d that f a i r l y large differences i n s o i l : s o l u t i o n r a t i o s were being compared since s o i l s were weighed on a fresh basis before correction to oven dry weight and the moisture contents of the s o i l s at 100 cm water varied between 29 and 60%. A search of the l i t e r a t u r e did not reveal a systematic comparison of s o i l : s o l u t i o n (0.15% CaCl2> r a t i o extraction of soluble s u l f a t e , although d i f f e r e n t r a t i o s are u t i l i z e d (Barrow, 1961; Bettany and Halstead, 1972; Tabatabai and Bremner, 1972; Walker, 1972; Williams and Steinburgs, 1959); therefore t h i s analysis was conducted. The Prest and Cardinal samples showed a very great increase i n sulfate extracted as the r a t i o was increased, whereas the remaining two samples did not show t h i s same increase (Figure 1). The large increase i n extraction of soluble sulfate from Prest and Cardinal samples was taken to be a contribution from adsorbed s u l f a t e . These two samples appeared to contain s i g n i f i c a n t amounts of adsorbed sulfate since a much greater amount of sulfate was extracted by phosphate b u f f e r , however the Oxbow and Stevens s o i l s did not show these wide di f f e r e n c e s . A l s o , the dpH - 29 -values for Prest and Cardinal s o i l s (Table 1) showed a greater r e l a t i v e a b i l i t y to adsorb s u l f a t e . From these res u l t s one can see the importance of considering the s o i l : solution r a t i o e f f e c t with calcium chloride extraction p a r t i c u l a r l y for samples containing adsorbed s u l f a t e . Although a low s o i l to solution r a t i o would more clos e l y simulate natural s o i l to solution conditions, i t would be more convenient to use a higher s o i l : s o l u t i o n r a t i o to avoid errors a r i s i n g from the addition of s o i l water when analysing fresh s o i l samples. A closer examination of the various methods of extracting sulfate from fresh s o i l samples indicated that the calcium chloride extractable sulfate was the most clo s e l y related to microbial a c t i v i t y , however sodium acetate appeared to be a very close a l t e r n a t i v e . A comparison of the four sulfate extracts of the four s o i l s with one another and also with inorganic nitrogen are shown i n Table 3 by c o r r e l a t i o n c o e f f i c i e n t s of paired r e s u l t s through the experiment with the closed incubation. As might be expected, the sulfate extractable by the four solutions was highly correlated, since calcium chloride i s expected to extract soluble sulfate while sodium acetate, phosphate buffer and sodium bicarbonate extract soluble and variable proportions of adsorbed and - 30 -20 Cardinal Oxbow Prest Stevens A & x — — x • •• o o E 15-CL CL 10-3-Z3 tn CD o £ 5" CO / A O — I 1 1 1 1 1 1 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Log (extractanf: soil ratio) FIGURE 1. Sulfate sulfur extracted from four s o i l s with 0.15% calcium chloride at various s o i l : s o l u t i o n r a t i o s - 31 -TABLE 3. Linear correlation coefficients (r) of sulfate extracts and inorganic nitrogen at 0, 1, 2, 4, 8 and 14 week intervals during incubation at 30°C and 100 cm water Extract Extractable sulfate with Calcium Phosphate Sodium Sodium chloride buffer bicarbonate acetate Sulfate: Phosphate buffer Sodium bicarbonate Sodium acetate 0.75*** 0.69*** 0.97*** 0.70*** 0.97*** 0.9 8*** Nitrate 0.76*** 0.41' 0.42* 0.4 2* Ammonium 0.9 5*** 0.74*** 0.7 8**' 0.49* Nitrate and ammonium 0 .9 7*** 0 .6 3' 0.58** 0.59' '-Significant (95%). **Highly s i g n i f i c a n t (99%). ***Very highly s i g n i f i c a n t (99.9%). - 32 -organic sulfate (Ensminger and Freney, 1966). However, comparing the extracts with carbon dioxide evolved (Table 2) and with inorganic nitrogen, differences i n the extracts become evident. Both calcium chloride and sodium acetate have r e l a t i v e l y high c o r r e l a t i o n c o e f f i c i e n t s with carbon dioxide evolution, however correlations of these two extracts with inorganic nitrogen (carbon dioxide and nitrogen mineralized were very highly c o r r e l a t e d , r = 0.95) were higher with calcium chloride than with sodium acetate. It i s concluded, therefore, that the best extraction f o r sulfate as a measure for microbial mineralization would be the calcium chloride extract of fresh s o i l samples. The paper by Bremner and Douglas (19 71) questioning the use of p l a s t i c films appeared a f t e r the experiment was started with the closed incubation. Calculations were made as to the maximum oxygen consumption (Prest sample during the f i r s t week of incubation) assuming a respiratory quotient of u n i t y , and using d i f f u s i o n data for polyethylene (Stotzky et al. , 19 62) which showed that the oxygen content i n the r e l a t i v e l y large volume of the jar would not decline more than one percentage. Since the jars were aerated at 1, 2,4, 8 and 14 week in t e r v a l s and carbon dioxide was absorbed by NaOH, the system was considered s u f f i c i e n t l y - 33 -aerobic under the experimental conditions. The aeration should be s i m i l a r to a stoppered method which was considered . aerobic (Tabatabai and Bremner, 1972). No hydrogen s u l f i d e gas (which may evolve under anaerobic conditions) was detected with any of the s o i l s through the entire incubation. To further check the incubation system, the open ja r incubation previously described was used and r e s u l t s of calcium chloride extractable s u l f a t e , inorganic nitrogen and pH of fresh samples at 1, 2, 4 and 8 week interva l s were compared to s i m i l a r r e s u l t s of the closed system. Paired t tests did not reveal s i g n i f i c a n t d i f f e r e n c e s . However, i t was noted that there was greater v a r i a b i l i t y with the analyses of samples of the open incubation, probably r e f l e c t i n g the greater moisture f l u c t u a t i o n through the period and change i n bulk density of the samples by packing during d a i l y water additions. It was concluded from the r e s u l t s that the closed incubation system employed resulted i n aerobic s o i l conditions which f a c i l i t a t e d both the preservation of stable moisture conditions and the measurement of the carbon dioxide evolved as a monitor of microbial a c t i v i t y . - 34 -CONCLUSION It was established that analyses of fresh s o i l samples i n an incubation experiment were imperative to get a measure of microbial mineralization of s o i l s u l f u r . Since the process of drying samples r e s u l t s i n a release of sulfate (Williams, 1967), dried samples could not be used to measure the degree of sulfur mineralization a r i s i n g from microbial a c t i v i t y , unless the amount was constant through-out the experiment. Although wetting and drying may be an important factor i n releasing sulfate (Barrow, 1961), i t s eff e c t could not be considered constant at a l l stages of the incubation with the four samples i n th i s study. Further, of a variety of sulfate extracting s o l u t i o n s , i t was found that 0.15% calcium chloride extractable sulfate values most closel y r e f l e c t e d microbial a c t i v i t y , even i n the cases where s o i l s contained adsorbed s u l f a t e . The s o i l : s o l u t i o n r a t i o may influence the amount of sulfate extracted by calcium chloride and although a 1:1 r a t i o may more c l o s e l y simulate the s o i l s o l u t i o n , the 1:5 r a t i o was convenient and suitable for assessing microbial changes. A closed incubation system, where carbon dioxide evolution was monitored by sodium hydroxide absorption, was shown to be s u f f i c i e n t l y aerobic under the incubation - 35 -conditions used and could be a convenient method for future use provided microbial a c t i v i t y was not unduly high. - 36 -REFERENCES Barrow, N.J. 1960. A comparison of mineralization of nitrogen and of sulphur from decomposing organic materials. Aust. J. Agric. Res. 11: 960-969. Barrow, N.J. 1961. Studies on the mineralization of sulphur from s o i l organic matter. Plant and S o i l 26: 205-223. Bart, A.L. 19 69. Some factors a f f e c t i n g the extraction of sulphate from selected Lower Fraser Valley and Vancouver Island s o i l s . M.Sc. Thesis. University of B r i t i s h Columbia. 89 pp. Beaton, J.D., G.R. Burns and J. Platou. 19 68. Determination of sulphur i n s o i l s and plant material. Technical B u l l e t i n No. 14. The Sulphur I n s t i t u t e , Washington. Bettany, J.R. and E.H. Halstead. 19 72. An automated procedure for the nephelometric determination of sul f a t e i n s o i l extracts. Can. J . S o i l S c i . 52: 127-129. Bremner, J.M. and L.A. Douglas. 19 71. Use of p l a s t i c films for aeration i n s o i l incubation experiments. S o i l B i o l . Biochem. 3: 289-296. Chao, T.T., M.E. Harward and C S . Fang. 1965. Exchange reactions between hydroxyl and sulphate ions i n s o i l . S o i l S c i . 109: 310-318. Douglas, L.A. and J.M. Bremner. 1971. An evaluation of the barium peroxide method of maintaining aerobic conditions and determining carbon dioxide evolved during incubation of s o i l s treated with organic materials. S o i l B i o l . Biochem. 3: 289-296. Ensminger, L.E. and J.R. Freney. 1966. Diagnostic techniques for determining s u l f u r d e f i c i e n c i e s i n crops and s o i l s . S o i l S c i . 101: 283-290. - 37 -F e i g l , F. 1956. Spot tests for inorganic a n a l y s i s . E l s e v i e r Publishing Co., New York. 616 pp. Freney, J.R. 1967. Sulfur-containing organics. In S o i l Biochemistry. A.D. McLaren and G.H. Peterson, (Eds.) Marcel Dekker, Inc., New York. pp. 220-259. Freney, J.R., G.E. M e l v i l l e and C.H. Williams. 1971. Organic sulphur fracti o n s l a b e l l e d by addition of 3 5S-sulphate to s o i l . S o i l B i o l . Biochem. 3: 133-141. Johnson, CM. and H. N i s h i t a . 1952. Microestimation of sul f u r i n plant materials, s o i l s and i r r i g a t i o n waters. Anal. Chem. 24: 736-742. Jordan, H.V. and C E . Bardsley. 1958. Responses of crops to sulphur on southeastern s o i l s . S o i l S c i . Soc. Am. Proc. 18: 259-264. Kilmer, V.J. and D.C. Nearpass. 1960. The determination of available sulfur i n s o i l s . S o i l S c i . Soc. Am. Proc. 24: 337-340. Kowalenko, C G . and L.E. Lowe. 1972. Observations on the bismuth s u l f i d e colorimetric procedure for sulfate analysis i n s o i l . Comm. S o i l S c i . Plant Analysis 3: 79-86. Stotzky, G., R.D. Goos and M.I. Timonin. 1962. Microbial changes occurring i n s o i l as a r e s u l t of storage. Plant and S o i l 16: 1-8. Tabatabai, M.A. and J.M. Bremner. 197 2. D i s t r i b u t i o n of t o t a l and available s u l f u r i n selected s o i l s and s o i l p r o f i l e s . Agron. J . 64: 40-44. Walker, D.R. 197 2. S o i l s u l f a t e . I. Extraction and measurement. Can. J . S o i l S c i . 52: 253-260. Williams, C.H. 1967. Some factors a f f e c t i n g the mineral-i z a t i o n of organic sulphur i n s o i l s . Plant and S o i l 26: 205-223. Williams, C.H. and A. Steinbergs. 1959. S o i l sulphur fractions as chemical indices of available sulphur i n some Australian s o i l s . Aust. J . A g r i c . Res. 10: 340-352. - 38 -Z o t t l , H. 1960. Dynamik der St i c k s t o f f m i n e r a l i z a t i o n i n organischen Waldbodenmaterial. Plant and S o i l 13: 166-182. - 39 -CHAPTER 4 MINERALIZATION OF SOIL SULFUR AND ITS RELATION TO SOIL CARBON, NITROGEN AND PHOSPHORUS INTRODUCTION A previous report (Chapter 3) established the importance of understanding the li m i t a t i o n s of methods when applied to examination of microbial mineralization of s o i l s u l f u r . It was shown that i t i s imperative to extract fresh incubated s o i l samples rather than a i r drying p r i o r to analysis, and that calcium chloride extractable sulfate most clo s e l y followed microbial a c t i v i t y . Using the pro-posed techniques, the microbial mineralization of s o i l s u l f u r i s further considered i n r e l a t i o n to other s o i l c h a r a c t e r i s t i c s and extracts. The objective of th i s study i s to measure the microbial mineralization of su l f u r i n four d i f f e r e n t s o i l samples as a function of several parameters frequently used to characterize s o i l samples. I d e n t i f i c a t i o n of these parameters (carbon, nitrogen and phosphorus content; enzyme a c t i v i t y ; pH) i s fundamental to our understanding of mineralization processes and i s valuable for i d e n t i f y i n g future areas of study. - 40 -METHODS AND MATERIALS The s o i l samples, a n a l y t i c a l and incubation methods used i n t h i s study are those reported previously (Chapter 3). Discussion of incubation r e s u l t s i s with reference to the closed system, unless otherwise stated. Additional a n a l y t i c a l and extraction procedures were used to measure the following parameters: pH by glass electrode measure-ments on 1:5 suspensions of s o i l to water or s o i l to 1 N KC1; ar y l s u l f a t a s e a c t i v i t y (Tabatabai and Bremner, 1970b); l i p i d phosphorus content (Kowalenko and McKercher, 19 70); available phosphorus by extraction with 0.5 M sodium bicarbonate at pH 8.5 (Jackson, 1965); organic carbon by the Walkley-Black procedure (Jackson, 1965); t o t a l nitrogen by the Kjeldhal method (Bremner, 1965); t o t a l phosphorus by an i g n i t i o n method (Odynsky, 1936); t o t a l s u l f u r by alkaline oxidation (Tabatabai and Bremner, 1970a); di r e c t estimate of carbon-bonded sulfur using modifications to an o r i g i n a l method (Lowe and DeLong, 1962) adapted from work by Freney et al. (1970). Characterizing analyses (Table 1) were done on a i r dry samples but the s u l f a t e , nitrogen, phosphorus, pH, enzyme, and phospholipid analyses at 0, 1, 2, 4, 8 and 14 week incubation i n t e r v a l s , were carried out on fresh samples. - 41 -RESULTS AND DISCUSSION Some of the chemical c h a r a c t e r i s t i c s of the four s o i l samples used i n t h i s study are shown i n Table 1. The samples were chosen to give a range of s o i l c h a r a c t e r i s t i c s and t h i s i s i l l u s t r a t e d by the range of carbon, nitrogen, phosphorus and pH values. A l s o , as noted i n a previous report (Chapter 3), the Stevens sample was from an area which responds to sulfur f e r t i l i z a t i o n and both Cardinal and Prest samples contain adsorbed inorganic s u l f a t e . Differences between the d i r e c t method of estimating carbon-bonded sulfur and that calculated by difference between t o t a l and Hl-reducible may indicate s t a b i l i t y of cer t a i n sulfur compounds due to cert a i n s o i l c h a r a c t e r i s t i c s . The modification to the o r i g i n a l method (Lowe and DeLong, 1962) resulted i n about a three-fold increase i n recovery of carbon-bonded sulfur i n a l l four s o i l s i n d i c a t i n g that the difference value i s l i k e l y carbon-bonded rather than sulfate s u l f u r . Microbial a c t i v i t y measured by carbon dioxide evolution and n i t r a t e and ammonium accumulation over the 14 week incubation were sim i l a r to those i l l u s t r a t e d by Barrow (1961). The t o t a l amounts of carbon and nitrogen mineralized are shown i n Table 2, and are compared with o r i g i n a l amounts - 42 -TABLE 1. Some chemical analyses of soil samples S o i l sample Analysis Total organic C, % Total N, ppm N Total P, ppm P Total organic P, ppm P Total S, ppm S HI reducible S, ppm S C-bonded S, ppm S Direct D i f f e r e n c ea pH, water pH, 1 N KC1 Cardinal Oxbow Prest Stevens 3.27 890 260 56 300 98 110 202 4.8 4.6 1.97 4.08 2.91 1740 595 232 369 165 99 204 8.3 7.8 3100 975 204 438 139 212 299 5.4 4.4 1710 907 174 214 38 96 176 6.5 5.6 aT o t a l s u l f u r minus HI reducible s u l f u r . - 43 -TABLE 2. Mineralization of oarbon and nitrogen from four soils during 14 weeks incubation C09-C a r,-? Mineralized 0 ^ Ori g i n a l ^ -& or _ + % of inorganic N evolved o r i g i n a l N(N0~ +NH ) t o t a l , M n -...„ +. S o i l ppm organic C ppm H N (N03 +NH^ ) ppm Cardinal 1064 3.3 20 2.2 8 Oxbow 1250 6.3 107 6.2 10 Prest 2706 6.6 252 8.1 34 Stevens 817 0.4 73 4.3 7 - 44 -present. Both Oxbow and Prest mineralized a high proportion of carbon and nitrogen, but the Stevens sample mineralized a much smaller proportion of i t s organic carbon and a r e l a t i v e l y high amount of i t s nitrogen. The Cardinal sample was unable to convert ammonium nitrogen into n i t r a t e and there may have been several possible reasons. Low pH, i n t e r f e r i n g organic compounds, lack of adequate phosphorus or lack of n i t r i f y i n g bateria were p o s s i b i l i t i e s for i n h i b i t i o n of n i t r i f i c a t i o n . Although the i n i t i a l pH of the sample was low, the pH of the Prest sample during some periods of the incubation was lower than Cardinal values, so th i s does not appear to be the important c o n t r o l l i n g factor i n t h i s case. Similar to Barrow's (1961) r e s u l t s , the pH although f l u c t u a t i n g throughout the incubation period showed a general d e c l i n e . The Oxbow sample did not fluctuate as much as the others, probably because of i t s higher buffer capacity with the presence of carbonate. Correlation of pH with extractable sulfate was a l l very highly correlated (r values between 0.6 5 and 0.70) but whether t h i s i s a cause or an eff e c t r e l a t i o n s h i p could not be determined from the r e s u l t s . - 45 -A r e l a t i v e l y wide range of available phosphorus (bicarbonate extractable) was present i n the four s o i l s (Figure 1). In a l l cases there appeared to be immobilization of phosphorus p a r t i c u l a r l y i n the Stevens samples where a high amount of available phosphorus was o r i g i n a l l y present. A very poor c o r r e l a t i o n (r = 0.15) resulted on comparing mineralized phosphorus with carbon dioxide evolved. This may be a r e f l e c t i o n of high microbial demands for phosphorus. However, thi s p a r t i c u l a r extractant, which should s o l u b i l i z e various inorganic fractions including adsorbed and soluble phosphorus, may not have been the best indicator i n r e l a t i o n to microbial a c t i v i t y , as was found when various sulfate extractants were correlated with carbon dioxide evolution (Chapter 3). Lipid-P showed a general decline as microbial a c t i v i t y progressed in d i c a t i n g that some organic phosphorus components may be a source of phosphorus, however, the o r i g i n a l amount of t h i s phosphorus component was small (ranging from 2.4 to 6.1 ppm P). Changes i n l i p i d - P were very highly (99.9%) negatively correlated (r = -0.69) with carbon dioxide evolution, which could have resulted from the l i p i d s serving as a carbon or a phosphorus source f o r microbes. Available phosphorus extracted by - 46 -IOCH 8 0 -6 0 -zz o JZ CL O JZ Q_ 2 0 -0 Cardinal Oxbow Prest Stevens A A • • o o \ \ \ \ b <y :<—x— . X 1 1 1— — i — i 1— — — i — i — i — —1 1 1 1 —1 1 8 14 Time (weeks) FIGURE 1. Sodium bicarbonate extractable phosphorus of four s o i l samples during 14 week incubation ' - 47 -bicarbonate throughout the incubation was s i g n i f i c a n t l y correlated with calcium chloride extractable sulfate (r = 0.48) but was not s i g n i f i c a n t with phosphate b u f f e r , acetate or bicarbonate methods. The low r values might be expected, since there was poor c o r r e l a t i o n of available phosphorus with microbial a c t i v i t y . Correlations of l i p i d - P with extractable sulfate were very highly s i g n i f i c a n t l y correlated (r between 0.80 and 0.85) r e f l e c t i n g a possible l i n k to s u l f u r through r e a d i l y available carbon present i n these compounds. Arylsulfatase a c t i v i t y d r a s t i c a l l y declined i n a l l four s o i l s as microbial a c t i v i t y progressed (Figure 2). Tabatabai and Bremner (19 72) reported mineralizable sulfur (10 week value) was not s i g n i f i c a n t l y correlated with i n i t i a l a r y l s u l f atase a c t i v i t y with 12 Iowa samples. Cooper (1972) indicated a possible implication of arylsulfatase a c t i v i t y with an i n i t i a l rapid release of sulfate upon wetting some Nigerian s o i l s and showed a decline i n a c t i v i t y during a dry period i n some f i e l d sampling. Preliminary r e s u l t s have indicated that the enzyme i s associated with the s o l i d f r a c t i o n of the s o i l since the enzyme was not extracted with the enzyme assay acetate b u f f e r , even with a sonic - 48 -2 0 0 $ 140* \ TJ a> g 120" a> Cardinal - - A Oxbow X— X Prest • • • • Stevens o— — o 100 a. o c i a. 8 0 3. Time (weeks) FIGURE 2. Arylsulfatase a c t i v i t y of four s o i l samples during 14 week incubation - 49 -dispersion (L.E. Lowe, unpublished data). The exact role of t h i s enzyme i n the s o i l could not be evaluated with current information, but i t did appear from these r e s u l t s that a r y l s u l f a t a s e a c t i v i t y was of secondary importance i n sulfate release during incubation of these s o i l s . Correlation (r = 0.49) of ary l s u l f a t a s e a c t i v i t y throughout the incubation with calcium chloride extractable sulfate was just s i g n i f i c a n t (95% l e v e l ) . Changes that occurred i n levels of sulfate i n the four extracts of two representative samples are shown i n Figures 3 and 4. Only Prest and Stevens samples are i l l u s t r a t e d showing two types of patterns which occurred. The Cardinal sample resembled Prest by having considerably less sulfate extracted with calcium chloride than the remaining three extractants, the differences between them were considered to include adsorbed and organic s u l f a t e . Oxbow resembled Stevens where there were much smaller differences among the four extraction methods. In a l l cases with bicarbonate, phosphate b u f f e r , and acetate extractions of s u l f a t e , somewhat inconsistent fluctuations were prominent during the i n i t i a l incubation period (0-4 weeks). The poor correlations of extractable sulfate using these three methods - 50 -IOO 80-• • / \ \ / o \ Calcium chloride x x Acetate A Phosphate buffer o o Bicarbonate • -a VP-. *a.... 8 •a o -A —J 14 Time (weeks) FIGURE 3. Sulfate sulfur extracted by four solutions from Prest s o i l sample during 14 week incubation Calcium chloride x-Acetate A A Phosphate buffer o -o Bicarbonate • • 1 2 4 8 14 Time (weeks) FIGURE 4. Sulfate s u l f u r extracted by four extracting solutions from Stevens sample during 14 week incubation - 52 -with microbial a c t i v i t y may have been due to these f l u c t u a t i o n s . These fluctuations may r e f l e c t the appearance of organic sulfates during the microbial adjustment to the new environmental conditions. Several attempts to separate the organic sulfate contribution i n the extracts were unsatisfactory. Methods using Sephadex G-25, barium pre-c i p i t a t i o n and charcoal adsorption had c e r t a i n l i m i t a t i o n s . An ion retardation r e s i n (AG 11A8 from Bio-Rad Laboratories) separation which gave a minimum estimate of organic sulfate indicated s i g n i f i c a n t quantities of organic sulfate (5-4 8% of t o t a l sulfate extracted) were present i n the bicarbonate and phosphate buffer extracts of the four s o i l s . Although there appears to be a r e a l contribution by organic sulfate to measurements of extractable s u l f a t e , the degree of t h i s contribution and the structure of the organic sulfate involved i s s t i l l unknown. Since i t i s u n l i k e l y that the contribution of organic sulfate remains constant throughout the incubation i n t e r v a l s , i t i s not possible to calculate a net mineralization of sulfur i n a simple manner which attri b u t e s a l l extracted sulfate to anionic s u l f a t e . Williams (1967) used potassium phosphate buffer as the extractant and although the trends could be considered a - 53 -correct i n t e r p r e t a t i o n , absolute values for net mineral-i z a t i o n may not necessarily be true. The 0.15% calcium chloride extract may also contain some organic sulfate which would be measured by the hydriodic acid reduction method, but current methods are not s a t i s f a c t o r y to measure the small quantities which may be present. Walker (19 72) did reveal d i f f e r e n t r e s u l t s i n a few s o i l s tested using t h i s extractant when quantitative r e s u l t s with a barium p r e c i p i t a t i o n method were compared with hydriodic reduction, which may be due to organic s u l f a t e . A previous report (Chapter 3) showed that the calcium chloride extract of fresh samples through the incubation was the most suitable method for following s u l f u r mineral-i z a t i o n . The accumulation of sulfate using t h i s extractant for four s o i l s used i s shown i n Figure 5. The pattern of sulfate release i s much more consistent i n t h i s extract than the other three extracts. The Prest sample l i k e l y r e f l e c t s i t s very high microbial a c t i v i t y . However, as was noticed with the other extracts, major changes occur i n the i n i t i a l four week period, which was quite s i m i l a r to that which was previously reported (Barrow, 1961). - 54 FIGURE 5. Calcium chloride extractable sulfate sulfur of four s o i l samples during 14 week incubation - 55 -Correlation of nitrogen mineralization with carbon dioxide carbon evolved previously established the r e l a t i o n -ship with microbial a c t i v i t y . In turn, very highly s i g n i f i c a n t correlations were found with sulfate mineralized (calcium chloride extract) and nitrogen mineralized (r = 0.98) as well as to changes i n ammonium nitrogen (r = 0.90) and n i t r a t e nitrogen (r = 0.79), the higher value with ammonium rather than n i t r a t e may indicate a r e l a t i o n to ammonification rather than n i t r i f i c a t i o n . Whether sulfur mineralization i s d i r e c t l y related to nitrogen mineralization or i n d i r e c t l y through carbon mineralization could not be discerned. The l a t e r may be the case since Williams (1967) noted that toluene suppressed mineralization of su l f u r but not of nitrogen. Relationships of su l f u r and nitrogen mineralization with carbon dioxide evolution are represented graphically i n Figures 6 and 7. Two preliminary incubation tests were made with the Stevens sample to consider amendments which could influence the mineralization of s u l f u r . Williams (1967) showed that adding calcium or magnesium carbonate increased mineralization of s u l f u r i n some s o i l s used, however i n the case of the Stevens sample i n t h i s study a s i g n i f i c a n t difference - 56 -280 n 240-Cardinal Oxbow Prest Stevens A A x x o • O -o cl200-"O a> £ 160-o a> 120-c: a> cn O 4 8 12 16 20 24 28 Carbon dioxide carbon evolved (ppm x 100) FIGURE 6. Nitrogen mineralized i n r e l a t i o n to carbon dioxide evolved from four s o i l samples during 14 week incubation - 57 -—51 i 1 1 1 ' 1 1 1 » 1 1 1 • 1 0 4 8 12 16 20 24 28 Carbon dioxide carbon evolved (ppm x 100) FIGURE 7. Sulfur mineralized i n r e l a t i o n to carbon dioxide evolved from four s o i l samples during IH week incubation - 58 -(paired t test) was not evident between an untreated and 0.5% calcium carbonate treated sample. Stewart and Whitfield (1965) suggested that a high N:S r a t i o i n a s o i l would r e s u l t i n immobilization of s u l f u r . S u f f i c i e n t nitrogen was added to a sample of the Stevens s o i l to change the N:S r a t i o from 8.1 to 2 0 using ammonium n i t r a t e and incubated under the described conditions. Sulfur mineralization (calcium chloride extractant) was followed at 1, 2, 4, and 8 week i n t e r v a l s . Immobilization of sulfur occurred at 4 and 8 week samplings since no sulfate was detected in the extract. The microbial a c t i v i t y was s l i g h t l y depressed by the high nitrogen a d d i t i o n , but because there was nearly s i m i l a r a c t i v i t y and that sulfate content fluctuated i n the i n i t i a l four weeks, the immobilization was considered microbial. With the four s o i l s used i n t h i s study, the net sulfur mineralized, expressed as a percentage of several s u l f u r f r a c t i o n s , was largest with the hydriodic acid reducible f r a c t i o n (Table 3). Also of note was that the two s o i l s (Stevens and Cardinal) which resulted i n l i t t l e or no sulfate accumulation afte r 11 weeks incubation have a greater proportion of t h e i r s u l f ur i n the carbon-bonded - 59 -TABLE 3. Net sulfur mineralized in 14 weeks expressed as percentage of various sulfur fractions Total HI C-bonded S Sample Total S organic Sa reducible S (difference) Cardinal 0 0 0 0 Stevens 0.2 0.2 1.0 0.2 Oxbow 1.5 1.6 3.4 2.7 Prest 4.4 4.8 13.9 6.4 aT o t a l S corrected for' inorganic sulfur present i n phosphate buffer extract. - 60 -form. One would anticipate from a chemical consideration, that since inorganic sulfate release from organic sulfate would require hydrolysis whereas the carbon-bonded sulfur would require oxidation steps, the hydriodic acid reducible form of sulfur would be r e l a t i v e l y more important i n s o i l s u l f u r mineralization on a short term basis. However, even 3 5 with S-sulfate incorporation data (Freney et al. , 1971) i t i s d i f f i c u l t to come to any conclusions whether one f r a c t i o n i s more important than another i n t h i s regard because of the lack of understanding of the mechanisms involved. More research i n th i s area would indeed be u s e f u l . Tabatabai and Bremner (197 2) were unable to r e l a t e mineralizable sulfate (10-week value) with t o t a l s u l f u r , sulfate s u l f u r , organic carbon, t o t a l nitrogen, mineralizable nitrogen (10-week value) or arylsulfatase a c t i v i t y , using 0.1 M lithium chloride extraction of s u l f a t e , although they found s i g n i f i c a n t correlations of t o t a l s u l f u r with organic carbon and t o t a l nitrogen for surface and subsurface samples. Since only four s o i l s were used i n t h i s study, s t a t i s t i c a l c orrelations of th i s type were not s u i t a b l e , but Table M-indicates that mineralizable sulfur (lH-week value) could be influenced by C:N and C:S r a t i o s . The table also points out - 61 -TABLE 4. Comparison of mineralizable sulfur (14 weeks) with two soil to solution ratios for extraction of soluble sulfate (ppm S), C:N and C:S ratios I n i t i a l S0 4-Sa  with r a t i o of Sample Mineralizable S (ppm) 1:1 1:5 C:S C :N Cardinal 0 0.6 5.0 103 37 Stevens 0.4 0.9 1.6 115 17 Oxbow 5.6 1.9 2.4 57 11 Prest 19.3 9.3 10.6 84 13 aAnalyses on o r i g i n a l a i r dried samples using 0.15% calcium c h l o r i d e . - 62 -t h a t t h e s o i l t o s o l u t i o n r a t i o f o r e x t r a c t i o n o f s o l u b l e s u l f a t e ( C h a p t e r 3) may a l s o be an i m p o r t a n t c o n s i d e r a t i o n i n p r e d i c t i n g r e l e a s e o f s u l f a t e , p a r t i c u l a r l y f o r samples c o n t a i n i n g adsorbed s u l f a t e . The v a l u e f o r m i n e r a l i z a b l e s u l f u r i s not e x p e c t e d t o be g r e a t l y d i f f e r e n t when t a k e n between about the e i g h t h and f o u r t e e n t h weeks o f i n c u b a t i o n , s i n c e t h e changes i n e x t r a c t a b l e s u l f a t e a r e r e l a t i v e l y s m a l l (see F i g u r e 5 ) . CONCLUSION R e l a t i o n s h i p s o f s u l f u r m i n e r a l i z a t i o n , as i n d i c a t e d by s u l f a t e r e l e a s e d u r i n g an a e r o b i c i n c u b a t i o n o f s o i l s , were shown w i t h m i n e r a l i z a t i o n o f carbon and n i t r o g e n . I t i s i m p e r a t i v e t o use a s u i t a b l e e x t r a c t i n g a g e n t , so t h a t the r e s u l t s w i l l not be masked by t h e p r e s ence o f adsorbed and, i n p a r t i c u l a r , o r g a n i c s u l f a t e . M i n e r a l i z a t i o n o f s o i l s u l f u r was r e l a t e d t o carbon d i o x i d e r e l e a s e and hence m i c r o b i a l a c t i v i t y . Whether s u l f u r m i n e r a l i z a t i o n i s d i r e c t l y r e l a t e d t o n i t r o g e n m i n e r a l i z a t i o n , t h a t i s , s u l f u r and n i t r o g e n i n t h e same compounds o r whether t h e y a r e l i n k e d i n d i r e c t l y by c a r b o n c o u l d not be e v a l u a t e d , but was c o n s i d e r e d t o be a t l e a s t i n p a r t due t o an i n d i r e c t r e l a t i o n s h i p . A r y l s u l f a t a s e a c t i v i t y i n s o i l i s s t i l l not - 63 -understood and i t s d e c l i n i n g a c t i v i t y through the i n c u b a t i o n made i t appear to be of minor importance i n t h i s p a r t i c u l a r study. Phosphorus appeared to be used i n l a r g e amounts during m i c r o b i a l a c t i v i t y , and poor r e l a t i o n s h i p s to m i c r o b i a l a c t i v i t y may have been because of a d e f i c i e n c y of the element or the e x t r a c t i o n method was not s u i t a b l e . The d e c l i n e of l i p i d - P w i t h no concurrent r e l e a s e of i n o r g a n i c phosphorus f u r t h e r supports the hypothesis of a phosphorus d e f i c i e n c y . Phospholipids appeared to be a r e a d i l y a v a i l a b l e source of carbon f o r the microorganisms. I t i s now apparent th a t f u t u r e experiments should be c a r e f u l l y designed with due regard to the c r i t i c a l e f f e c t the a n a l y t i c a l method has on the r e s u l t s observed. The type of e x t r a c t i n g s o l u t i o n used, the s o i l to s o l u t i o n r a t i o s e l e c t e d , and the use of d r i e d or f r e s h samples f o r a n a l y s i s profoundly a f f e c t s the degree of apparent m i n e r a l i z a t i o n of s u l f u r . Work on s u l f u r m i n e r a l i z a t i o n reported i n the l i t e r a t u r e should be c r i t i c a l l y re-examined and new work planned i n the l i g h t of these c o n c l u s i o n s . - 64 -REFERENCES Barrow, N.J. 1961. Studies on the mineralization of sulphur from s o i l organic matter. Aust. J . A g r i c . Res. 12: 306-319. Bremner, J.M. 1965. Total nitrogen. In Methods of S o i l Analysis. Agron. 9, Part I I , 1149-1178. Cooper, P.J.M. 1972. A r y l sulfatase a c t i v i t y i n northern Nigerian s o i l s . S o i l B i o l . Biochem. 4: 333-337. Freney, J.R., G.E. M e l v i l l e and C.H. Williams. 1970. The determination of carbon bonded su l f u r i n s o i l . S o i l S c i . 109: 310-318. Freney, J.R., G.E. M e l v i l l e and C.H. Williams. 1971. Organic sulphur fractions l a b e l l e d by addition of 35s-sulphate to s o i l . S o i l B i o l . Biochem. 3: 133-141. Jackson, M.L. 1965. S o i l chemical a n a l y s i s . Prentice-H a l l , Inc. Englewood C l i f f s , New Jersey. 498 pp. Kowalenko, C G . and R.B. McKercher. 1970. An examination of methods for extraction of s o i l phospholipids. S o i l B i o l . Biochem. 2: 269-273. Lowe, L.E. and W.A. DeLong. 1963. Carbon bonded sulphur i n selected Quebec s o i l s . Can. J . S o i l S c i . 43: 151-155 . Odynsky, W. 193 6. S o l u b i l i t y and d i s t r i b u t i o n of phosphorus i n Alberta s o i l s . S c i . A g r i c . 16: 652-664. Stewart, B.A. and C.J. W h i t f i e l d . 1965. Effects of crop residue, s o i l temperature, and sulfur on the growth of winter wheat. S o i l S c i . Soc. Am. Proc. 29: 752-755. Tabatabai, M.A. and J.M. Bremner. 197 0a. An a l k a l i n e oxidation method for determination of t o t a l s u l f u r i n s o i l s . S o i l S c i . Soc. Am. Proc. 34: 62-65. - 65 -Tabatabai, M.A. and J.M. Bremner. 197 0b. Arylsulfatase a c t i v i t y of s o i l s . S o i l S c i . Soc. Am. Proc. 34: 225-229. Tabatabai, M.A. and J.M. Bremner. 1972. D i s t r i b u t i o n of t o t a l and available s u l f u r i n selected s o i l s and s o i l p r o f i l e s . Agron. J . 64: 40-44. Walker, D.R. 197 2. S o i l s u l f a t e . I. Extraction and measurement. Can. J . S o i l S c i . 52: 253-260. Williams, C.H. 1967. Some factors a f f e c t i n g the mineralization of organic sulphur i n s o i l s . Plant and S o i l 26: 205-223. - 66 -CHAPTER 5 THE EFFECT OF NITROGEN ADDITIONS ON THE MINERALIZATION OF SOIL SULFUR DURING INCUBATION INTRODUCTION The importance of a nitrogen:sulfur balance i n plant n u t r i t i o n has been studied and recognized as being important for maximum production as well as quality of production (Beaton, 1966; Dijkshoorn and Van Wijk, 1967; Stewart and Carsons, 1969). An important source of sulfur for plant n u t r i t i o n i s the s o i l , where both organic and inorganic forms are present. The organic f r a c t i o n i s an important source i n many surface s o i l s and mineralization has been shown to be influenced by nitrogen:sulfur r a t i o s i n experiments with various organic compounds (Barrow, 1960; Stewart, Porter and V i e t s , 196 6b) and crop residues (Stewart, Porter and V i e t s , 1966a; Stewart and W h i t f i e l d , 1965; Whitehead, 1970). Numerous reports indicate that there i s a f a i r l y close r e l a t i o n s h i p between the mineral-i z a t i o n of su l f u r and nitrogen from s o i l s (Barrow, 1958; Nyborg, 1968; Stewart, 1966; White, 1959; Williams 1968), however poor correlations have been reported for - 67 -mineralizable sulfur with other s o i l analyses including t o t a l s u l f u r , sulfate s u l f u r , organic carbon, t o t a l nitrogen, mineralizable nitrogen and arylsulfatase a c t i v i t y (Haque and Walmsley, 197 2; Tabatabai and Bremner, 1972). The poor correlations may be due to di f f e r e n t rates at which su l f u r and nitrogen components are mineralized i n the s o i l and t h i s was supported by Swift and Posner (1972) with work on nitrogen, phosphorus and sulfur contents of s o i l humic acids that were fractionated to various molecular weights. The constancy of sulfur i n various fractions was suggested to have been due to si m i l a r sulfur and carbon mineralization r a t e s . Previous work (Chapter 4) indicated that some of the discrepancies that may arise may be a res u l t of the p a r t i c u l a r techniques used, such as the type of sulfate extractant and method of analysing for s u l f a t e . The exact r e l a t i o n s h i p of nitrogen to sulfur mineralized was unresolved and carbon was suggested as a l i n k between the two. Since there appeared to be a rel a t i o n s h i p between sul f u r and nitrogen mineralization i n the s o i l , changes that occur i n mineralization of sulfur i n s o i l when nitrogen additions at level s that may be used i n f e r t i l i z e r - 68 -applications was studied. An incubation procedure, i n the absence of photosynthetic a c t i v i t y , was applied to study the influence that nitrogen applications may have on the mineralization of s o i l s u l f u r . METHODS AND MATERIALS The incubation method and conditions were those previously reported (Chapter 3) using the closed system. The nitrogen additions were made by a suitable solution being added to the water to bring the moisture content to 100 cm water tension. Carbon dioxide evolved was absorbed with IN NaOH for the eight week incubation period, and the absorption solution was changed and analysed at 1, 2, 4 and 8 weeks. At the end of eight weeks, pH i n 1:5 s o i l to water solution and sulfate extracted by 0.15% calcium chloride was determined on fresh samples. A l l res u l t s were calculated to oven dry weight. The nitrogen sources used were NH^OH, KN03, NH^H^O^ , NH4N03 and (NH2)2C0 at levels of 50, 100 and 150 ppm nitrogen. Samples where no nitrogen additions were made were also included. The s o i l samples (Oxbow and Prest) used i n th i s study were previously described (Chapters 3 and 4). These two samples were chosen because of t h e i r - 69 -higher extractable sulfate l e v e l s , hence measurements would be more accurate. Thus, the experiment was a f a c t o r i a l design with two s o i l s (Prest and Oxbow), using f i v e sources of nitrogen at four rates (0, 50, 100 and 15 0 ppm N). Extractable sulfate and pH were measured at the beginning and a f t e r eight weeks incubation. RESULTS AND DISCUSSION A number of ways are possible i n a consideration of the influence of nitrogen additions on s u l f u r mineralized from s o i l s , and an incubation method using an analysis of extractable sulfate a f t e r a certain time was used i n t h i s experiment. A previous report (Chapter 4) showed that large fluctuations of extractable sulfate occurred during the i n i t i a l four weeks of incubation, and that at eight weeks and beyond the pattern was more consistent. For t h i s reason i t was decided to examine the influence of nitrogen additions by measurements afte r eight weeks incubation. An extraction with calcium chloride was used since i t most clo s e l y followed microbial a c t i v i t y shown by carbon dioxide evolved and nitrogen mineralized. The r e l a t i v e l y high amounts of nitrogen did not appear to be at a l e v e l high enough to influence the hydriodic acid - 70 -reduction (see Chapter 1) and a l l samples contained the same amount of extractable sulfate (as compared to t h e i r respective samples containing no added nitrogen) at the beginning of the incubation. The r e s u l t s of the extractable sulfate present i n the samples a f t e r eight weeks incubation are shown i n Table 1. The two s o i l s show very large differences i n sulfate extractable at t h i s time and that each i n d i v i d u a l s o i l behaved d i f f e r e n t l y to the nitrogen additions. The Oxbow sample showed a general decrease of sulfate with nitrogen additions and Prest was the inverse, except with the addition of potassium n i t r a t e . The o r i g i n a l sulfate extractable by calcium chloride was 6.1 ppm sulfur for Oxbow and 32.2 ppm sulfur for Prest. A s t a t i s t i c a l analysis using a f a c t o r i a l design method revealed very highly s i g n i f i c a n t differences (F, 0.005) due to the nitrogen and s o i l samples. The o v e r a l l differences between rates were not s i g n i f i c a n t possibly because of the r e l a t i v e l y small differences and the two samples behaving opposite to one another. However, interactions of nitrogen source with r a t e , nitrogen source with s o i l , rate with s o i l , and s o i l , source and rate a l l together were very highly - 71 -TABLE 1. Calcium chloride (0.15%) extractable sulfate of soil samples treated with various sources at several rates after eight weeks incubation at 100 cm moisture tension at 30°C Nitrogen source Rate (ppm N) Extractable sulfate s u l f u r (ppm) Oxbow Prest None 0 11.9 51.9 NH^OH 50 100 150 8.8 7.4 6.2 52.2 53 . 0 52.8 KNO, 50 100 150 8.2 6.0 8 . 0 53.0 48.6 43 .1 NH4H2P04 50 100 150 5.8 7.4 8.4 55.2 61.4 64.1 NH4N03 50 100 150 9.1 7 . 2 7 . 0 55.0 54.2 60.0 (NH2)2CO 50 100 150 6.9 7.0 7.4 52.8 55 .0 52.8 - 72 -s i g n i f i c a n t . This indicated that each s o i l behaved d i f f e r e n t l y with the nitrogen sources and that the rate within each treatment was s i g n i f i c a n t . Analysis of variance of the i n d i v i d u a l r e s u l t s revealed that the precision of the r e s u l t s f o r the i n d i v i d u a l samples was s u f f i c i e n t to allow the small differences of the treatment to be s i g n i f i c a n t . S i m i l a r l y , an analysis of variance of r e s u l t s of pH and carbon dioxide evolved a f t e r the incubation indicated very highly s i g n i f i c a n t r e s u l t s due to the nitrogen source, s o i l , rate (the rate was s i g n i f i c a n t at only F, 0.01 for carbon dioxide evolved) and a l l interactions of s o i l , source and r a t e . The variance between the r e p l i c a t e s of the pH analyses were also s i g n i f i c a n t indicating that some of the differences were s u f f i c i e n t l y small so that they could not be distinguished from the i n d i v i d u a l r e p l i c a t e s . These s i m i l a r i t i e s between the various variances of extractable s u l f a t e , pH and carbon dioxide evolved would suggest that these may be r e l a t e d , hence the c o r r e l a t i o n c o e f f i c i e n t s (simple, p a r t i a l and multiple) were calculated (Table 2). The r e s u l t s of simple correlations would indicate that pH and carbon dioxide evolved are r e l a t e d . This r e l a t i o n s h i p i s l i k e l y due to - 73 -TABLE 2. Correlation oo extractable sulfate (Y evolved (X^) and pH after an eight wee two soils treated efficients (r and R) of ) with carbon dioxide (Xg) of results k incubation of with nitrogen n , . • S o i l sample Correlation £ • c o e f f i c i e n t Oxbow Prest Oxbow and Prest Simple: r 1 2 0.318*** -0.346*** -0.981*** 0.148* 0.562*** 0.993: A A . ry i r „ 0.334*** -0.249*** -0.982 P a r t i a l : r , 0a 0.047 0.524* 0.802** y l . 2 r 0 ,b 0.306 -0.071 -0.323 y2.1 Multiple: R , 0 0 .337 0 .565** 0.994** y.12 '"'Significant (95%) **Highly s i g n i f i c a n t (99%). *ft*Very highly s i g n i f i c a n t (99.9%) NOTE: Si g n i f i c a n t considered at 95% and 99% for p a r t i a l and multiple c o r r e l a t i o n s . C o r r e l a t i o n between extractable sulfate and carbon dioxide evolved for fixed pH. ^Correlation between extractable sulfate and pH for fixed carbon dioxide evolved. - 74 -the formation of ammonium and n i t r a t e ions by microbial a c t i v i t y , which would e f f e c t the pH. These differences varied f o r each of the s o i l samples. There would appear to be some influence of microbial a c t i v i t y and pH on the sulfate formed, although these would vary with the in d i v i d u a l s o i l sample. Overall, i t appears that the sulfate formed was influenced by the microbial a c t i v i t y and the r e s u l t i n g pH of t h i s a c t i v i t y . Thus the ef f e c t of the nitrogen sources at the various rates on the two samples was a r e s u l t of the influence they had on the microbial a c t i v i t y as shown by the carbon dioxide evolved and the r e s u l t i n g by-products as shown by the pH. This, influence was d i f f e r e n t for each s o i l sample that was used, hence other s o i l c h a r a c t e r i s t i c s would influence the exact expression of microbial a c t i v i t y on the sulfu r mineralized. It i s of interest to consider the influence that the c a r r i e r of the nitrogen may have on the sulfate released. The two that are very s t r i k i n g are KNO^ and NH^H^PO^ treatments of the Prest sample. Potassium n i t r a t e resulted i n a decline i n the amount of sulfate extractable a f t e r the incubation period and i t was noted that carbon dioxide evolved decreased (which was opposite to that of other treatments for that sample). This was thought to be due - 75 -to the c a t i o n , K , which possibly had some influence on + + + NH^ i n t e r a c t i o n s . Some influences of K on NH^ uptake has been indicated i n higher plants (Monem Balba and Shabanah, 1972). Relatively large increases i n the amount of extractable sulfate resulted from NH^f^PO^ treatments which may have been a r e s u l t of an ef f e c t on microbial a c t i v i t y , as a r e l a t i v e l y high amount of carbon dioxide carbon was evolved with t h i s treatment. Indeed, phosphorus interactions with sulfur have been shown i n experiments of plant response to f e r t i l i z e r treatments (Bouma, 1971; Eppendorfer, 1971; Jones et a l . , 1970; Oke, 1969). Phosphate applications may also have an ef f e c t on sulfate adsorption (Williams, 1966) and t h i s sample was shown to contain adsorbed sulfate (Chapter 3). CONCLUSION The data showed that nitrogen applications to a s o i l sample can influence the amount of sulfate released and that the exact r e s u l t was dependent on the p a r t i c u l a r s o i l , nitrogen source and rate that was used. In one s o i l (Prest) increases i n su l f u r mineralized were generally observed and i n the other (Oxbow) decreases. It appeared that the influence of added nitrogen on sulfate extracted - 76 -was a r e s u l t of an e f f e c t on microbial a c t i v i t y as indicated by changes i n carbon dioxide evolved and the r e s u l t i n g pH of the s o i l . In addition to the i n t e r a c t i o n of the source and the rate on the s u l f u r mineralized, the p a r t i c u l a r nitrogen c a r r i e r such as potassium or phosphate may be important i n the reaction. Differences due to the oxidation state of nitrogen added (whether as NO^  or NH^) were not evident. The exact reason why one s o i l behaved d i f f e r e n t l y from the other could not be determined, and a greater d i v e r s i t y of samples would be needed to evaluate the reasons f o r t h i s . The influence of nitrogen additions on s u l f u r n u t r i t i o n of plants may be important i f mineralization of s o i l organic forms i s an important source. This influence may be p a r t i c u l a r l y important i n s o i l s containing r e l a t i v e l y low amounts of soluble and adsorbed s u l f a t e , where mineralization of organic sul f u r may be important. It would be d i f f i c u l t to d i f f e r e n t i a t e the importance of t h i s e f f e c t i n s o i l s where only small net amounts of mineralized s u l f u r are evident, unless there i s a greater understanding of the metabolic process involved. U t i l i z a t i o n of l a b e l l e d s u l f u r i n experiments and knowledge of the s o i l organic s u l f u r components would be very informative. It was - 77 -evident that much more work can be done on the mineralization of su l f u r i n s o i l p a r t i c u l a r l y the processes affected by a g r i c u l t u r a l practices such as f e r t i l i z a t i o n , organic matter (straw, manure) incorporation and pesticides usage would be important considerations. - 78 -REFERENCES Barrow, N.J. 195 8. E f f e c t of the nitrogen and sulfur content of organic matter on the production of ammonium and sulphate. Nature 181: 18 06-18 07. Barrow, N.J. 196 0. The ef f e c t of varying the nitrogen, sulphur, and phosphorus content of organic matter on i t s decomposition. Aust. J . A g r i c . Res. 11: 317-330. Beaton, J.D. 1966. Sulfur requirements of ce r e a l s , tree f r u i t s , vegetables, and other crops. S o i l S c i . 101: 267-282 . Bouma, D. 1971. Effects of phosphorus and sulphur on dark r e s p i r a t i o n of subterranean clover leaves. Aust. J . Ag r i c . Res. 22: 723-730. Dijkshoorn, W. and A.L. Van Wijk. 1967. The sulphur requirement of plants as evidenced by the sulphur-nitrogen r a t i o i n the organic matter. A review of published data. Plant and S o i l 26: 129-157. Eppendorfer, W.H. 1971. Effects of S, N and P on amino acid composition of f i e l d beans (Viaia faba) and responses of the b i o l o g i c a l value of the seed protein to S-amino acid content. J . S c i . Fd. A g r i c . 22: 501-505. Haque, I. and D. Walmsley. 197 2. Incubation studies on mineralization of organic sulphur and organic nitrogen. Plant and S o i l 37: 255-264. Jones, M.B., P.W. Lawler and J.E. Ruckman. 1970. Differences i n annual clover responses to phosphorus and s u l f u r . Agron. J . 62: 439-442. Monem Balba, A. and M.R. Shabanah. 1972. The e f f e c t of calcium carbonate and potassium sulfate on the e f f i c i e n c y of ammonium as tested by barley seedlings. Plant and S o i l 37: 3 3-39. Nyborg, M. 1968. Sulfur deficiency i n cereal grains. Can. J . S o i l S c i . 48: 37-41. - 79 -Oke, O.L. 196 9. Sulfur n u t r i t i o n of legumes. Expl. A g r i c . 5: 111-116. Stewart, B.A. 1966. Nitrogen-sulphur relationships i n plant t i s s u e s , plant residues and s o i l organic matter. S o i l Chemistry and F e r t i l i t y . Meeting of Commission II and IV of the International Society of S o i l Science, Transactions, Aberdeen. (G.V. Jacks, ed.) 131-138. Stewart, B.A. and R.B. Carsons. 196 9. Nitrogen-sulphur relationships i n wheat, maize and beans. Agron. J . 61: 267-271. Stewart, B.A., L.K. Porter and F.G. V i e t s , J r . 1966a. Ef f e c t of sulfur contents of straw on rates of decomposition and plant growth. S o i l S c i . Soc. Am. Proc. 30: 355-358. Stewart, B.A., L.K. Porter and F.G. V i e t s , J r . 1966b. Sulfur requirements for decomposition of c e l l u l o s e and glucose i n s o i l . S o i l S c i . Soc. Am. Proc. 30: 453-456. Stewart, B.A. and C.J. Whitefield. 1965. Effects of crop residue, s o i l temperature, and sulfur on the growth of winter wheat. S o i l S c i . Soc. Am. Proc. 29: 752-755. Swift, R.S. and A.M. Posner. 197 2. Nitrogen, phosphorus and sulphur contents of humic acids fractionated with respect to molecular weight. J . S o i l S c i . 23: 50-57 . Tabatabai, M.A. and J.M. Bremner. 1972. D i s t r i b u t i o n of t o t a l and available s u l f u r i n selected s o i l s and s o i l p r o f i l e s . Agron. J . 64: 40-44. White, J.G. 195 9. Mineralization of nitrogen and sulphur i n sulphur-deficient s o i l s . New Zealand J . A g r i c . Res. 2: 255-258. Whitehead, D.C. 197 0. Carbon, nitrogen, phosphorus and su l f u r i n herbage plant roots. J . Br. Grassland Soc. 25: 236-241. - 80 -Williams, C.H. 1966. Nitrogen, sulphur and phosphorus, t h e i r interactions and a v a i l a b i l i t y . S o i l Chemistry and F e r t i l i t y . Meeting of Commissions II and IV of the International Society of S o i l Science, Transactions, Aberdeen. (G.V. Jacks, ed.) 92-111. Williams, C.H. 1968. Seasonal fluctuations i n mineral sulphur under subterranean clover pasture i n southern New South Wales. Aust. J . S o i l Res. 6: 131-13 9. GENERAL SUMMARY AND CONCLUSIONS Relatively l i t t l e research has been done on s u l f u r mineralization i n s o i l s and as a r e s u l t there i s a dearth of knowledge of the processes that are involved. The primary reason for t h i s limited information i s that suitable a n a l y t i c a l methods and procedures are not available o r , i f a v a i l a b l e , have not been c r i t i c a l l y evaluated with respect to the v a l i d i t y of r e s u l t s that are obtained. Therefore, a considerable portion of t h i s thesis was devoted to an examination of pertinent methods and procedures before the processes involved i n sulfur mineralization were examined. Improvements i n the q u a n t i f i c a t i o n of sulfate i n s o i l materials were accomplished by modifications to the hydriodic acid reduction method through an examination and application of the bismuth colorimetric f i n i s h instead of the methylene blue f i n i s h . A simple method was applied to control the nitrogen gas flow within the desired range fo r the procedure. Concentration of sample solutions by predrying p r i o r to the reductive step enhanced the s e n s i t i v i t y of the a n a l y t i c a l method when using the bismuth colorimetric f i n i s h and increased the v e r s a t i l i t y of application to samples low i n sulfur compounds. Elimination of a gas wash - 82 -s t e p r e s u l t e d i n a u s e f u l r e d u c t i o n i n t h e a n a l y t i c a l t i m e . N i t r a t e p r e s e n t i n l a r g e q u a n t i t i e s was s h o w n t o i n t e r f e r e w i t h t h e h y d r i o d i c a c i d r e d u c t i o n o f s u l f a t e t o s u l f i d e , h o w e v e r , t h e q u a n t i t y n e e d e d f o r t h i s e f f e c t (6 mg n i t r a t e i n t h e s a m p l e a l i q u o t ) w o u l d o n l y b e i m p o r t a n t i n e x t r e m e c a s e s . T h e s e o b s e r v a t i o n s w e r e i m p o r t a n t f o r r e d u c t i o n i n a n a l y t i c a l t i m e a n d u n d e r s t a n d i n g c e r t a i n l i m i t a t i o n s o f t h e p r o c e d u r e . T h e c h r o m o t r o p i c a c i d m e t h o d d e s c r i b e d i n t h e s e c o n d c h a p t e r was f o u n d t o b e a v e r y c o n v e n i e n t m e t h o d f o r d e t e r m i n i n g n i t r a t e i n s o i l e x t r a c t s . I t h a d t h e a d v a n t a g e s o f s p e e d , e a s e o f o p e r a t i o n , s e n s i t i v i t y a n d r e l a t i v e f r e e d o m f r o m i n t e r f e r e n c e s b y m a t e r i a l s common i n t h e e x t r a c t s . T h e r e s u l t s o n s o i l e x t r a c t s b y t h e m e t h o d c o m p a r e d v e r y f a v o r a b l y w i t h t h e p h e n o l d i s u l f o n i c a c i d m e t h o d o f d e t e r m i n i n g n i t r a t e , a n d b e c a u s e o f i t s r e l a t i v e f r e e d o m f r o m i n t e r f e r e n c e , was m o r e v e r s a t i l e w i t h r e s p e c t t o a n a l y s i s u s i n g v a r i o u s e x t r a c t a n t s . I n c u b a t i o n i n a c l o s e d s y s t e m was e x a m i n e d a n d f o u n d c o m p a r a b l e t o a e r o b i c i n c u b a t i o n i n a n o p e n s y s t e m ; i n a d d i t i o n t h e c l o s e d s y s t e m p e r m i t t e d m a i n t e n a n c e o f c o n s t a n t w a t e r c o n t e n t , h o m o g e n e o u s s a m p l i n g , a n d a n a l y s i s - 83 -of evolved carbon dioxide. Of four methods of extracting sulfate from the four s o i l samples, the 0.15% calcium chloride extract was most cl o s e l y correlated to carbon dioxide evolved and nitrogen mineralized. This e x t r a c t , then, was considered the most suitable when considering s u l f u r mineralized by microbes. A i r drying samples i n t r o -duced further sulfur transformations, which were not consistent through various stages of the incubation period; hence, analysis of fresh samples was found to be mandatory. Some of the differences could have been due to changes i n the s o i l to solution extraction r a t i o , since the moisture contribution was calculated a f t e r the sulfate analysis of samples weighed at a constant weight. The four s o i l samples used i n the study were each at d i f f e r e n t moisture contents. However, despite the r e s u l t i n g extraction r a t i o s used i n t h i s study, much better correlations of sulfur mineralization were found with microbial a c t i v i t y when using fresh samples than drying the samples. The s o i l to solution r a t i o e f f e c t i s a factor that should not be neglected i n other considerations. Changes i n the amount of sulfate extracted by various s o i l to solution r a t i o s were greatest i n samples containing adsorbed s u l f a t e , - 84 -possibly i n d i c a t i n g that a portion of t h i s sulfate f r a c t i o n i s extracted besides soluble s u l f a t e . R e l a t i v e l y large fluctuations i n the extractable sulfate occurred i n the early stages of the incubation and appeared to l e v e l off by eight weeks, so res u l t s during the f i r s t four weeks would not be recommended for single analyses and subsequent correlations with other f a c t o r s . The fourth chapter was an attempt to i d e n t i f y r e l a t i o n -ships of the su l f u r mineralized i n four s o i l s with other s o i l f a c t o r s . The sulfate measured i n calcium chloride extracts of fresh samples was used for the comparisons because of relationships shown i n the previous chapter. F a i r l y close relationships of su l f u r mineralized were evident with carbon dioxide evolved, nitrogen formed and r e s u l t i n g s o i l pH. Poorer correlations were found with phosphorus extracted by a bicarbonate method and a r y l -s u l f atase a c t i v i t y . The enzyme a c t i v i t y declined consider-ably through the incubation and from t h i s i t was assumed to be of l i t t l e importance i n sulfate release under these conditions. Relationships of sulfur mineralized with phospholipid phosphorus were unclear and i t was suggested that phospholipids may be a source of phosphorus and carbon for microbial a c t i v i t y . There did appear to be close relationships among carbon, nitrogen and su l f u r - 85 -m i n e r a l i z a t i o n , however more work i n t h i s area i s considered necessary and p o t e n t i a l l y u s e f u l . The influence of the organic s u l f u r form, whether carbon-bonded or the ester form, may be an important consideration i n predicting the amount of sulfur mineralized i n the s o i l during microbial a c t i v i t y . Organic sulfates were shown to be present i n extracts using phosphate buffer and bicarbonate, however, a completely s a t i s f a c t o r y separation was not achieved. A method for measuring organic sulfate i n extracts would be very useful i n interpreting some res u l t s i n t h i s study which could not be f u l l y explained. Was the organic sulfate contribution to the sulfate extracted constant throughout the incubation period? Were the large f l u c t u -ations of sulfate extracted from the sample during the i n i t i a l stages of incubation related to the organic sulfates i n the extracts? Did the organic sulfate i n the extracts (especially those other than 0.15% calcium chloride) contribute to t h e i r poor correlations with microbial a c t i v i t y ? Although many d e f i n i t e conclusions could not be drawn from the r e s u l t s , c e r t a i n areas for further f r u i t f u l research are indicated for examining s o i l s u l f u r transformations. An examination of the e f f e c t that nitrogen additions, s i m i l a r to forms and rates that might be used during - 86 -f e r t i l i z a t i o n , had on sulfu r mineralized a f t e r an incubation was reported i n the l a s t chapter. Two s o i l s were used with f i v e d i f f e r e n t nitrogen sources at four rates, and differences i n su l f u r mineralized were noted. These d i f f e r -ences were variable, depending on the s o i l and the nitrogen source applied. The e f f e c t appeared to be primarily an influence of the nitrogen source on microbial a c t i v i t y as shown by changes of carbon dioxide evolved and also of i t s influence, d i r e c t or i n d i r e c t , on the microbial environment as shown by changes i n pH. The r e s u l t s provide some evidence of the importance of considering the sulfu r supplying power of the s o i l when nitrogen applications are made. This w i l l depend on the form of the ap p l i c a t i o n , the environmental conditions as they a f f e c t microbial a c t i v i t y , the importance of s o i l microbial a c t i v i t i e s for the supply of su l f u r for plant growth and the nature and supply of organic s u l f u r i n the p a r t i c u l a r s o i l . This report has been, of necessity, of an exploratory nature, however some p r a c t i c a l implications could be drawn from the information that was presented. Important suggestions were made with respect to methodology and some of the information presented appears important i n evaluating some information previously reported i n the l i t e r a t u r e . - 87 -Several p o t e n t i a l l y useful aspects for further research a c t i v i t i e s are evident from t h i s work, which would illuminate the processes involved i n sulfur transformations in the s o i l . Some of these aspects would include work on methods f o r quantitative and q u a l i t a t i v e evaluation of s o i l organic s u l f a t e s , organic s u l f u r compounds i n the s o i l and t h e i r contribution to sulfu r mineralization, and con-sidering interactions of s u l f u r with other elements during mineralization. - 88 -APPENDIX 1 EVALUATION OF METHODS FOR DIRECT DETERMINATION OF CARBON-BONDED SULFUR IN SOIL INTRODUCTION DeLong and Lowe (1962) were the f i r s t to report a method of determining the carbon-bonded sulfur content of s o i l m aterial. The procedure involved a digestion of s o i l using Raney n i c k e l in a l k a l i and a subsequent release of hydrogen s u l f i d e from the s o l u t i o n . It was stated that t h i s procedure released most forms of organically combined s u l f u r , except the s u l f u r of a l k y l sulfones and ester s u l f a t e s , and many inorganic forms such as elemental s u l f u r , acid-soluble sulfides and some of the s u l f u r of more refractory s u l f i d e s but not inorganic s u l f a t e . Hence, for s o i l materials containing no elemental s u l f u r , where s u l f i d e could be estimated separately, t h i s procedure could be used with confidence to measure the carbon-bonded sul f u r i n these f r a c t i o n s . An examination of several s o i l s and t h e i r extracts (acid-extractable, a l k a l i - e x t r a c t a b l e and non-extractable - 89 -frac t i o n s ) showed that 90% of the carbon-bonded su l f u r could be recovered from organic samples and from a podzol B2^ sample, but 16-48% could not be accounted for i n three mineral surface samples used (Lowe and DeLong, 1963). Losses by formation of hydrogen s u l f i d e or other v o l a t i l e sulfur-containing materials and conversion of carbon-bonded to other forms which were not measured, were suggested, with the l a t t e r case i t was known that heating sulfonic acids i n acid solution could, i n some cases, give r i s e to free sulfate and the oxidation effects of hot a l k a l i n e solutions could have caused conversion of a l k y l sulfoxides or s u l f i d e s to a l k y l sulfones which would not be recover-able. Indications were that most of the carbon-bonded su l f u r may be humic acid associated since a larger amount was measured i n the a l k a l i - e x t r a c t a b l e than acid-extractable and also suggested that i t was more resi s t a n t to degradation. Further examination of s o i l samples showed that the dominant organic form appeared to be s u l f a t e , while i n organic s o i l s the organic sulfate and carbon-bonded fractions were quantitatively s i m i l a r (Lowe, 1964). Incomplete recovery of the s u l f u r i n the mineral s o i l s suggested an " i n e r t " s u l f u r f r a c t i o n which was p a r t i a l l y extracted by f a i r l y d r a s t i c procedures. Carbon-bonded sulfur decreased - 90 -with depth i n a l l p r o f i l e s studied and represented 17 and 9% of the t o t a l s u l f u r of the Chernozemic A, and B h m horizons and 2 6 and 15% i n the Grey Wooded L-H and B horizons, respectively (Lowe, 1965). The nitrogen:carbon-bonded su l f u r r a t i o s (40-60) were higher than nitrogen: t o t a l s u l f u r and nitrogen:HI reducible s u l f u r . Freney et al. (1970) noted that there was incomplete recovery (36-97%) of methionine s u l f u r from Australian s o i l s using the d i r e c t estimation of carbon-bonded s u l f u r , therefore they examined the procedure with respect to possible interferences. Destruction of organic matter with sodium hypobromite did not improve the recovery. They examined possible interference by the mineral portion of the s o i l and found that iron and manganese caused some interference but k a o l i n i t e , montmorillonite, s i l i c o n , calcium, magnesium, titanium, chromium, copper and vanadium when present i n normal amounts i n s o i l did not i n t e r f e r e . They further examined the mechanism of interference of iron and manganese and subsequently found that using a larger amount of c a t a l y s t , sodium hydroxide and increasing the reduction and extraction times increased recovery, successfully overcoming i n t e r -ference by iron and manganese. However, the recovery of a l l the organic sulfur as either s u l f i d e or sulfate i n the - 91 -presence of the i n t e r f e r i n g elements, and the marked interference by f e r r i c iron a f t e r a l k a l i n e reduction showed that they did not i n t e r f e r e with the s p l i t t i n g of the carbon-sulfur bond. The low r e s u l t s appeared to have been caused by the oxidation of s u l f i d e to sulfate when the reaction mixture was a c i d i f i e d . They f i n a l l y concluded that the r e a c t i v i t y with Raney n i c k e l was not s p e c i f i c since i t would not cleave the carbon-bonded su l f u r of a l i p h a t i c sulfones or sulfonic acids to s u l f i d e . Cysteine, for example, may be converted to cysteic a c i d , cystathionine, cysteine-S-sulfonate and other s u l f u r compounds by reaction with sodium hydroxide. Comparisons were therefore made of the o r i g i n a l method for carbon-bonded s u l f u r determinations and modifications to i t using Freney et al. (1970) data. The analyses were done on several s o i l s and s o i l materials. METHODS AND MATERIALS The samples used represent three organic s o i l samples (Lulu muck, Metchosin, H horizon under Sitka spruce) and four mineral samples used for sulfur studies (Cardinal Oxbow A, , Prest A, and Stevens A, ) . - 92 -The method for di r e c t determination of carbon-bonded su l f u r was that described by Lowe and DeLong (1963). The sample was placed i n a large digestion f l a s k (200 ml) with approximately 0.1 g nickel-aluminum a l l o y together with 5 ml 5% NaOH and 25 ml d i s t i l l e d water. The fl a s k was then clamped to the condenser and the nitrogen gas connected to the side arm. The mixture was heated for 3 0 minutes at about 8 0°C with nitrogen gas flowing. Foaming was e f f e c t i v e l y reduced using Antifoam A S i l i c o n spray (Dow Corning) and did not aff e c t the r e s u l t s . After d i g e s t i o n , the fl a s k was cooled, 5 ml 1:1 HCl was added through the top of the condenser, the U tube connected and hydrogen s u l f i d e collected and measured using sodium hydroxide and bismuth (Kowalenko and Lowe, 197 2) a f t e r heating the fl a s k for 3 0 minutes. Modifications from data reported by Freney et al. (197 0) were applied to the above procedure. Changes to the method were increasing the amount of catalyst to 1 g, volume of NaOH to 20ml, time of i n i t i a l digestion to three hours and volume of 1:1 HCl to 3 0 ml. Sulfur fractions were extracted from s o i l with Chelex 100 r e s i n (sodium form) using 10 g s o i l , 3 g re s i n and 48 ml water with shaking time of 1 hour. A chloroform and an aqueous extract were extracted using the Bligh and Dyer - 93 -method, which was previously used to extract s o i l phospholipids (Kowalenko and McKercher, 1971). Analyses were conducted on suitable aliquots of these extracts. RESULTS AND DISCUSSION The re s u l t s showed that the modified procedure values were considerably higher than with the o r i g i n a l (Table 1). From the data provided by Freney et al. (1970) i t would appear that the modified procedure would be a r e s u l t of a more complete measure of the carbon-bonded sulfur that i s present i n the s o i l , however, would not be a measure of a l l carbon-bonded sulfur possibly present. S i m i l a r l y , the modified method resulted i n higher values for carbon-bonded sulfur i n several s o i l extracts (Table 2). The analyses with the o r i g i n a l method required predrying of the chloroform to get any r e s u l t , possibly a function of incomplete reaction because of i m m i s c i b i l i t y . It appeared that even with s o i l extracts the modified procedure was more complete, probably because interferences may have been overcome by the modifications. Similar r e s u l t s on the aqueous portions of the Bligh and Dyer extract may be that i n t e r f e r i n g substances were not present. However, the large difference of the two methods - 94 -TABLE 1. Carbon-bonded sulfur (ppm S) of soils determined by two direct methods Lowe and DeLong Modified S o i l method method Lulu 6,541 ±61 14,540 ±54 Metchosin 2,159 ±12 4,205 ±45 0-31 (Sitka) 121 ±9 327 ±24 Cardinal 34 ±1 110 ±0 Oxbow 27 ±1 99 ±2 Prest 72 ±1 212 ±4 Stevens 30 ±3 26 ±0 Total 8,984 ±88 19,519 ±129 - 95 -TABLE 2. Carbon-bonded sulfur (ppm S) in several soil extracts measured by two direct determination methods S o i l Extract Lowe and DeLong Modified method method Metchosin Chelex 100 34 ±4 100 ±5 Bligh and Dyer: Chloroform Aqueous 3 ±0 1.5 ±0.5 18 ±5 1.5 ±0.5 Lulu Bligh and Dyer: Chloroform 23 ±0' Aqueous 23 ±3 56 23 ±5 + 3 aChloroform predried. - 96 -for Chelex r e s i n extracts may be because t h i s extract contains substances which i n t e r f e r e with the o r i g i n a l method which were overcome by the modifications. CONCLUSION Modifications, taken from data from work done by Freney et al. (197 0) on an o r i g i n a l method for the di r e c t determination, resulted i n a method that generally yielded increased amounts of carbon-bonded s u l f u r . This increase was taken to be a r e s u l t of overcoming i n t e r -ferences by iron and manganese present i n the s o i l . A disadvantage of the modified method i s that the time of analysis i s greatly increased. Conservation of time for analysis of carbon-bonded sulfur i n certain s o i l extracts could be achieved by using the o r i g i n a l method, however, care must be taken as to the p a r t i c u l a r extract that i s analysed. Although the modified method could not be considered to give a complete estimation of a l l carbon-bonded su l f u r present, the method could be useful i n characterizing a certain f r a c t i o n of i t . Further research i n t h i s area would be u s e f u l , and a great variety of modifications are possible ( P e t t i t and van Tamelen, 1962). - 97 -REFERENCES DeLong, W.A. and L.E. Lowe. 1962. Note on carbon-bonded sulphur i n s o i l . Can. J . S o i l S c i . 42: 223. Freney, J.R., G.E. M e l v i l l e and C.H. Williams. 1970. The determination of carbon bonded sulphur i n s o i l . S o i l S c i . 109: 310-318. Kowalenko, C G . and L.E. Lowe. 1972. Observations on the bismuth s u l f i d e colorimetric procedure f o r sulfate analysis i n s o i l . Comm. S o i l S c i . Plant Analysis 3: 79-86. Kowalenko, C G . and R.B. McKercher. 197 0. An examination of methods for extraction of s o i l phospholipids. S o i l B i o l . Biochem. 2: 269-273. Lowe, L.E. 1964. An approach to the study of the sulphur status of s o i l s and i t s application to selected Quebec s o i l s . Can. J . S o i l S c i . 44: 176-179. Lowe, L.E. 1965. Sulphur fractions of selected Alberta s o i l p r o f i l e s of the Chernozemic and Podzolic orders. Can. J . S o i l S c i . 45: 297-303. Lowe, L.E. and W.A. DeLong. 1963. Carbon bonded sulphur i n selected Quebec s o i l s . Can. J . S o i l S c i . 43: 151-155. P e t t i t , G.R. and E.E. van Tamelen. 1962. Desulfurization with Raney n i c k e l . Organic Reactions 12: 356-529. - 98 -APPENDIX 2 OBSERVATIONS ON AN ALKALINE OXIDATION METHOD FOR DETERMINATION OF TOTAL SULFUR IN SOILS INTRODUCTION A variety of procedures have been used for determining t o t a l s u l f u r i n s o i l s (Beaton et at. , 1968), however none have been universally accepted. Two of the main problems have been lack of precision and accuracy, and s u i t a b i l i t y f o r routine work. Recently, a procedure developed by Tabatabai and Bremner (1970a) using alkaline oxidation and hydriodic acid reduction has shown promise as a suitable method for estimating t o t a l s u l f u r i n s o i l s . Comparisons indicated that the method gave more precise r e s u l t s than some other methods used i n the past (Tabatabai and Bremner, 1970b). The Leco Sulfur Analyser did not appear to be a suitable procedure for t h i s analysis (Tabatabai and Bremner, 197 0b; Bremner and Tabatabai, 1971). The purpose of t h i s report i s to examine the a l k a l i n e oxidation method for determining t o t a l s u l f u r i n s o i l s , - 99 -i t s s u i t a b i l i t y for operation i n t h i s laboratory, some possible modifications and comparison to Leco Sulfur Analyser values. METHODS AND MATERIALS The s o i l samples used i n t h i s study are described i n Table 1. They represented a variety of samples, both organic and inorganic, from a variety of areas, and included two subsurface horizons. Total s u l f u r was measured on the s o i l samples using the method outlined by Tabatabai and Bremner (1970a). The procedure for oxidation was as follows: Weigh the s o i l sample into a digestion f l a s k using 100 mesh s o i l (or 6 0 mesh i f s o l i d gypsum i s not present), add three ml of sodium hypobromite s o l u t i o n , s w i r l to mix, l e t i t stand for f i v e minutes and swi r l again. Stand the flasks upright i n a sand bath at 250-260°C, heat the solution to dryness and then 3 0 minutes more. Remove the f l a s k from the heat, cool for f i v e minutes, add one ml water and heat the f l a s k to bring a l l residue into suspension. Cool the f l a s k s , add one ml formic acid and analyse the oxidized s u l f u r f o r sulfate sulfur by hydriodic acid - 100 -TABLE 1. Soil samples used in total '• s u l f u r oontent studies Sample Type Horizon Location % Total Lulu Muck H Lulu Island -Metchosin Organic H Van. Island -0-3 Sitka H Port Renfrew 11.7 0-6 Hemlock H Jordan River 14.4 0-8 Oak Ah Uplands Park 5 . 61 0-14 Douglas f i r H-Ah Stamp F a l l s Park 4.02 0-19 Lodge pole pine FH Manning Park 6 . 91 0-22 Cedar Ah St. Francis Park 4.05 0-24 Maple Ah St. Francis Park 4.81 0-28 Grass Ah F t . St. John (Laundry) 5.00 0-31 Grass Ah F t . St. John (Mytron) 6.40 0-32 Grass Ah F t . St. John (Peoria) 4.80 0-37 Aspen Ah Beatton River 4.80 0-41 Ponderosa H-Ah Princeton 4.80 0-44 Sphagnum H Mi l l i g a n Creek 14.60 Cardinal Podzolic Bt h Steelhead 3 .08 Oxbow Orthic b l k . Ah Hepburn, Sask. 2 .11 Prest Humic gleysol Ah MacMillan Island 3.69 Stevens Orthic b l k . Ah B r i d e s v i l l e 2 .47 Stevens Orthic b l k . C B r i d e s v i l l e 0.11 - 101 -reduction. Sulfate sulfur was measured using the bismuth colorimetric f i n i s h (Kowalenko and Lowe, 197 2) so sample weights containing either 0-200 or 0-40 ppm sulfur could be used. Samples that were analysed using the Leco Sulfur Analyser were according to i n s t r u c t i o n s , however, samples containing high amounts of organic matter were preashed i n the presence of magnesium oxide i n a muffle furnace at 2 00°C for one half hour, then 3 00°C f o r one half hour and f i n a l l y 400°C for one hour. Carbon-bonded su l f u r was determined by a modified d i r e c t determination method (Appendix 1) and hydriodic acid reducible sulfur by the bismuth method (Kowalenko and Lowe, 197 2). RESULTS AND DISCUSSION The u t i l i z a t i o n of the bismuth s u l f i d e colorimetric f i n i s h to sulfate analyses resulted i n reduced time of d i s t i l l a t i o n of the procedure (Kowalenko and Lowe, 1972). S i m i l a r l y , the adoption of th i s method showed a reduction in time i n the t o t a l s u l f u r analysis with the alkaline oxidation method. Measurements of sulfur released by - 102 -hydriodic acid from the apparatus at 10, 20, 3 0 and 5 0 minute i n t e r v a l s revealed 8 9% of the t o t a l s u l f u r was released i n 10 minutes and complete d i s t i l l a t i o n at and beyond 2 0 minutes. A minimum of 2 0 minutes could be used when the bismuth s u l f i d e method i s used with t h i s apparatus. A l s o , the bismuth method showed great v e r s a t i l i t y i n choice of concentration ranges of s u l f u r analysed and even the upper range of 2 00 ppm s u l f u r could be extended by d i l u t i o n with blank material (Dean, 1966). The l i m i t of absorption i n 20 ml IN NaOH of su l f u r as hydrogen s u l f i d e was 315 ppm. Kowalenko and Lowe (197 2) showed that s e n s i t i v i t y was increased by predrying sample aliquots using hydriodic reduction, however, i n the al k a l i n e oxidation method for t o t a l s u l f u r determination the oxidized material was suspended in one ml water and a c i d i f i e d with one ml formic acid p r i o r to a n a l y s i s . A comparison of predried standard material with standard material dissolved i n one ml water and a c i d i f i e d with one ml formic acid showed no s i g n i f i c a n t d i f f e r e n c e s , hence predrying i n t h i s procedure was not p a r t i c u l a r l y advantageous. Lavkulich and Wiens (197 0) have used sodium hypochlorite for the destruction of organic matter. Because of i t s - 103 -r e l a t i v e ease of handling, sodium hypochlorite was t r i e d as an alternative for sodium hypobromite oxidation. Sodium hypochlorite (7% available) was diluted to give a concentration s i m i l a r to that used of sodium hypobromite i n the oxidation. Comparison of oxidations made with these two reagents i s shown i n Table 2. The res u l t s show that there was a s i g n i f i c a n t amount of sulfur present i n the sodium hypochlorite which must be accounted for p r i o r to computing to a gram s o i l b a s i s. A l s o , the sodium hypochlorite oxidation values were not always as high as with sodium hypobromite method p a r t i c u l a r l y the mineral s o i l sample (0-31) and precision was not always as good (Lulu). During the f i r s t stages of hydriodic acid reduction, a brown vapor appeared with the hypochlorite oxidized material but not with the hypobromite oxidized material, possibly i n d i c a t i n g an iodine e f f e c t s i m i l a r to that noted for perchloric acid treated material (Johnson and N i s h i t a , 1952). A difference was also observed when formic acid was added during the a c i d i f i c a t i o n , where a white s o l i d formed with hypobromite material but not with hypochlorite material. Comparisons of t o t a l s u l f u r determined by the Leco Sulfur Analyser and the a l k a l i n e oxidation method are shown in Table 3. Total sulfur values calculated by summing the - 104 -TABLE 2. Comparison of total sulfur (ppm) of soil samples by sodium hypobromite and sodium hypochlorite oxidations Sample NaOBr NaOCl Reagent 2 ±2 29 +3 Lulu 22,186 ±578 23,333 ±2,6 1 9a Metchosin 7,345 ±647 6,648 ± 284a 0-31 1,213 ± 35 564 ± l la aCorrection made for sulfur present i n reagent. - 105 -TABLE 3. Comparison of soil sulfur values (ppm S) by two direct determinations and summation method for a few of the samples Direct methods Sample Leco NaOBr Lulu 18 ,522 ±8 7a 22 ,186 ±578 Metchosin 6 ,069 ±4 8a 7 ,345 ±647 0-3 1 ,216 ± 0a 1 ,814 ±128 0-6 1 ,461a L 1 ,285 ± 29 0-8 1 ,106 ± oa 1 ,213 ± 10 0-14 399 ±3 0a 226 ± 7 0-19 732 ± 6a 731 ± 10 0-22 515 ±11 537 ± 10 0-24 549 ±25 642 ± 10 0-28 431 ± 6 553 ± 0 0-31 344 + 50 1 ,213 ± 35 0-32 451 ±25 737 ± 17 0-37 89 184 ± 5 0-41 555 ± 0a 545 ± 8 0-44 5 ,162 ±5 2a 8 ,941 ±348 Cardinal 95 ± 9 300 ± 7 Oxbow 274 ±23 369 ± 9 Prest 382 ±30 438 ± 10 Stevens 95 ±11 214 ± 10 Stevens (C) 21 + 7 28 ± 4 Sum HI and C-bonded S 20,205 ±333 5,659 ±602 646 ± 24 208 ± 5 264 ± 4 351 ± 5 13 4 ± 3 28 ± 6 aPreashed. - 106 -hydriodic acid sulfur and carbon-bonded sulfur determined on several samples was also included and t h i s value should be considered a minimum estimate (Freney et al. , 1970). The r e s u l t s show that the a l k a l i n e oxidation values are probably more reasonable since Lulu, 0-31 and Stevens samples have a higher HI plus C-bonded S value than the Leco value. CONCLUSION The al k a l i n e oxidation method for the determination of t o t a l s u l f u r i n s o i l and s o i l materials was found to be suitable for use i n t h i s laboratory. Although not as fast as the Leco Sulfur Analyser, t h i s method has the advantage of being more v e r s a t i l e (extracts can be measured as well as s o i l s d i r e c t l y ) and giving a value that i s probably more consistently accurate. The bismuth s u l f i d e colorimetric method was a suitable alternative to the methylene blue method o r i g i n a l l y used for measuring the s u l f u r , and e f f e c t i v e l y reduced the time of t h i s part of the procedure. Sodium hypochlorite was not a suitable oxidant as an alternative to sodium hypobromite. - 107 -REFERENCES Beaton, J.D., G.R. Burns and J . Platou. 1968. Determination of s u l f u r i n s o i l s and plant material. Technical B u l l e t i n No. 14. The Sulphur I n s t i t u t e , Washington. Bremner, J.M. and M.A. Tabatabai. 1971. Use of automated combustion techniques for t o t a l carbon, t o t a l nitrogen, and t o t a l s u l f u r analysis of s o i l s . Instrumental Methods for Analysis of So i l s and Plant Tissue. S o i l S c i . Soc. Am. Madison, U.S.A. Dean, G.A. 1966. A simple colorimetric f i n i s h for the Johnson-Nishita m i c r o d i s t i l l a t i o n of s u l f u r . Analyst 91: 530-532. Freney, J.R., G.E. M e l v i l l e and C.H. Williams. 1970. The determination of carbon bonded sulfur i n s o i l . S o i l S c i . 109: 310-318. Johnson, CM. and H. N i s h i t a . 1952. Microestimation of sulfur i n plant materials, s o i l s and i r r i g a t i o n waters. Anal. Chem. 24: 736-742. Kowalenko, C G . and L.E. Lowe. 197 2. Observations on the bismuth s u l f i d e colorimetric procedure for sulfate a n a l y s i s . Comm. S o i l S c i . Plant Analysis 3: 79-86. Lavkulich, L.M. and J.H. Wiens. 1970. Comparison of organic matter destruction by hydrogen peroxide and sodium hypochlorite and i t s e f f e c t on selected mineral constituents. S o i l S c i . Soc. Am. Proc.1 34: 755-758. Tabatabai, M.A. and J.M. Bremner. 197 0a. An alkaline oxidation method for determination of t o t a l s u l f u r i n s o i l s . S o i l S c i . Soc. Am. Proc. 34: 62-65. Tabatabai, M.A. and J.M. Bremner. 1970b. Comparison of some methods for determination of t o t a l s u l f u r i n s o i l s . S o i l S c i . Soc. Am. Proc. 34: 417-420. - 108 -APPENDIX 3 CHEMICAL ANALYSES OF FOUR SOIL SAMPLES DURING AN INCUBATION EXPERIMENT TABLE 1. Soil analysis results of four samples at various intervals during an incubation experiment using a closed system 0 , Interval (weeks) bample Extract treatment 0 1 2 4 8 14 A. Cardinal sample Sulfate (ppm S): Calcium chloride Fresh 4 .7 2. 6 10. 6 6 .9 6 .6 4. 7 Dried 3 .5 5 . 1 4. 5 3 .4 2 . 9 1. 7 Acetate Fresh 65 . 9 76 . 4 96. 7 69 .1 77 .1 68. 7 Dried 68 .2 67 . 3 62. 5 83 .8 60 .4 61. 0 Phosphate buffer Fresh 90 .9 94. 1 88. 0 102 .8 95 .5 72. 5 Dried 85 .0 81. 5 71. 3 95 .0 66 .0 86. 0 Bicarbonate Fresh 99 .0 88 . 8 101. 2 103 .9 93 .6 79. 5 Dried 99 .0 97. 0 81. 6 102 . 9 94 .2 68 . 0 Sulfatase a c t i v i t y (yg p-nitrophenol/ Fresh 27 .8 7. 1 9. 3 5 .8 2 .6 6. 5 g/hr) Dried 10 .5 1. 5 5. 0 3 .7 0 . 0 0. 0 Phosphorous (ppm): Bicarbonate Fresh 4 .3 3. 9 4. 2 3 .1 4 .4 1. 8 Dried 3 .5 1. 8 1. 8 4 .0 2 . 9 2. 6 Phospholipid-P Fresh 3 . 6 3. 6 2. 8 3 .5 3 .8 2 . 9 Nitrogen (ppm): Ammonium-N Fresh 7 .2 14. 2 18. 1 18 .7 22 .4 27 . 7 Dried 6 .0 11. 0 12. 4 16 .8 20 .0 26. 2 Nitrate-N Fresh 0 .7 0. 7 0. 7 0 .7 0 .7 0. 5 Dried 0 .0 0. 5 0. 0 0 .5 1 .0 0. 4 pH Fresh 4 .88 5 . 75 5. 08 5 .75 6 .02 5 . 28 Moisture (%) Fresh 30 . 2 29. 7 27 . 4 27 .1 25 .3 25. 1 - 109 -TABLE 1. (Continued) Extract B. Oxbow sample Sulfate (ppm S): Sample treatment Interval (weeks) 11 Calcium chloride Fresh 2 .5 1 .8 11. 0 6 .1 7 . 0 8 .1 Dried 7 .5 4 .3 4. 6 5 .9 6 .4 10 .5 Acetate Fresh 5 .4 15 . 9 23 . 3 8 .7 13 .3 14 . 2 Dried 10 .1 10 .5 8 . 6 12 .5 11 .5 14 .3 Phosphate buffer Fresh 9 . 9 15 .5 15 . 4 18 .7 13 .8 6 .6 Dried 17 .5 17 .5 12. 0 18 .3 8 .8 23 .8 Bicarbonate Fresh 7 .8 33 . 0 9. 7 16 .0 13 .6 12 .2 Dried 13 .5 17 .6 12. 2 13 . 2 11 .0 12 .4 Sulfatase a c t i v i t y (ug p-nitrophenol/ Fresh 50 .1 33 .0 26 .7 1 .4 1 .1 0. 0 g/hr) Dried 48 .3 7 .5 5 .0 5 .5 3 .0 2 . 6 Phosphorus (ppm): Bicarbonate Fresh 15 .6 15 . 9 16 .6 13 .6 13 .9 16. 0 Dried 13 .5 14 .2 14 .6 16 .3 16 .4 17 . 8 Phospholipid-P Fresh 3 .5 3 .4 0 .7 1 .1 1 .3 0. 5 Nitrogen (ppm): Ammonium-N Fresh 5 .7 3 .4 3 .4 2 .2 4 .0 4. 2 Dried 3 .0 3 . 0 3 .0 5 .7 2 .8 3 . 2 Nitrate-N Fresh 4 .3 26 .5 33 . 2 61 . 9 94 .5 112. 4 Dried 8 .0 26 .3 33 .5 56 .0 86 .5 110. 8 pH Fresh 8 .71 8 .60 8 .41 8 .47 8 .62 8 . 33 Moisture (%) Fresh 29 .5 27 .4 27 .6 26 .3 24 . 9 23. 2 - 110 -TABLE 1. (Continued) Extract C. Prest sample Sulfate (ppm S): Sample _ treatment 0 Interval (weeks) 14 Calcium chloride Fresh 13 .4 20 .2 31 . 0 33 .8 32 .4 32 .7 Dried 18 .5 18 .9 20 .1 20 .8 20 .1 23 . 6 Acetate Fresh 19 .7 48 .2 54 .6 47 .8 52 .5 52 . 0 Dried 28 .9 32 .0 31 .7 37 .4 35 .7 38 .5 Phosphate buffer Fresh 40 .9 82 .5 97 .2 75 .8 71 .9 57 .6 Dried 48 .8 50 .0 43 . 0 50 .8 42 .0 65 .5 Bicarbonate Fresh 42 .5 39 . 2 60 .7 78 . 8 68 .6 61 .8 Dried 49 .0 46 .8 45 .1 47 .2 46 .8 52 .6 Sulfatase a c t i v i t y (yg p-nitrophenol/ Fresh 199 .5 125 .3 88 .2 9 .0 9 .4 3 .4 g/hr) Dried 95 .3 16 .0 10 .7 7 .2 7 .2 5 .8 Phosphorus (ppm): Bicarbonate Fresh 32 .8 35 .5 41 .4 31 .4 31 .3 30 . 8 Dried 19 .0 16 .4 16 . 9 18 . 9 18 .9 21 .2 Phospholipid-P Fresh 6 .1 6 .7 7 .3 5 .4 4 .6 2 . 4 Nitrogen (ppm): Ammonium-N Fresh 30 .3 124 .7 158 .0 131 .5 124 . 2 149 . 2 Dried 29 .5 73 .5 66 .7 72 .5 73 .5 79 .7 Nitrate-N Fresh 4 .2 23 . 9 44 . 9 71 .9 118 .5 136 . 6 Dried 3 .5 16 .5 40 .5 52 .0 74 .0 101 .3 pH Fresh 5 .52 5 .84 5 .57 5 .62 5 .56 5 .41 Moisture (%) Fresh 59 .0 59 .1 58 .8 58 .4 56 .6 55 .0 - I l l -TABLE 1. (Continued) Extract D. Stevens sample Sulfate (ppm S): Sample treatment Interval (weeks) 1 14 Calcium chloride Fresh 1 .6 0 .4 9 .3 3 .6 3 .6 2. 0 Dried 5 .5 2 .5 2 .6 3 .5 2 .7 2. 5 Acetate Fresh 2 .1 7 .6 4 .6 7 .0 3 .6 4. 4 Dried 5 .1 4 . 0 2 .7 5 .6 4 .1 3 . 9 Phosphate buffer Fresh 14 .3 22 .3 12 . 2 15 .9 5 .4 5. 7 Dried 16 .3 10 .8 8 . 0 9 .8 9 .3 10. 3 Bicarbonate Fresh 4 .1 13 .1 12 .1 13 .8 8 . 9 4. 2 Dried 5 .5 8 .0 12 . 2 8 .8 9 .0 9. 6 Sulfatase a c t i v i t y (yg p-nitrophenol/ Fresh 34 .4 18 .7 15 . 0 1 .6 3 .4 0. 9 g/hr) Dried 21 .3 3 .8 7 . 0 2 .8 1 .5 1. 9 Phosphorus (ppm): Bicarbonate Fresh 88 .6 92 .7 87 .5 67 . 2 69 .8 78 . 8 Dried 64 .5 62 .1 64 .7 60 .3 67 . 5 70. 2 Phospholipid-P Fresh 2 . 4 1 . 2 3 .3 0 . 8 2 .5 0. 6 Nitrogen (ppm): Ammonium-N Fresh 4 .1 8 .9 4 .1 2 . 5 4 . 9 7. 3 Dried 9 .0 6 .5 3 .2 3 .2 2 .8 4. 8 Nitrate-N Fresh 2 .5 12 .6 25 .6 37 .4 57 .2 72. 2 Dried 2 .3 12 .0 20 .2 32 .0 45 .3 67 . 3 PH Fresh 6 . 69 6 .18 5 . 98 6 .17 6 .17 5 . 63 Moisture (%) Fresh 38 . 9 38 .5 38 . 2 37 . 0 35 .0 33 . 8 E. Stevens sample plus nitrogen (2570 Mg N (as N H 4 : NO 3 ) added/g so Sulfate (ppm S): Calcium chloride Fresh 1 .6 3 .1 0 .3 0 .0 0 .0 --pH Fresh 6 .69 6 . 03 5 .78 5 .81 5 .82 — - 112 -TABLE 2. Cumulative carbon dioxide carbon (mg/g soil) evolved from four soils during a fourteen week incubation Sample Interval (weeks) 14 Cardinal 0.2570 0.3891 0.5399 0.7655 1.0647 Oxbow Prest 0.2546 0.4249 0.6523 0.9265 1.2451 0.7077 1.0064 1.4295 1.9884 2.7058 Stevens 0.1479 0.2375 0.3727 0.5747 0.8174 Stevens plus nitrogen3 0.1287 0.1785 0.2940 0.3732 L2570 yg N (as NH NOg) added/g s o i l , - 113 -TABLE 3. Soil analysis results of soil samples determined on fresh material at various intervals during an incubation experiment using an open system with daily water additions Interval (weeks) Sample Cardinal Cardinal plus 1% Oxbow Oxbow Prest Extract Sulfate (ppm S): Calcium chloride Nitrogen (ppm N): Ammonium-N Nitrate-N pH Sulfate (ppm S): Calcium chloride Nitrogen (ppm): Ammonium-N Nitrate-N pH Sulfate (ppm S): Calcium chloride Nitrogen (ppm) Ammonium-N Nitrate-N pH Sulfate (ppm S): Calcium chloride Nitrogen (ppm): Ammonium-N Nitrate-N 0 4.7 8.7 8.9 7.5 3.9 7 0 14, 1, 16 1, 19 1, 23 0 4.7 10.3 7 0 12, 1. 8.0 17 .6 0.8 9.2 21, 1, 24 2 2.5 2.1 5.2 6.7 5 . 4, 3 22 5 40, 4 43, 6 51 4.88 5.70 5.64 5.60 5.04 6.1 9 3 4.88 6.03 5.81 5.74 5.96 6.2 8.71 8.85 8.82 8.64 0 8.46 13.4 21.5 23.9 37.1 30.6 30 4 150.8 8.4 189 30 153 78 129.8 76.8 pH 5.52 6.23 5.89 5.58 5.35 - 114 -TABLE 3. (Continued) Interval (weeks) Sample Stevens plus 0 CaC0„ Extract Sulfate (ppm S): Calcium chloride Nitrogen (ppm): Ammonium-N Nitrate-N 1.6 .1 ,5 0.5 7.8 15.6 0.3 6.0 24.8 8.5 5 33 3.3 7.8 34.8 pH 6.69 8.32 8.26 8.23 7.77 - 115 -APPENDIX 4 EXAMINATION OF VARIOUS METHODS FOR SEPARATION OF ORGANIC FROM INORGANIC SULFATE IN SOIL EXTRACTS, INTRODUCTION In many studies of s o i l s u l f u r , i t i s essential to d i s t i n g u i s h between organic and inorganic sulfate i n extracts. However, a completely s a t i s f a c t o r y solution to t h i s problem has not been determined, whether using a barium p r e c i p i t a t i o n or a hydriodic acid reduction method of q u a n t i f i c a t i o n (Freney, 1958; Hesse, 1957). It appears that barium p r e c i p i t a t i o n of only inorganic sulfate i n the presence of organic sulfate i s assumed i n biochemical systems (Baddiley et al. , 1957; Dodgson, 1961) however s o i l extracts provide a sp e c i a l problem. Organic matter present i n most s o i l extracts could possibly act as protective c o l l o i d s or be co-precipitated during a barium chloride treatment, r e s u l t i n g i n erroneous r e s u l t s (Beaton et al., 1968). A l s o , various anions may i n t e r f e r e with p r e c i p i t a t i o n methods and t h e i r removal often r e s u l t s i n decreased - 116 -s e n s i t i v i t y because of d i l u t i o n as, for example, use of exchange r e s i n s . The degree of s t a b i l i t y toward hydrolysis of the organic sulfates during such treatments i s not known. The use of Sephadex for an organic-inorganic separation has been successfully used (Lowe, 1966); however lack of knowledge of the molecular sizes of organic sulfate esters i n s o i l extracts and t h e i r mobility i n Sephadex columns re s u l t s i n only considering t h i s determination as minimal for organic s u l f a t e . The purpose of t h i s r e p o r t , then, i s to consider various methods of quantitatively separating organic from inorganic sulfate i n s o i l extracts; to estimate the v a l i d i t y of the separation, the r e l a t i v e ease of operation and s e n s i t i v i t y for measuring extracts containing r e l a t i v e l y small concentrations of s u l f a t e . METHODS AND MATERIALS The s o i l s used i n t h i s study were samples for a sulfur mineralization study and included one Gle y s o l i c ( P r e s t ) , one Podzolic subsurface (Cardinal, B ) and two Chernozemic r h (Oxbow and Stevens) samples. The sulfate extractants used i n t h i s study were 0.5 M sodium phosphate buffer at pH 7.0 - 117 -(Bart, 196 9) and 0.5 M sodium bicarbonate at pH 8.5 (Kilmer and Nearpass, 1960). Methods of separation involved the use of a Sephadex G-25 column, eluted with 0.02 M sodium tetraborate and sulfate determined on various fractions using hydriodic acid reduction (Kowalenko and Lowe, 1972). A colorimetric barium p r e c i p i t a t i o n method (Carlson et al. , 1967) was u t i l i z e d on extracts that were p u r i f i e d with either a R + cation exchange r e s i n (Rexyn 101 (H ) , 40-100 mesh) or application of a sodium peroxide method (Bettany and Halstead, 1972). A measurement of the organic sulfate was t r i e d using hydriodic acid reduction afte r the inorganic sulfate was precipitated by a barium chloride a d d i t i o n . Adsorption of organic sulfate was attempted using either charcoal or polyvinylpyrrolidinone, with a measurement of inorganic sulfate being made by hydriodic acid reduction on the unadsorbed portion. Unadsorbed organic sulfate was measured when extracts were passed through an ion retardation r e s i n column (AG11A81 s e l f adsorbed form). With the l a s t method (retardation r e s i n ) , the extracts were f i r s t washed through with water, then the column cleaned up with 2 N NaOH and then water. To further monitor the movement of the organic matter through the 1 Bio-Rad Laboratories - 118 -column, organic carbon was measured on predried volumes using the Walkley-Black method (Jackson, 1965), carbohydrate monitored by an anthrone method (Brink et al. , 1960) and changes i n the organic f r a c t i o n by determining E4:E6 r a t i o s (Campbell et al., 1967). RESULTS AND DISCUSSION The Sephadex method of separating organic from inorganic sulfate from extracts of the Cardinal sample was not s a t i s f a c t o r y . This estimate was considered low for organic sulfate quantities since colored organic matter i n the f r a c t i o n was observed where the inorganic sulfate was expected. A l s o , there was not a clearcut separation of f r a c t i o n s , indicated both by colored material and sulfate and hence i t was d i f f i c u l t to estimate the order of magnitude this error involved. The various methods u t i l i z i n g barium p r e c i p i t a t i o n were also unsatisfactory since both the r e s i n and sodium peroxide p u r i f i c a t i o n s of s o i l extracts did not remove a l l of the colored material. Since the colorimetric determin-ation was not a straight l i n e standard curve, corrections could not be applied. The cation exchange r e s i n that was - 119 -used contributed to some sulfate contamination, hence must be taken into consideration. A l s o , the p u r i f i c a t i o n resulted i n a d i l u t i o n of the sulfate i n the extracts to a point where only extracts containing high i n i t i a l concentration of sulfate could be measured within the s e n s i t i v i t y of the colorimetric procedure. A simple procedure of barium chloride p r e c i p i t a t i o n of inorganic sulfate and subsequent measure of unprecipitated organic sulfate was obviously unsatisfactory since most of the colored organic matter i n the extracts p r e c i p i t a t e d . Adsorption of organic materials on charcoal was not complete and also sulfate contamination and adsorption were possible. Sulfate contamination was shown to be possible even from " p u r i f i e d " charcoal when materials such as bicarbonate and phosphate buffer are present but when a low s a l t concentration solution was used, sulfate was adsorbed by the charcoal. Therefore i t was considered exceedingly d i f f i c u l t to know exactly how inorganic sulfate would behave i n various solutions when treated with charcoal. Polyvinylpyrrolidinone was an unsatisfactory adsorbant for these extracts where the pH was neutral or higher and a c i d i f i c a t i o n was avoided because of p o s s i b i l i t i e s - 120 -of hydrolysis or c o - p r e c i p i t a t i o n of inorganic sulfates with materials that may p r e c i p i t a t e . F i n a l l y , the ion retardation r e s i n was examined as a method for separating organic from inorganic s u l f a t e . It was found that inorganic sulfate was very strongly adsorbed by the r e s i n and was desorbed with d i f f i c u l t y using 2 N NaOH. This strong reagent for desorption also eluted contamination inorganic sulfate from the r e s i n . However, t h i s strong adsorption of inorganic sulfate was not considered a major problem provided organic sulfates were not also as strongly adsorbed. The only problem t h i s strong adsorption made was a cross-check on the recovery of t o t a l s u l f a t e from the r e s i n , since contamination sulfate was also released. The extracts showed a very sharp, clear v i s u a l f r a c t i o n of colored organic material. However, organic compounds not v i s u a l l y evident could s t i l l have been eluted a f t e r t h i s f r a c t i o n . The Walkley-Black determination of organic carbon i n various fractions eluted o f f the column was somewhat inconclusive i n showing that a l l the organic carbon came of f i n one single discrete f r a c t i o n , because of i t s lack of s e n s i t i v i t y . It d i d , however, indicate that a major amount (4 6-10 0%) of the organic carbon was i n t h i s unadsorbed f r a c t i o n . The - 121 -anthrone test indicated that some carbohydrates may have been s l i g h t l y retarded and may not have been present i n the colored f r a c t i o n . A comparison of E4:E6 r a t i o s of approximately equal amounts of material both treated and untreated by the r e s i n , indicated that there were s l i g h t changes i n the colored material a f t e r passing through the r e s i n . In some of the treated f r a c t i o n s , t h i s change may have been due to f i l t e r i n g of organic coloids by the r e s i n rather than retardation of certai n components, since supercentrifuging also made changes i n the E4:E6 r a t i o s . Some s o l i d material was noticed at the bottom of the c e n t r i -fuge tubes. I t was concluded that the ion retardation separation, much l i k e the Sephadex G-25 method, could be used to give a minimum estimate of organic sulfate i n s o i l e x t r a c t s. Cross-checking the recovery of added sulfate was d i f f i c u l t because of strong adsorption and contamination, which was not a problem with the Sephadex method. The retardation r e s i n did have an advantage of speed, ease of operation and s e n s i t i v i t y since a r e l a t i v e l y small organic f r a c t i o n could be collecte d and r e a d i l y dried for sulfate a n a l y s i s . Organic sulfate contents of the four s o i l s extracted by two d i f f e r e n t reagents are shown i n Table 1. The res u l t s - 122 -TABLE 1. Percentage organic sulfate sulfur in soil extracts using an ion retardation resin separation Extract Sample Bicarbonate Phosphate buffer Cardinal 5.2+0.2 7.7 ±1.7 Oxbow 48.4 ±4.5 41.1 Prest 28.0 ±3.5 28.3 Stevens 20.4 ±0.4 45.0 - 123 -show that there i s a f a i r l y s i g n i f i c a n t amount of organic sulfate present i n these extracts. The Cardinal sample did appear to be somewhat low and Sephadex separation indicated there may be a larger portion present. Part of th i s discrepancy may be due to some hydrolysis of organic sulfates by reagents or the r e s i n . CONCLUSION An examination of various methods of separating organic from inorganic sulfate i n s o i l extracts revealed that present methods are inadequate i n providing an accurate value. Various methods involving barium p r e c i p i t a t i o n were not s a t i s f a c t o r y for s o i l extracts for a variety of reasons including c o - p r e c i p i t a t i o n and lack of s e n s i t i v i t y . Charcoal was an unsatisfactory adsorbant of organic sulfate since the charcoal could contribute to contamination sulfate or under certain conditions i t could adsorb inorganic s u l f a t e . Polyvinylpyrrolidinone was inadequate as an adsorbant i n t h i s study, probably because the pH was too high. Both Sephadex and an ion retardation r e s i n method were found to give a minimum estimate of organic sulfate - 124 -in e x t r a c t s , but each had cert a i n inadequacies. The Sephadex separation was not considered suitable for extracts from the Cardinal sample because a discrete organic separation did not r e s u l t and colored organic material appeared i n the inorganic f r a c t i o n . This dispersion of organic matter over a large e l u t i o n volume contributed to d i l u t i o n of s u l f a t e , making analysis tedious for extracts with low concentrations of s u l f a t e . The r e t a r d -ation r e s i n yielded a discrete organic f r a c t i o n , but completeness of separation from inorganic sulfate could not be confirmed. A cross-check of the r e s u l t s could not be done by measuring t o t a l recovery of sulfate o r i g i n a l l y applied because of contamination and d i f f i c u l t y of desorbing inorganic s u l f a t e . The retardation r e s i n method did indicate f a i r l y s i g n i f i c a n t amounts of organic sulfate (5 to 48%) i n bicarbonate and phosphate buffer extracts of the four s o i l s used i n t h i s study. - 125 -REFERENCES Baddiley, J . , J.G. Buchanan and R. Letters. 1957. Synthesis of adenosine - 5! sulphatophosphate. A. Degradation product of an intermediate i n the enzymatic synthesis of s u l f u r i c e s t e r s . J . Chem. Soc. (1957), 1067-1071. Bart, A.L. 1969. Some factors a f f e c t i n g the extraction of sulphate from selected Lower Fraser Valley and Vancouver Island s o i l s . M.Sc. Thesis. University of B r i t i s h Columbia. 8 9 pp. Beaton, J.D., G.R. Burns and J . Platou. 1968. Determination of sulphur i n s o i l s and plant material. Technical B u l l e t i n No. 14, The Sulphur I n s t i t u t e , Washington. Bettany, J.R. and E.H. Halstead. 197 2. An automated procedure for the nephelometric determination of sulfate in s o i l e xtracts. Can. J . S o i l S c i . 52: 127-129. Brink, R.H., J r . , P. Duback and D.L. Lynch. 196 0. Measurement of carbohydrates i n s o i l hydrolysates with anthrone. S o i l S c i . 89: 157-166. Campbell, C.A., E.A. Paul, D.A. Rennie and K.J. McCallum. 1967. A p p l i c a b i l i t y of carbon-dating method of analysis to s o i l humus studies. S o i l S c i . 104: 217-224. Carlson, R.M., R.A. Rosell and W. V a l l e j o s . 1967. Modification to increase s e n s i t i v i t y of barium chloranilate method for s u l f a t e . Anal. Chem. 39: 688-690. Dodgson, K.S. 1961. Determination of inorganic sulfate i n studies on the enzymatic and non-enzymatic hydrolysis of carbohydrate and other sulfate esters. Biochem. J . 78: 312-319. Freney, J.R. 1958. Determination of water-soluble sulfate i n s o i l s . S o i l S c i . 86: 241-244. Hesse, P.R. 1957. The ef f e c t of c o l l o i d a l organic matter on the p r e c i p i t a t i o n of barium sulphate and a modified method for determining soluble sulfate i n s o i l s . Analyst 82: 710-712. - 126 -Jackson, M.L. 1965. S o i l chemical a n a l y s i s . P r e n t i c e - H a l l , Inc. Englewood C l i f f s , New Jersey. 498 pp. Kilmer, V.J. and D.C. Nearpass. 1960. The determination of available sulfur in s o i l s . S o i l S c i . Soc. Am. Proc. 24: 337-340. Kowalenko, C G . and L.E. Lowe. 1972. Observations on the bismuth s u l f i d e colorimetric procedure for sulfate analysis i n s o i l . Comm. S o i l S c i . Plant Analysis 3: 79-86 . Lowe, L.E. 1966. The separation and determination of organic and inorganic sulfate i n s o i l extracts. Can. J . S o i l S c i . 46: 92-93. - 127 -APPENDIX 5 CARBON DIOXIDE AND pH ANALYSES OF TWO SOIL SAMPLES TREATED WITH VARIOUS NITROGEN SOURCES AND RATES AFTER EIGHT WEEKS INCUBATION IN A CLOSED SYSTEM Nitrogen source Rate (ppm N) Carbon dioxide evolved (mg C/g s o i l ) Oxbow Prest pH (1:5 s o i l to water) Oxbow Prest None NH^OH KNO. NH4H2P04 NH4N03 (NH2)2CO 0 0.9084 1.8690 8 .68 5 .58 50 0.7500 1.9668 8 .86 5 .44 100 0.7452 2.0349 8 .70 5 .51 150 0.7311 2.0520 8 .68 5 .48 50 0.7788 1.8819 8 .57 5 .54 100 0.7179 1.8906 8 .57 5 .58 150 0.6787 1.7394 8 .59 5 .50 50 0.8760 2.0412 8 .42 5 .55 100 0.7491 2.0892 8 .16 5 .44 150 0 .7185 2.1162 7 .84 5 .49 50 0.7500 2.0349 8 .63 5 .48 100 0.7782 1.9131 8 .58 5 .44 150 0.7476 1.8723 8 .39 5 .51 50 0.7923 2.0118 8 .51 5 .50 100 0.7593 2.0166 8 .48 5 .48 150 0.7800 2.0045 8 .38 5 .51 

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