T. & r. n J. t O " f- . Fag"; l‘ ‘1 “3-." .l l. ..O\\ , L 4- ”on,” =- r~ .12, This is to certify that the dissertation entitled Aluminum Stress Effects on Microbial Activity in Soil Ecosystems presented by Matthew R. Vila has been accepted towards fulfillment of the requirements for Ph.D. degree in Soil Science Date 2/2/84 012771 MS U is an Affirmative Action/Equal Opportunity Institution ‘}V1ESI_} RETURNING MATERIALS: Place in book drop to LJBRARJES remove this checkout from .—;—. your record. FINES will be charged if book is returned after the date stamped below. ALUMINUM STRESS EFFECTS ON MICROBIAL ACTIVITY IN SOIL ECOSYSTEMS BB MATTHEW REY VILA A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Crop and Soil Science 1984 (:3 COPYRIGHT BY MATTHEW REY VILA l98h ABSTRACT ALUMINUM STRESS EFFECTS ON MICROBIAL ACTIVITY IN SOIL ECOSYSTEMS BY MATTHEW REY VILA The assessment of aluminum (Al) stress effects on microbial act- ivity (C0; evolution rate) was conducted for soil ecosystems and art- ificial soil miktures. The possible biological interactions of Al and Al in the presence of ferric iron (Fe’) were examined. Also. the act- ivity of several soil enzymes were assayed for comparison between A1 stressed and non-Al stressed soils. The results from these studies suggest that in the presence of. F53 , A1 stress is intensified. This investigation also demonstrated an increase in biomass associated A1 in the presence of F33. The significance of this finding is discussed relative to Al stressed Oxi- sols and soils in general with the occurence of soluble Fe in these enviroments. The results of the enzyme activity studies reflect a possible phosphate limited environment for the Al stressed soils. DEDICATION This dissertation is dedicated to my wife Susanne who’s love. patience. and help made my duration of this work an enjoyable one. ii ACKNOHLEDGEMENTS I would like to thank my major Professor. Dr. Vernon H. Meints for his support and the freedom with which he allowed me to explore soil science. Also. appreciation is expressed for his allowing me to experience soil science first hand by working in the Soil Testing Laboratory at Michigan State University during my tenure there. A special thanks is also due to Dr. Boyd 6. Ellis for his enlightening contributions to this work and for his instructive observations of me as a student. Finally. a special thanks to Dr. Alfred Haug for my course work in biophysics which I found invaluable. Thanks also to Dr. Haug for the many hours of discussion on this dissertation topic and for rasing my intial interest in this subject matter. iii TABLE OF CONTENTS Page LIST OF TABLES ................................................ vi LIST OF FIGURES ............................................... vii INTRODUCTION .................................................. 1 LITERATURE REVIEW ............................................. 2 MATERIALS AND METHODS ......................................... 11 Soil respiration study ................................ 11 Growth study .......................................... 14 Soil descriptions ..................................... ‘15 Bacteria and culture methods .......................... 15 Biomass estimates ..................................... 16 Enzyme activity assays ................................ l7 Pyrophosphatase .................................. l7 Phosphatase ...................................... l7 Sulfatase ........................................ i7 Dehydrogenase .................................... 17 Aluminum assimilation study ........................... 18 Soil chemistry assays ................................. 19 RESULTS AND DISCUSSION ........................................ 21 Artificial soil study ................................. 21 Natural soil study .................................... 41 Soil enzyme assays .................................... 55 Pyrophosphatase .................................. 55 Phosphatase ...................................... 55 SUlfatase IIIIIIOUIIOIIIIIO...DUI-ICIUIICIIIIIIIII 57 iv Dehydrogenase .................................... SUMMARY AND CC’NCLUSIONS .IIIIIIIIIOIOIIOIIIIOIOIIOIIIIIOIIIOII. BIBLIOGRAPHY IIIOIIIDIIIIIIIICIUOIIIIIIOUICOCI.0......-III-III. LIST OF TABLES Tables Page i. Monod model parameters for carbon dioxide evolution from artifiCial sail DOUG-Ill.-I‘ll-IIIIIIIOIIIIIIIIIIIIIIS23 2. Soil chemistry and biological assays ..........................42 3. Monod model parameters for carbon dioxide evolution from natural 50115 IIIIII-.IIIIIIIIIOIIIIIIIIIIOIIIOIIIIIII44 vi LIST OF FIGURES Figure Page 1. sail respiration fIBSk IIIll-Ill...GUCCI-IIIIIIOIIOIIOIIIICO.12 2a. Al stressed Bacillus mggatgrium (3-12) carbon dioxide evolution rates in artificial soil ...............22 2b. Al stressed Rhingium see. (1-110) carbon dioxide evolution rates in artificial soil .......................22 3a. Fe induced Al stressed Bacillus mggatgrium (B-12). carbon dioxide evolution rates in artificial soil ...............25 3b. Fe induced A1 stressed Bhizobium gag, (I-liB). carbon dioxide evolution rates in artificial soil ...............25 3c. FetIlllEDTA-Al stressed §g§1l1g§_mgggtggium (3-12). carbon dioxide evolution rates in artificial soil ...............30 l 3d. FetllllEDTA-Al stressed gningium_§g L (1-110). carbon dioxide evolution rates in artificial soil ...............30 3e. FetllllNTA-Al stressed Basillgg_mggatg;1um (3-12). carbon dioxide evolution rates in artificial.soil ...............32 3f. FetllllNTA-Al stressed Bh152212m_322; (1-110). carbon dioxide evolution rates in artificial soil ...............32 39. Fe(III)Citrate-Al stressed Ba;111gg_mgggtgnium (B-iZ): carbon dioxide evolution rates in artificial soil ........33 3h. Fe(III)Citrate-Al stressed Rhigobium gee, (I-ilB). carbon dioxide evolution rates in artificial soil ...............33 4a. AI-biomass association with Bacillu§_mgggtgrium (3-12) ......35 4b. Al-biomass association with Rhigogium gee, (1-110) ..........35 5a. Growth study. Al stressed B§§i11u3_mgggtgnigm (3-12) ........37 5b. Growth study. Al stressed RhizggiumLsagL (1-110) ............37 5c. Carbon dioxide production related to growth. Bacillus mggaterium (B-12) and Rhizobium see. (1-110) .............40 6a. Low Al soil carbon dioxide evolution rates ..................43 6b. Al stressed soil carbon dioxide evolution rates .............43 7a. Fe amended low A1 soils carbon dioxide evolution rates ......46 vii 7b. 8a. 8b. 9a. 9b. 10a. 10b. lla. 11b. 12a. 12b. 13a. 13b. 14a. 14b. 15a. 15b. 16a. 16b. 17a. 17b. Fe amended Al stressed soil carbon dioxide evalution rates Ill-IIIIIIIICIIIIIII-Ill.III-IIIDIIIIIOIII46 Low Al soils biomass C. biomass C vs exch. Al ...............47 Al stressed soil biomass Cs biomass C vs exch. Al ...........47 Fe amended low Al soil biomass C. biomass C vs EXChI A1 COO-ICCICIICICI.I.IIOCIIIIIIOI-IOIIIOIOIIIIIIIIII49 Fe amended Al stressed soil biomass C. biomass C vs EXCh. Al OIII-CIIIIIII0.0-.-IOIIIIIUIOIOIIIIOI.III-IIO-00.49 Mg amended low Al soil biomass C. biomass C vs EXCh. Al CHIC-ICIIII-CIOIIICII'D-IIIOIIIIIIIIIIIIIOI'lllfllsa Mg amended Al stressed soil biomass C. biomass C vs BXChI A1ICIIICCOIIIIUCCIICIOOIIIII-IICIIIIIOIIIIIIIIIIIIOSB Low Al soils biomass C, biomass C vs soil organic C .........52 Al stressed soil biomass C. biomass C vs soil organic C .....52 Fe amended low Al soils biomass C. biomass C vs soil organiccCl...-GUCCI-CUIOIIICIIIIII.IIIICIOCCIIIIOII0.0.053 Fe amended Al stressed biomass C. biomass C vs soil organiccIIOIIOOUIICIIII-IIIIIIUIIIICIIOOIIOUIOCI.ICOCO-.53 Mg amended low Al soils biomass C. biomass C vs soil organiccIIIOOOII...III-IICCIUCIIICIIIIIIIIODIOIIIII0000054 Mg amended Al stressed soil biomass C. biomass C vs soil organic C ................................................54 Low A1 soil pyrophosphatase activity ........................56 Al stressed soil pyrophosphatase activity ...................56 Low Al soil phosphatase activity ............................58 A1 stressed soil phosphatase activity .......................58 Low Al soil sulfatase activity ..............................6@ Al stressed soil sulfatase activity .........................60 Low A1 soil dehydrogenase activity ..........................61 Al stressed soil dehydrogenase activity .....................61 viii INTRODUCTION The world wide occurrence of aluminum (Al) stressed acid soils warrants the study of Al stress effects on biological systems. These soils are most frequently found in the tropics and subtropics. and generally are classified as oxisols. The temperature and humidity of these regions are ideal for crop production. The common occurence of high exchangeable Al in soils of these regions often limits production. High concentrations of soluble Al and iron (Fe) species are characteristic of these soils along with kaolinitic clays and low pH. Currently. the specific effects of Al on agricultural and ecological systems are not well understood. A general understanding of soil microbial activity under Al stress would help explain the problems associated with rhizosphere associations between plants and their respective microorganisms. This study was conducted to assess the effects of Al stress on microbial activity in acid aluminum soils. A survey of the current literature revealed a subtle suggestion that Fe might be implicated in the intensification of Al stress effects on microbial activity. In the literature. the involvement of Fe”3 in Al stress response was not addressed directly. Contradictory views as to whether microbial activity in acid soils is even subject to Al inhibition are found in the literature. This study addressed the hypothesis that soluble Fe+3 will intensify Al stress effects on microbial activity. LITERATURE REVIEW Aluminum stress effects on crop production related to acid soils have been extensively studied (Mattson and Hester. 1933; Chernov. 1947; Harward and Coleman. 1954; McLean et. al.. 1965; Evans and Kamprath. 197B; Hoyt and Turner. 1975; Thomas. 1975; Reid. 1976). However. only recently have the effects of Al stress on the soil microflora been investigated. While several investigations into the‘ effects of Al stress on microbial activity have been conducted. results in the literature have been inconclusive as to the specific effects of Al on microbial metabolism (Keyser and Munn. 1979a .and‘ Keyser and Munn. 1979b). To understand the biological effects induc- ed by Al stress. knowledge of the chemical state of Al in the medium used for experimentation is most important to the determination of bioactive forms of Al. In mineral soils. hydrogen (H) and A1 are the two cations prim- arily responsible for soil acidity. Chernov (1947) made an early association between trivalent cations (i.e. Al and Fé’) and acid soils. Chernov realized the instability of proposed H saturated soils and suggested that Al and Fe saturate exchange sites in acid soils. Jenny (1961) reported that the weak acid properties of acid clays in fact result from a complete or partial saturation of exchange sites by various hydrolyzed Al species. Another characteristic of Al controll- ed acidity is that H saturated clays respond more like strong acids than Al saturated soils which behave more like weak acids (Bohn. et. 31.9 1979)e Trivalent A1 occurs in soil in a variety of forms. These forms of soil Al include various degrees of hydroxylated monomeric and poly- meric species (Bohn. et.al.. 1979). In acid soil where pH ranges from 5.0 to 3.0. monomeric Al may occur as hydrated forms of Afa. AlOHn’ . Al(OH):_and ARCH); in solution. Gibbsite or gibbsite-like minerals are suggested to be the major forms of Al which precipitate when Al excedes its solubility product (Bohn. et.al.. 19793 and Linsay. 1979). Polymeric forms of Al occur between hydroxylated species forming large units with a general formulation of (Al(0Hh‘(H o;;:::)n where n is the number of Al ions per unit polymer. Polymerization is promoted by colliod surfaces present in solution and suggests proximity enhance- ment. Recent studies suggest that monomeric as well as polymeric species may be involved as sources of exchangable Al (Turner. 1967: Vieth. 1978). Previously. it was held that only monomeric species had the overall mobility to be exchangeable. Exchangeable Al is the form most often associated with the phytotoxic effects of Al stressed soils (Barnhisel and Bertsch. 1982; and Reeves and Sumner. 1970). A ques- tion still remains as to whether polymeric exchangeable Al polymerizes prior to or after exchange extraction (Barnhisel and Bertsch. 1982). Hargrove and Thomas (1981) have examined plant growth in soils amended with Al-citrate. Al-EDTA. Al-fulvate. Al(OH),_Cl and no Al. There were no differences among Al-citrate. Al-EDTA. Al-fulvate. and no A1 rela- tive to plant growth. However. soils amended with Al(OH)LCl demonstr- ated severe phytotoxic effects. Hargrove and Thomast1981) also demon- strated a strong negative relationship between exchangeable soil Al and plant growth. Plants grown in acid soils often appear to exhibit a variety of nutrient problems. In acid soils with high Al percent saturation values. calcium (Ca) as well as magnesium (Mg) are usually displaced by A1 at exchange sites. Potassium (K) levels are also depressed in Al stressed soils. Molybdenum (Mo) availability is decreased under low pH conditions and often provides for Mo deficiency problems for nitrogen-fixing legumes (Bohn. et.al.. 1979; and Jackson. et.al.. 1963). A variety of micronutrients become increasingly soluble. often to phytotoxic levels. in acid soils (Brady. 1974). Iron and manganese (Mn) toxicities often occur under highly solublizing acid conditions. Zinc (Zn) toxicity also may occur in acid soils. although this is a rare phenomena (Vitosh. et. al.. 1981). Under acid-Al soil condi- tions. phosphorous (P) added to soils may become rapidly unavailable due to fixation and precipitation with Al compounds present (Hsu and Rennie. 1962: Hsu. 1965; Parfitt. 1977: and Sims and Ellis. 1982). All of the effects on nutrient availability just described con- fuse the issue of Al toxicity problems in acid soils. These effects often occur in association with acid-Al stressed soils. The assess- ment of the direct effects of Al on organisms living in the soils has been unsucessful. Complications arising from the various nutritional problems mentioned make observation of the direct relationships be- tween Al and soil organisms difficult to resolve. A review of the literature indicates Al stress conditions are inhibitory but not usually lethal to microbial populations (Keyser and Munns. 1979a; Cooper and Morgan. 1979a; and Munns and Keyser. 1981). Hartel and Alexander (1983) claim that Al in acid soils is of no general consequence to the activity and the survival of cowpea Rhizobia strains in soils. Munn and Keyser (1981) concluded that under prolonged Al stress spontaneous mutation toward Al tolerant strains did not occur. These authors also demonstrated by synchronous culture methods that cell division is greatly delayed but that Al was not generally lethal to Rhizobia cell survival. Studies examining Ca deficiencies in Rhizobia have also been con- ducted. Amendments of Ca to Al stressed cultures of Rhizobia strains demonstrated no relief from the Al toxicity experienced by the cultures (Keyser and Munns. 1979a). Keyser and Munns (1979b) also‘ examined the effects of Mn toxicity under Al stress conditions. They found no enhancement of the toxic effects of Al stress on Rhizobia strains in the presence of soluble Mn. The effect of Al stress and low P availability relative to Rhizobia strains has been examined. While a low P concentration did limit growth in Rhizobia strains. the effects of Al and acidity were found to be much more severe (Keyser and Munns. 1979b). The effect that allophane clay has on the growth of Egchggiga goli has been examined. In a study by Cooper and Morgan (1979b). it was demonstrated that allophane at pH 5.0 did not exhibit Al stress responses when amended to EL_ngi cultures. However. sol- uble Al added at 0.2 umol Al/mL demonstrated a significant reduction in cell respiration and cell division. Zwarun and Thomas (1973)_ demonstrated that exchangable Al alone had little effect on microbial activity. but that soluble Al did reduce viability for cultures of Pseudomonas stutzeri . Furthermore. Zwarun and Thomas (1971) found no effects on a Bacillus sp. exposed to Al-saturated clays with only exchangable Al available. From a review of the literature. it appears that in bacterial cultures where exchangable A1 is the source of Al. only a minimal effect. if any. is noticeable. Primary effects come from additional amendments of soluble Al (Zwarun and Thomas. 1971.19733 and Cooper and Morgan 1981b). Cooper and Morgan (1981b) suggested that in clay systems the HT given off by microbial growth is absorbed by the clay. These investi- gators noted that when pH was monitored in simultaneous treatments. decreases in pH due to growth of §.§oli were reduced in the presence of allophane. while the metabolic rates were the same with or without allophane. A slight enhancement was noted in the metabolic rate as the amount of allophane was increased (Cooper and Morgan. 1979a. and 1979b). This enhancement is in agreement with the observations of Stotzky and Rem (1966) concerning microbial interactions with clays. A review of the literature revealed no references to the specific physiological effects of Al on microorganisms. However. in- direct references to one area of microbial physiology were made in several articles. These subtle comments point to an involvement of soluble Fe” in the intensification of Al stress on soil microorganisms. In an extensive review of Fe transport. Arceneaux and Byers (1976). cite an experiment by Davis and Byers (1971) in which Al was used as an inhibitor for a permease-like Fe uptake mechanism. In this experiment. Al was thought to coprecipitate Fe:3 . originally FeCls . making it unavailable to the transport-permease system. The organ- isms used were Bacillus megaterium mutants which lacked the siderochr- ome chelates to supercede the permease system. When exogenous sidero- chrome for that organism was amended to the system. Fe“ transport re- sumed immune to the presence of A1 at 4x10"5 fl concentration (Davis and Byer. 1971). Arceneaux and Byers (1976) cite examples which demonst- rate that microorganisms which are able to take up one kind of micro- bial siderochrome can usually utilize a variety of Fe-chelates produc- ed by other microorganisms. Such microbial produced chelates include citric acid. a variety of catechols. and hydroxamic acid polymers. In Egghg:;§§_ggll . Bacillus megatgrium . Aergbagter agrgggngg . and Bacillus gyhtilis . it has been demonstrated that high Fe?3 concentrations (10' -10fl M.) repress synthesis of the enzyme system which inturn synthesizes siderochrome chelate. Under high Fe concen- tration. membrane bound carriers transport ng into the cell (Downer. et.al..1970). Under Al stress. Rhizogium jagonlgum demonstrated some relief from Al-stress when Fe(III)-EDTA replaced an equilvalent concentration of Fe"3 as FeC13(Keyser and Munns. 1979b). There is no immediate ex- planation for this effect except that EDTA might be chelating soluble Al. This explanation is doubted by the investigators. and it is not supported by a relatively low stability constant for an Al-EDTA complex at pH 4.5. and a high stability constant for Fe(III)-EDTA ‘(Sillen and Martell. 1974; and Mortvedt. et.al.. 1974). Finally. Cooper and Morgan (1979a. 1979b) noted in experiments with Al-stress- ed E, 5011 that one treatment at an intermediate Al concentration de- monstrated a greater stress response than treatments at higher Al con- centrations. In these treatments. E. coli were subjected to alloph- ane clay and soluble Al. The investigators stated that Fe"3 released by allophane occurred in the intermediate Al stress experiment. They suggested that the Fé‘ caused a precipitation of bacterial cells which resulted in the greater than expected stress response (Cooper and Morgan. 1979a. and 1979b). The chemical properties of Al and Fe'.3 under acid conditions are very similar. In general. F5‘ and Al exhibit similar solubility and hydration characteristics. It has been suggested that Fee’under acid conditions can precipitate microorganisms in solution (Tenny and Stumm. 1965). Cooper and Morgan (1979a) found flocculation of E; coli in the presence of allophane clay and/or Al. but they did not at- tribute the Al complexing phenomena to a reduction in microbial act- ivity. At the pH of 4.5. F53 and Al have similar binding affinities for soil organic matter (Bloom. 1981). However. under situations where the specific association of Fe"3 or A1 with organic matter is by chelation; Fe.a out-competes Al for the chelate. This observation is supported by Féa’high stability constant for compounds like EDTA when compared with Al (Log K=25.0 for Fe.3 . 16.1 for Al) (Bohn et. al..1979). This difference is attributed to the ability of transition elements like Fefato orient their electronic configurations to optomi- ze the ligands general configuration. Aluminum being much more rigid in its electronic structure is not as competitive (Bohn et. al.. 1979). This redistribution of electronic structure for Fe and not for Al may be the reason for the observations by Davis and Byers (1971) that bacterial siderochromes selectively chelated Fe(III) out -5 , of a solution containing 4x10 M Al. In both classes of microbial chelates. hexadentate cages of six oxygens hold Fe\ securely (Silver. 1978). i The membrane bound carriers for Fe would appear to be a reason- able site for physiological inhibition by Al. No literature is avail- able relative to Al uptake by Fe"3 membrane assimilation mechanisms. Evidence suggests that membrane carriers for inorganic ion species may be relatively non-specific for ions sharing certain similar properties. Most likely. these carriers are optimized toward a spec- ific ion. However. similar to enzyme systems. these carriers might interact with other non-optimal ions sharing similar ionic properties. These interactions with less optimal ion species are not unlike enzy- matic transformations of substrate analogs which differ only slightly from their nominal substrates for a given enzymatic reaction. Evidence for a similar process in ionic solute assimilation can be found in the MgfiLtransport system. The M5’- transport system has been shown to be optimal for qu'. but competetively inhibited by a variety of divalent cations. These competetive divalent cations include cd"-. CJT . and Nil (Silver. 1978). To date. no highly specific uptake mec- hanism exists to explain how these trace elements are assimilated by microorganisms. Silver (1978) suggested that these cations are taken up in a sufficient quantity through competetive assimilation through the Mal'transport system. Excessive extracellular Mml'or Cdi'have been shown to enter E. coli creating cytotoxic levels through the Mg 'transport system (Silver. 1978). Ionic properties such as ionic radius. ion charge. calculated activities. and ionic potentials are so similar that it is not ID surprizing that competetive uptake occurs for these divalent trace +L metal ions in the Mg transport system (Silver. 1978; and Bohn et. al.. 1979). It seems reasonable that a similar competition between +3 +3 . +3 . soluble Fe and A1 might occur for the membrane bound Fe carrier as i + the ionic properties of Fe. and A1 3are quite similar (Bohn et. al.. 1979). MATERIALS AND METHODS SOIL RESPIRATION STUDY: The respiration studies were conducted utilizing the alkali absorption method for carbon dioxide. The general methods for the carbon dioxide assay were adapted along the guidelines set forth by Van Cleve. et. al.. (1979). Ten grams of soil were dispensed into a 50 mL erlynmeyer flask. the appropriate substrates added. the CO; cache put in place. and the flask stoppered (See Fig. 1). The CO; catche consisted of a 2.0ml plastic cup filled with 1.0m1 of 0.4 g NaOH solution. The catche was attached to a 30 gauge wire which was held pinched between the stopper and the flask. At the end of an experiment. the 1.0 mL of NaOH was removed from the flask and added to 5.0 mL of 10% BaClz solution. This solution was titrated with 0.1 N HCl (standardized with T.H.A.M.). The carbon dioxide evolved is reported as nmol C/g soil/h. The formula to obtain the carbon dioxide evolution rate is as follows;- nmol C/g Soil/h =((B-A)xNx1000)/g Soil/Total time(h) B = ml of acid titrated to blank A = ml of acid titrated to active sample N = the normality of the acid The artificial soil was prepared by saturating a montmorillon- 11 Sto Holding wire pper NaOH container Flask Soil Fig. 1. Soil respiration flask. 13 ite clay (commercial grade 'vol-clay‘) and leaching it with a 1.0 fl AlCl} solution. The clay was washed with distilled water until no free Cl. was detectable with AgSO‘. and then mixed with sand to achieve a 5% clay mixture by weight. This system was found to contain 4.44 umoles of exchangeable Al per gram of mix. The sand-clay mixture ex- hibited a pH of 4.6 1 0.2. while the pure sand gave a pH of 4.7 : 0.1. Throughout this study. the pH of these artificial soils did not vary more than 0.2 pH units. Measurements for pH were made before and after each experiment. All respiration experiments were conducted at 25 1 0.5 C. Ten grams (air dried wt.) of artificial soil were added to a flask. and brought to 30% moisture content through the innocul- ation with microbial cell slurry’s and substrate amendments. All ex- periments were run for 18 hours. For natural soils. 10 9 (air dried wt.) were added to the incubation flasks. and were treated in a manner similar to the artificial soils. Amendments to the artificial soils included 1.0 mL of microbial cell slurry. 1.0 mL of carbon substrate solution. and 1.0ml of additional amendments or sterile distilled water. For the natural soils. the same amendments were used except the cell slurry was replaced by 1.0 mL of sterile distilled water 24 hours prior to the start of the experiment. The natural soils consisted of Al stressed and non-stressed soils. The carbon substrate solutions used for the artificial soils consisted of a i=1 mixture of glucose (Mallinckrodt) and yeast extract (Difco. Lot-652609). This solution was analyzed for percent C and ad- justed to give a final concentration of 4. 8. 12. 16. and 20 umol sub- 14 strate C/g soil when 1.0 mL of the substrate solution was added to 10 g of soil. For the natural systems. only glucose at 4. 8. 12. 16. and 20 umol substrate C/g soil was used. Cell slurries for the artificial soils were prepared by centrifuging broth cultures. washing the pellet in distilled water. centrifuging and washing the pellet again. and re- suspending the pellet in distilled water. Percent carbon determina- tions were made on the cell slurry. and the slurry was diluted to yield 200 ug microbial C/g soil when 1.0 mL of the suspension was add- ed to 10 g of soil. The Fe amemdments were made prior to the addition of the carbon substrate for both artificial and natural soils. Iron was amemded to the soils at 0.01. 0.1. 1.0. and 1.3 umol Fe lg of soil as FEC13. The respiration experiments using Fe(III) chelates included EDTA. NTA. and citric acid as the complexing agents. Each of the PET chel- ates (Fe(III)EDTA. Fe(III)NTA. and Fe(III)Citrate) were brought to three concentrations in solution. 10" M. 104M. and 10‘M. These iron solutions were amended to the artificial soil system at 1.0 mL/10 g of soil. Utilization of the chelated forms of F6‘ allowed for control of soluable Fe"3 . GROWTH STUDIES: The effects of different F33 and Al treatments on the growth rates of Bacillus megaterigm (B-12) and Rhizobium sea. (I-110) were conducted turbimetrically on a Bosch and Lomb Spectronic-20 spectro~ photometer using optical side-arm culture flasks. Growth rates were 15 monitored for 48 hours and maintained at approximately 25 C. The con- trol flask consisted of a sodium acetate buffer. 0.02 M . pH 4.6; and glucose-yeast extract (131). at 8.0 umol C/mL. The subsequent treat- ments included the control media above plus A1 (3.8 x 104'M ) or Fe (1.0x10qfl Fe(III)Citrate)» giving an Fe“ molar activity of . approximately 10." M . The treatments included the control. acetate buffer plus glucose-yeast extract (GY). GY with Fe+3 amended (GY+Fe) and GY with an initial Al amendment (GY+Al). Two other treatments included were GY+Fe with Al amended after 18 hours of growth (GY+Fe > A1) and GYfAl with Fe+3 amended after 18 hours of growth (GY+A1 > Fe). SOIL DESCRIPTIQNS: The soils used in this study included the following low Al soils; IB. (Owosso) a fine-loamy. mixed. mesic. Typic Hapludalf; IC. (Capac) a fine-loamy. mixed. mesic. Aeric Ochraqualf; ASIA. (Barry) a fine-loamy. mixed. mesic. Typic Argiaquoll; FSlA. (Boyer) a coarse- loamy. mixed. mesic. Typic Hapudalf; and CK-19. (Brookston) a fine- loamy. mixed. mesic. Typic Haplaquoll. The Al stressed soil was repre- sented by the group IA - VA. (Kalamazoo sandy loam) a fine-loamy. mix- ed. mesic. Typic Hapludalf. BACTERIA AND CULTURE METHODS: Bacillus megaterium (B-12) was obtained from the culture col— lection of the Dept. of Microbiology and Public Health at Michigan State University. Stock cultures of Bacillus megaterium (B-12) were 16 kept on nutrient agar slants (Difco). The slow growing Rhizobium see. (I-llO) was obtained from the laboratory of Dr. Frank Dazzo. Dept. of Microbiology and Public Health. Michigan State University. The Rhizobium strain was maintained on mannitol-yeast extract agar slants with mannitol. 10 g/L; yeast extract (Difco). 1.0 g/L; KZHP04o3HzO. 0.65 g/L; MgSOqo7H O. 0.2 g/L; NaCl. 0.1 9/L; and special agar (Nobel. Difco). 15 g/L. Broth culture media for cell slurry production for both B. megaterium (B-12) and Rhizobium see. (I-110) contained 5.0 g/L glucose and 5.0 g/L yeast extract (Difco) incubated at 25 C. for 24 hours for B. mggatgrium (B-12). and 48 to 72 hours for the Rhizobium sag. (I-110). BIOMA ESTIMAT S: The cell slurry biomass estimate. carbon content. was determined by transfering 1.0 mL of the washed cell suspension to a container holding 10ml of 0.5 N NazCr‘O,. To this solution. 10 mL of concentrated H1504 was added. The mixture was allowed to digest for 30 minutes. and then was read on a Bousch and Lomb Spect-20 spectro- photometer at 645nm. Glucose solutions of known carbon content were used for calibration. Natural soil biomass estimates were conducted according to the respiration method of Anderson and Domsch (1978). In this method. nat- ural soils were amended with 0.5. 2.7. 5.5. 8.3. and 11.1 umol glucose/g soil. The soils were monitored for Coz’evolution as describ- ed above. Incubation was conducted for 2 hours at 22 i 0.5 C. 17 ENZYME ACTIVITY ASSAYS; Pyrophosphatase: The procedure of Tabatabai (1982) for the assay of pyrophosph— ate activity was modified. The procedure used here differed in that no buffer was added. and the incubation temperature was maintained at 25 1 0.5 C. Activity was reported as umol P/g soil/h. Phosphatase: The procedure of Tabatabai (1982) was modified. The modificat- ions included substituting distilled water for the Modified Universal Buffer. and incubation at 25 g 0.5 C. para-nitrophenol phosphate was the substrate used for this assay. Enzyme activity was reported as umol nitrophenol/g soil/h. Sulfatase: The procedure of Tabatabai (1982) was used and modified. Modifications included the substitution of distilled water for the acetate buffer. and incubation at 25 1 0.5 C. The substrate utilized in this assay was p-nitrophenol sulfate. Activity was reported as umol nitrophenol/g soil/h. Dehydrogenase: One gram of soil (air dry weight) was transfered to a 30 mL 18 test tube. One milliliter of substrate solution (yielding triphenyl tetrazolium chloride (TTC) at 90 umol TTC/g soil) was added to the soil and incubated 24 h. at 25 C. Extraction with 10ml 95% methanol. was conducted by mixing the methanol and soil. then pouring the suspension into a funnel with No.42 filter paper (Whatman). and final- ly washing the sample on the filter paper with an additional 10 mL of 95% methanol. The extracted triphenyl formazan was then analyzed col- orimetrically on a Bousch and Lamb Spect-20 at 545nm. Soils with no TTC added were incubated and extracted for subtaction of background. AL M M A SIMI AT ON S Y: Bacillus mggalcricm (B-12) and Bhiz99120_222; (I-110) were grown in culture media containing 1.0 9 glucose and 1.0 g of yeast ex- tract (Difco) in 100ml of water. They were incubated at 25 C for 24 and 48 hours. respectively. The broth cultures were centrifuged and washed 3 times with 0.02 M acetate buffer at pH 4.6. The third wash- ing was decanted and the cell pellet resuspended in 30 mL of acetate buffer. There were five treatments for both B. megcterium (B-12) and Rhizocium see. (I-110). The treatments included a control with no A1 and four Al treatments. The four Al treatments were all brought to 1.11 umol Al/mL in solution. The first two of the four treatments were divided into azide and non-azide treatments. The next two treat- ments included 0.01 umol Fe/mL (FeCla) with azide and non-azide sub- treatments. The azide had a final concentration of 3.0xlOfl3 M . Bio- mass C was determined by wet oxidation as described earlier. 19 After incubation. the cell suspentions were centrifuged and washed 3 times with 0.02 M acetate buffer with a pH of 4.6. After the third centrifugation. the supernatant was decanted. the pellets resus- pended in 10 mL 30% HzOg. and digested for one hour. Next. 5.0 mL of concentrated HCl were added to the suspentions giving a final molarity of approximately 5.0 M HCl. The samples were allowed to digest for 48 hrs. They were assayed for aluminum on a SMI (Beckman) DC plasma emis- sion spectrophotometer at 308.2 nm. SOIL CHEMISTRY ASSAYS; Assays for pH. P. K. Fe. Mn. Zn. NO3. and Organic C were done in accordance with the methods put forth by Danke (1980) in Bgcccmgngcg ghgmiccl Soll Tcgt Prccedurgc for thg North antrgl Rccion Calcium and Mg were assayed from the same extract (ammonium acetate) that was obtained for K. Soil pH was determined by the water method. with the soil to water ratio 1:1 by soil dry weight (McLean. 1980). Potassium. Ca. and Mg were extracted from 2.5 g of air dry soil with 20 mL of 1.0 M ammonium acetate buffer at pH 7.0 (Carson. 1980). The extract was analyzed on a Technicon Autoanalyser II employing flame emission (propane) for K and Ca. and a colorimetric assay for Mg. Phosphorous was assayed using the Bray-Pl-Ascorbic acid method for orthophosphate (Knudsen. 1980). The micronutrients Fe. Mn. and Zn were extracted from 2.0 g of air dried soil with 20 mL of 0.1 M HCl (Whitney. 1980). The extracts ll 20 were assayed by atomic absorption on a Perkin-Elmer 290 atomic absorp- tion unit. Nitrate was assayed with an Orion nitrate ion selective probe. Twenty grams of air dry soil were extracted with 50 mL saturated CaSQ‘ . The slurry was then measured directly with the nitrate electrode (Carson. 1980). Exchangeable Al was extracted with 1.0 M KCl (5.0 9/50 mL) ac- cording to the procedure of Barnhisel and Bertsch (1982). Aluminum was assayed by the aluminon colorimetric method. Soil organic carbon was determined by chromic acid digestion.l In this method. 1.0 g of soil was transfered to a 30 mL test tube. Ten millimeters of 0.5 M Na.Cr.01 were next added to the test tube. and then 10 mL conc. stqlwas added causiously. The samples were digested for 24 hours. Five milliliters of the digest were decanted and dilut- ed with 10 mL of distilled water. After mixing. the sample was read at 645 nm on a Bousch & Lomb Spect-20. Soils of known organic C cont- ent were used as standards. RESULTS AND DISCUSSION Artificial Soil Study; An artificial soil system was developed to examine the effect(s) of F3‘ on bacteria-aluminum interactions. First. the effects of diff- erent exchangeable Al concentrations on microbial activity were examined in a sand-clay (95% sand. 52 clay) system. The results for the microorganisms Baclllcs mcgctgrium (B-12) and Rhizobium see. (I- 110) can be seen in fig. 2a and 2b respectively. These figures illustrate the effects of exchangeable Al on cell respiration in the form of carbon dioxide evolution rates versus substrate carbon concentration. With the organism B. mccaterlcm (B-12). the effect of exchangeable Al on the carbon dioxide evolution rate was dramatic as exchangeable Al was increased from 0 to 2.22 umol exchangeable Al/g soil. B.mcgacerlcm (B-12) demonstrated a decrease in both the maxim- al velocity and the initial velocity. Carbon dioxide evolution for B.megaterium (B-12) was reduced by 90 to 100 percent when the exch- angeable Al concentration was 2.22 umol exchangeable Al/g Soil (Fig. 2a). With Rhizobium see. (1-110). carbon dioxide evolution rates at 2.22 umol exchangeable Al/g soil were only reduced to 80 percent of that obtained with no exchangable Al present. The major effect on Rhizobium see. (I-110) carbon dioxide evolution kinetics was the slight reduction of the maximal velocities (Fig. 2b). The hyperbolic character of the data presented in figures 2a and 2b suggested that the best assessment of this microbial activity would come from Monod growth kinetics analysis (Spain. 1982). Table 1 gives the parameters derived from the data in figures 2a and 2b using the 21 Al-STRESSED Bmegaterium (812) m // 400 358 S 250 Will 8 293 ' 3’ 159 e 0 me E 8.37 E 1.11 8 13 1 2.22 (moi substrate C/g soil umol 91g soil Fig. 2a. Aluminum stressed WWW-12) with glucose-yeast extract (l:l) as C substrate. ' 8" /7‘ an L4 609 t 500 "a 483 I j murmur n1 a 3’ - 9 ° 2m 3 a 8.37 e 199 C o 1.11 0 4 8 12 16 as D 2.22 (mol Mate C/g soil ueol 91/3 soil Fig.2b. Aluminum stressed Rhicobium see. (I-llO) with glucose-yeast extract (l:l) as C substrate. “H Table 1. Monod model parameters for carbon dioxide evolution from artificial soils under aluminum stress. umol Al/g soil u (nmol C/g soil/h) K (umol C/g soil) X (Biomass. ug C/mL) umol exch. Al/g soil u (nmol C/g soil/h) K (umol C/g soil) X (Biomass. ug C/mL) Monod model equation: Bacillus megaterium (B-12) B: 0.37 1.11 2.22 473 537 323 51.9 1.58 4.40 2.17 27.3 200 200 2B0 200 Rhizobium see. (I-110) B 0.;7 1.11 .22 958 1226 1000 937 5.50 10.8 15.3 11.6 200 200 200 200 dX/dt = uX = u-(S/(K+S))°X S = umol substrate C/mL 'll 24 Monod growth model. Upon amendment of Fe¥3to Al stressed sand-clay artificial soil systems. both B. megaterium (B-12) and Rhizobium see, (I-ll0) demonstrated an intensified Al Stress response (Fig. 3a and 3b). The similarity in chemical properties of Al and FeP3 at pH 4.6 sugg- 3 uptake ests that microorganisms might accumulate Al through the F6" mechanisms. An important reason for examining the antagonistic effects of Fe on Al stress lays in the soil chemistry of Al stressed environments. Aluminum stressed soils exist primarily in the tropic and subtropic environments of the world. These soils are primarily oxisols in classification. They are typified as low pH. highly weathered soils containing kaolinitic clays and relatively high concentrations of Al and Fe oxides. Ecologically. the oxisol just described might repres- ent a similar antagonistic environment as exhibited in the experiments shown in Fig. 3a and 3b. In pure culture studies. several authors (De Carvalho. et.al.. 1981; and Hartel and Alexander. 1983) have suggested that slow growing Rhizobia species in the presence of soluble Al demonstrates no major inhibitory response. The data presented in figure 2b for Rhizobium SEE; (I-iiO) support these views. Aluminum on its own demonstrated little inhibitory effect on Rhizobium see. (I-ilo). From an ecological viewpoint. soluble F53 could be expected to be present in acid soils. Figures 3a and 3b illustrate the devastat- ing effect of soluble Fe"3 under Al stress conditions. When Fe+3 is insoluble or bound. bacteria can utilize a series of biologically produced chelating compounds to assimilate Fe Al+Fe B. negateriue (B-le) 439 359 330 i 259 “g 299 \ me new Fe m we \ . 0 <3 \. -: .. tea a 1.0x10 g 53 W o Lexie" a U 1.02.19" 0 0.37 1.11 2.22 U 1.3xie" moi Exch. ill/3 Soil umol m9 Soil Fig. 3a. Iron induced aluminum stressed B, megatericm (B-12) with glucose-yeast extract (1:1) as the C substrate at 8 umol C/g soil. Al+Fe Rhizobium spp. (1-110) h unn.msnmmte ‘1 nmol C/g 8011/ - a name" 1.0x157 1.0m“ a 0.37 1.11 2.22 1.33.10“ “ ueol Exch. Al/g Soil umol F913 Soil O EZIEJ c: a Fig. 3b. Iron induced aluminum stressed Rhizobium see, (I-110) with glucose-yeast extract (1:1) as the C substrate at 8 umol C/g soil. 26 . Since these are truely chelating compounds. it is suspected that their affinity for Al is low because of Al’s rigid electronic configuration. unlike the flexible configurations of Fe‘3 for ligand binding. Davis and Byers (1971) demonstrated the ability of Fe"3 chel- ating agents produced by B. megaterium to chelate Fe"3 coprecipated by 10 uM A1. with no apparent interference from Al. Emery (1974) has shown that certain siderochromes (biological Fe(III) chelating agents) which bind Cu(II) are not transported across microbial membranes. Selective binding of Cr(III) by Fe siderochromes have been demonstrat- ed. Leong (1971) showed that Salmonella tyghimurium assimilated Cr(III) chelated by bacterial siderochromes. The only other sidero- chrome system found which was interfered with by other metals was fer- richrome. Ferrichrome is only produced by fungi. but has been found to transport Al across the cell membranes of Uctllcgo secgrogena (Emery. 1974). All Fe chelate transport systems have been found to be repressible by high Fe?3 concentrations (Downer. et. al.. 1970; Silver. 1978). Frost and Roseberg (1975) have demonstrated a low affinity mem- brane carrier for uncomplexed Fefl . The carrier is not dependent on metabolic energy and is suspected to be a facillitating transport per- mease for Fe”. This permease is not repressible by high Fe+3 concentrations. and is presumed inducible by its substrate as are most permeases (Silver. 1978). The investigator suggests that the differences seen in the init- ial kinetics between B.me9aterium (B-12) and Rhizobium see. (I-110) under Fe induced Al stress lay in the morphological differences between the two microorganisms. Bacillus megaterium (B-12) is a gram I‘ll 27 (+) microorganism with the outer cell surface (cell wall and cell mem- brane) directly exposed to the local environment. Rhizobiumvgggc (I- 110) is a gram (-) microorganism with the cell-proper shrouded behind the outer membrane. Therefore. one could postulate a more direct and dynamic effect by antagonistic inorganic ions for gram (+) microorgan- isms. This concept is indeed supported by the data presented in Fig. 2a. 2b. 3a. and 3b. The most likely involvement of the outer membrane under Al stress is to function as a limiting diffusional barrier against Al associating with the cell proper for gram (-) microorgan- isms. Under Al stress. Rhizobium see. (I-110) cells seemed relatively immune to antagonistic effects by Al. The maximum C01 evolution rate decreased only slightly for Rhlzoclgm see. (I-110) (Fig. 2b). With the occurrence of Fe induced Al stress. the outer membrane protection may have been compromised (Fig. 3b). The investigator suggests that under Al stress. Al enters the periplasmic space. that a secondary diffusional barrier might arise. The periplasmic space may become saturated with loosely bound A1. A variety of phospholipids and prot- eins lining the periplasmic space could provide ample binding sites. Once saturated with Al the diffusion gradient for Al across the outer membrane could collapse offering a weak protective effect. Aluminum binding in the periplasmic space could even slightly concentrate Al to the extent of reversing the diffusion potential for Al across the out- er membrane. Suzuki. et. al. (1976) demonstrated that a gram (-) mut- ant. Escherichia coli . occurring with structural changes in an outer membrane lipoprotein was more inhibited by a variety of metal ions than was the wild type. Their study suggested that the E.coli mut- u 28 ants outer membrane no longer functioned as a impermeable barrier to the ions under examination. This mutant demonstrated a marked inhibi- tory response to the metal ions used in the study. This article lends support to the concept of the outer membrane of gram (-) microorgan- isms forming a protective barrier against general metal toxicities. Once this barrier is defeated the gram (-) organism is likely subject to the same general effects of metal toxicities as are gram (+) organ- isms. Duxbury and Bicknell (1983) have demonstrated that in soils subjected to a variety of toxic metals. the gram (-) organisms exhibited a greater tolerence to metal toxicity or stress than did the gram (+) organisms. This article lends support to the mechanism post- ulated for explaining the less dynamic response of anizcclcm_§ggc,(l- 110) to Al stress. This investigator suggests that when Fe is not present. the outer membrane of BELZQQiHm_§EEa (I-110) acts as a formidable barrier to Al under Al stress conditions. If increasing soluble Fe"3 induces the Ft;3 transport system. the permeability barrier of the outer membrane of Rhlgcclum see, (I-110) may collapse due to the import and the binding of Al due to Fe‘3 transport sites or import of other Al bound compounds. Figures 3a and 3b illustrate the effects of FeCl3 amendments on Al stressed microbial activity for the test organisms. The range of FeCl3 concentrations used was 0 to 10"M,. The average calculated range of molar activities for those Fe".a amendments was 10hoto 104'. depending on whether Fe;3 was assumed to be in equilibrium with amor- . phous Fe(OH); or soil Fe(OH), as defined by Lindsay (1979). When no *3 . . . .. free Fe was present. a fairly linear decreaSing response was exhibit- 29 ed by B. mecaterium (B-12) to increasing exchangeable Al concentra- tions (Fig. 3a). As the Fe concentration was increased. the Al str- ess response of B. mgcaterium (B-12) demonstrated a greater exponent- ial character. There were discernable differences among treatments. The same general trends were seen with Rhizcbium gee, (l-1l0) as were seen with B. mecaterium (B-12) (Fig. 3b). When no Fe” was pre- sent. the Al stress response curve took on a reverse '8' shape. The various Al stressed Fe+3 treatments exhibited a decreasing exponential character with no discernable differences among the Fe'.3 treatments (Fig. 3b). To further explore the effects of Fe*3 on Al stressed microbial activity. chelated forms of Fe"3 were amended to the Al stressed syst- ems. The forms studied included Fe(III)-EDTA. Fe(III)—NTA. and Fe(III)-Citrate. The use of chelated forms of Fe"3 allowed greater control of soluble Fe">3 in solution. For each chelate. treatments of 104. 10“. and 10'6 M total Fe(III)-chelate were added for a given treatment. The activity of Fe” then varied from chelate to chelate and from one total concentration to another. Aluminum stressed B, mgcatcriym (B-12) demonstrated a geat dif- ference in activity between the Al only control and Al plus Fe(III)- EDTA treatments (Fig. 3:). While there was a degree of variation among treatments. there were no discernable trends among the Fe(III)- EDTA treatments. Again. the Al only control for B, mcgaterium (B-12) responded with generally a linear decreasing response to increasing Al (Fig. 3c). The activity response to the Al plus Fe(III)-EDTA treat- ments was not clearly exponential in character. However. it did decr- ease rapidly at low Al concentrations and generally leveled off at Al+Fe-EDTA B.ee9ateriue (B—la) film 30 -— nmol C/g Soil/h RB ' \ 1.. \fl\ ”2 “tai— O 0.37 1.11 2.22 unlixdhlflngMl CRLC. Fe ACTIVITY - 0 -lb 0 10 o 10'” U 10“” mfla~afls Fig. 3c. Fe(III)EDTA induced aluminum stressed B. mecagerlum (B-12) with glucose-yeast extract (1:1) as the C substrate at 8 umol C/g soil. at pH 4.6. awe-cm n. :9... (His) m {d ‘L 53% \ r “3.... \ ‘a’m “L 'g \ umol been. Al/g Soil (291.6. to acuvm - a u 19"“ o 10'” l D 10"" mfla'afl. Fig. 3d. Fe(III)EDTA induced aluminum stressed Rhlzobium see. (I-110) with glucose-yeast extract (1:1) as the C substrate at 8 umol C/g soil. at pH 4.6. 31 1.11 and 2.22 mol Exch. Al/g soil. With Rhizobium see. (I-1l0) the Al only control exhibited a rev- ersed ”S' shape character. The Al plus Fe-EDTA treatments demonstrat- ed a strong exponential character in the decreasing inhibitory resp- onse. Iron(III)-EDTA should have given the lowest molar activity treatment of Fefgto which the test organisms were exposed. The stability constant for the Fe(III)-EDTA complex was 25 (Log K ). The next treatment involved the amendment of Fe(III)-NTA to the Al stressed artificial soil system. The stability constant for the Fe(III)-NTA complex was approximately 17 (Log K ). Bacillus , megaterium (B-12) exhibited a general linear decrease in activity in response to increasing exchangeable Al concentrations (Fig 3e). For the Fe(III)-NTA treatment. an exponential decrease in activity as de- scribed for Fe(III)-EDTA treatments was observed (Fig. 3e). The Fe(III)-NTA plus Al treatments demonstrated depressed activity in comparison with the Al only control (Fig. 3e). The data in figure 3f exhibited an exponential decrease in activ- ity for the Al plus Fe(III)-NTA treatments for Rhizobium see. (I-110). The decrease was similar to that described in figures 3b and 3d. Finally. the test organisms were treated with Fe(III)-Citrate under Al stress conditions (Fig 39 and 3h). The Fe(III)-Citrate treatments yielded curves of similar character as that obtained for the other Fe chelate treatments. Generally. B. mecaterium (B-12) exhibited a linear decrease in carbon dioxide evolution with respect to increasing exchangeable Al. Carbon dioxide evolution was further depressed in an exponential fash- ion in the presence of soluble Fag (Fig. 3a. 3:. 3e. and 39). Iron in -I' 32 aim-m i. eegateriie (13-12) 353 R .C .‘. 5 cam. Fe vacuum 9 X - a ,. \ d u 1e-l‘l. 3 an o 10'” a . o 0.37 1.11 2.22 U 10’” ueol Exch. RI/QiSOII solar act. Fig. 3e. Fe(III)NTA induced aluminum stressed B. mcgatcrlcm (B-12) with glucose-yeast extract (1:1) as the C substrate at 8 umol C/g soil. at pH 4.6. CRLC. Fe ACTIVITY .I1 0 18 -i) 10 -IO 18 0 0.37 1.11 umol Exch. All Soil Fig. 3f. Fe(III)NTA induced aluminum stressed Rhizcbium see. (I-110) with glucose-yeast extract (1:1) as the C substrate at 8 umol C/g soil. at pH 4.6. 2J2 33 AHFrCTbafli&.mmpflrum UlC.FeACHVNY a m n to“ U 13" -IO 8.37 1.11 umol Ekch. RI/ 8011 Fig. 39. Fe(III)Citrate induced aluminum stressed B. mggaterium (B-12) with glucose-yeast extract (1:1) as the C substrate at 8 umol C/g soil. at pH 4.6. 2J2 m e-Citrate R. .9... (1-110) CRLC. Fe ACTIVITY fl la-lO 10“ El 10'" 8.37 1.11 umol Exch. 91/ Soil Fig. 3h. Fe(III)Citrate induced aluminum stressed Rhizobium see. (I-110) with glucose-yeast extract (1:1) as the C substrate at 8 umol C/g soil. at pH 4.6. 2J2 34 the absence of Al generally exhibited a slightly enhancing effect or no effect towards microbial activity. With only Al present. Rhizobium see. (1-110) typically gave a decreasing carbon dioxide evolution response to increasing Al in the soil system. This decrease took the form of the reverse 98' curve de- scribed earlier. With soluble Fefl present. Al stressed Rhizobium sag; (I-110) exhibited a strong exponential decrease in CO, rate resp- onse to the increasing Al concentration (Fig. 3b. 3d. 3f. and 3h). To further substantiate the Fe-Al microbial interaction. an ex- periment with both microorganisms was conducted to establish Al assimilation. Both B. megaterium (B-12) and Rhlzccium see, (I-llO) were incubated in media in separate experiments with treatments of no Al (control). 1.11 umol Al/mL. Al only. and 1.11 umol Al/mL plus 0.01 umol Fefl lmL. With both organisms. Fefl was found to increase the amount of Al associated with the bacterial biomass (Fig. 4a and 4b). It is not possible to conclusively prove with this data that Al was assimilated into the cells. and not associated by means of surface interactions. However. if the association of A1 with these cells were due to surface interactions only. one might expect a dilution effect or no change in Al concentration associated with the biomass when compared to the Al-only and azide controls. Because there was a significant increase in associated biomass Al when compared to the azide controls. the investigator suggests that the increase is due to an uptake of Al associated with Fe“ assimila- tion. This conclusion is supported indirectly by the observations of Keyser and Munns (1979b) with Fe(III)-EDTA described earlier. and by the observations of Cooper and Morgan (1979a and 1979b) when they not— 35 BIWSS Al Emegaterium (813) Control RI A1+F e Treatment 'n 1H: (mol RI/g biomass TREATENT KEY 1:] with azide l- without azide Fig. 4a. Aluminum-biomass association experiment for g, mcgaterium (8-12). The concentration of aluminum was was 1.11 umol Al/mL. and iron was 0.018 umol Fe(III)/mL. BIOMASS m 9.5.... (1410) Control Al A1+Fe Treatment 1 mol (ll/3 biomass TREATENT KEY :1 with azide l- without azide . Fig 4b. Aluminum-biomass association experiment for 1; saga-110). The concentration of aluminum was 1.11 umol Alme. and iron was 0.018 umol Fe(III)/mL. :1: 36 ed increased aluminum stress in the presence of amorphous Fe . To futher assess the effects of Al and Al plus Fe on microbial growth. turbidity studies were conducted. There were five treatments for each of the test organisms. The first treatment was the control consisting of glucose and yeast extract (GY). The second treatment included GY and Fe(III)Citrate. with a calculated Feflsactivity of 1.0 x 104 M . The third treatment included GY and A1 at 3.8 x 10“ M . The fourth treatment was the same as the second except that at 18 hours Al was added giving a final Al concentration of 3.8 x 104M . The fifth and final treatment was the same as the third except that at 18 hours. Fe(III)-Citrate was added giving a calculated Féaactivity of 1.0 x 10J'M . All culture media were buffered with acetate buffer at pH 4.6 and incubated at 25 C for 48 h. Figure 5a illustrates the growth study results for B. mecaterlum (B-12) with the five treatments just described. Optical densities for the GY and GY + Fe reached their maximums by 30 h. Treatment GY + Fe > A1. with Al added at 18 h. reached maximum optical density by 42 h. Treatment GY + Al for B; megaterium (B-12) reached its submaximum at approximately 40 h. Treatment GY + Al > Fe . with Fe-Citrate added at 18 h exhibited the greatest inhibitory response of any of the treatments for B; megaterlum (B-12) in these experiments (Fig. a). The same treatments for Rhizobium see. (I-110) yielded a similar distribution of growth curves (Fig. 5b). The most interesting aspect of figures 5a and 5b are that the cells grown in the presence of 3.8 x 10-‘M Al and subse- quently amended with Fe(III)Citrate exhibited the most intense inhib- itory responses. In the presence of GY and Fe(III)Citrate. both organisms demonstrate an enhanced growth response relative to GY only. 37 e S x m nunnsnns 4o ".1 *- G? c 3 ,0 area 5 U 31'le 8' U swam I] went. Time (h) Al-Fe aeendeents Fig. 5a. Fe(III)Citrate induced aluminum stress time study for B, megaterlum (B-12). Glucose- yeast extract (GY). Fe source Fe(III)Citrate (Fe). and A1 source AlC13 (Al). Re We (1-110) mm W 35 Z 38 is d 95 x m lRBflMBflS .p ”i ' 5" i8 0 @er 5 D (39A! 3 3.. D swam 06121883364248 [1 GY+A1)Fe Time (h) Al-Fe amendments Fig. 5b. Fe(III)Citrate induced aluminum stress time study for Rhizobium see. (I-110). Glucose-yeast extract (GY). Fe source Fe(III)Citrate (Fe). and Al source AlCl. (Al). 38 The low rate of growth for the GY+Al>Fe treatment suggests an in- teraction between involving Al and Fe(III)Citrate which intensifies Al stress interactions with these microorganisms. There would seem to be three possible explanations for the Fe in- duced Al stress response. One explanation might include surface inter- actions with the microorganisms. Certainly. carboxyl and hydroxyl groups on cell surfaces provide ample binding sites for Al. What is hard to reconcile is the difference in the results between the GY+Fe}Al and GY+Al>Fe treatments (Fig. 5a and 5b). If the Fe induced Al stress response was due to precipitation or exchange phenomena. the difference in the net response of the last two treatments would not be expected. The use of chelated Fe"3 should preclude any major precipitation phenomena. One argument in favor of surface exchange phenomena might be related to the specific order of amendments. If Al is added first (GY+Al>Fe). then subsequent addition of Fe might dis- place Al from exchange sites causing hydrolysis of water and produc- tion of H+. lowering the pH. However. the systems under study were both buffered for pH and in equilibrium with Fe(III)Citrate. Between the buffering capacity of the acetate buffer and free citrate taking up HT. pH changes should have been negligible. The second explanation lays in the possibility of the formation and uptake of Al-Citrate. Undoubtly. formation of Al-Citrate will occur due to the equilibrium product of free citrate from the presence of Fe(III)Citrate. In the case of B. megaterium (B-12). it is well documented that this organism can assimilate and utilize Fe(III)Citr- ate for both the Fe and citrate components (Byers and Arceneaux. 1976). If Al-Citrate were to compete or be assimilated by other 39 means. a toxic accumulation of Al might occur intracellularly. The third explanation is similar to the second. The effects of Fe”.3 have in general been demonstrated to enhance microbial growth rates in this study in the absence of Al. The implicit increase in general metabolic rates in the presence of Fe may increase the cell- ular import of one or several nutrients which in the presence of alum- inum bind Al. Again. accumulation of intracellular Al could occur to toxic levels if such compounds were imported. Further complicating this explanation. this indirect effect could manifest itself in a non- linear fashion. Figure 5c demonstrates the relationship between accumulative CO, evolution and time for treatment GY for both test organisms. In summary. it has been demonstrated that under Al stress condi- tions. B.meg§terium (B-12) appears to demonstrate primarily a non- competitive inhibitory response. Rhizobicm sea. (I-110) exhibited only the slightest indication of non-competitive inhibition under Al stress. With Fe induced Al stress. B.megaterium (B-12) and Rhizocium see. (I-110) both demonstrated intensified inhibitory resp- onses. These results suggest that the assessment of Al stress on microb- ial activity must include analysis of factors which might appear sec- ondary to the stress effect. In this case. past ignorance of environ- mental factors such as the presence of Fe".3 in the natural environment have likely led to false assumptions about Al stress effects on microbial systems. Particularly. in oxisols which compose the majority of known acid Al soils. the effect of soluble Fed’likely to be present can not be overlooked in light of the data presented here. Several papers recently published (De Carvalho. et.al.. 1981; Hartel and URHIIEKNHIlmflmETKM N ll.- 40 / / 12182438364348 Tine (h) nmol c/mL §ou1ture media 'EBTCRENHSB B. negateriue B-la Rhizobium spp. I-118 Fig. 5c. Carbon dioxide production for both test organisms shown in figures 5a ' and 5b. Carbon dioxide production monitored were for the GY treatments only. This data can be used to relate CO; production to biomass production in figures 5a and 5b. 41 Alexander. 1983) have suggested that Al stress is vertually nonexist- ant for Rhizobium species. However. neither group of investigators took into account the effects Fe" on Al stress. NATURAL SOIL STUDIES: To examine the plausibility of Fe»3 induced Al stress occurring in nature. several natural soils including an Al stressed soil were stud- ied. The major portion of the study involved a comparison of the carbon dioxide evolution characteristics in a fashion similar the simulated soils discussed earlier. These studies included examination of the soils amended with only a carbon source (control). and the same soils amended with the same carbon source plus Fea . The carbon I source amended to the soils included 4. 8. 12. 16. and 20 umol substr- ate C/g soil. The substrate C source consisted of glucose. Iron amendments included 0.01. 0.1. 1.0. and 1.3 umol Fefi’lg soil treatments of Fenias FeCl,. The general chemical and biological-bio- chemical characteristics of the soils collected for this study are given in Table 2. Table 3 gives the Monod growth parameters derived from data in figures 6a and 6b for the soils collected for this study. Five of the soils sampled from around the state of Michigan were considered non-Al stressed. The general criteria used to define an Al stressed soil included a soil pH < 5.5 (soil to water. 1:1). and > 0.37 umol exchangeable Al per gram of soil. The Al stressed soils used in this study were located at the Kellogg Biological Station operated by Michigan State University. Hickory Corners. Michigan. The Al stressed soil samples were collected along a 200 m transect. acquiring 30 subsamples at each station. There were 5 stations. 40 m Table ASSAY PH Ca M8 Fe Mn an A1 P0 ORGC BMC BMFeC BMMQC Soil chemistry and biological assays Aluminum stressed soils IA 5.20 2.38 6.50 0.25 0.39 0.31 0.03 1.19 1.48 841 26.8 20.6 32.4 2.30 12.1 8.10 5.72 IIA 4.70 3.49 4.85 0.33 0.39 0.29 0.03 2.85 1.65 783 23.7 6.80 14.4 2.50 9.90 8.60 0.75 IIIA 4.70 3.87 8.15 1.08 0.30 0.31 0.05 2.22 0.68 867 23.7 24.7 23.7 2.70 8.10 9.40 0.75 IVA 4.50 3.10 4.85 0.58 0.39 0.31 0.03 2.96 0.68 841 13.9 14.9 15.4 0.90 9.40 9.40 ORGC-(umol soil organic C/g BMC-(umol biomass C/g soil) BMFeC-Fe(111) amended soil biomass C (umol biomass C/g soil) BMMgC-Mg amended soil biomass C (umol biomass C/g soil) PP-Pyrophosphatase activity (umol P/g soil/h) G-P -Phosphatase activity (umol p-nitrophen./g soil/h) ¢-S -Sulfatase activity (umol p-nitrophen./g soil/h). umol/g soil VA 4.60 2.97 8.15 2.75 0.39 0.15 0.02 5.44 0.52 700 18.0 5.20 15.9 1.00 8.10 10.7 soil) Low aluminum soils 13 6.20 0.85 28.1 7.88 0.48 0.20 0.02 0.11 0.81 783 26.3 27.3 13.9 1.50 18.8 8.10 DH-Dehydrogenase activity (umol TPF/g soil/h) 10 7.50 1.10 28.1 7.88 0.66 0.24 0.03 0.04 1.39 841 15.9 19.6 7.80 2.70 14.8 7.25 19.2 ASIA 5.90 4.36 5.45 1.67 0.55 0.02 0.15 2.03 992 27.3 30.8 10.3 3.20 37.1 FSIA 7.10 0.33 9.63 3.67 0.39 0.40 0.03 0.07 0.61 816 18.0 25.6 25.8 4.10 18.8 12.9 21.1 CK19 7.50 2.26 74.7 8.92 0.21 1.58 1725 36.5 39.1 59.2 4.10 9.40 10.8 55.2 L011 Al SOIL HICROBIfiL ACTIVITY L=I~ ‘ v v P “‘1 43 l“ nmol C/g soil/h .§§§§§§ “tn-o M 4 81216 38 . mol “strate ULsoil [j E] E) a W Al 01(19 8 16 0.04 FSlA 9.67 IR 8. 11 ASIA 8.15 mol POI/g soil Fig. 68. Carbon dioxide evolution rates from low aluminum soils. Carbon substrate used was glucose. Al-STRESSED SOIL 111612031“. ACT .C \ a-o on 0 W U) \ U .4 gm 0 4 81216 20 mol “strate 179 soil [31300 W Al IA 1.19 111A 2.22 NA 2.85 IVA 2.96 VA 5.44 UIOI Rl/g soil Fig. 6b. Carbon dioxide evolution rates from aluminum stressed soils. Carbon substrate used was glucose. 44 Table 3. Monod model parameters for carbon dioxide evolution from natural soils. Low Aluminum Stress Soils umol Al/g soil 0 0.04 0.07 0.11 0.15 u (nmol C/g soil/h) 3077 490 1433 736 800 K (umol C/g soil) 0.31 5.05 6.74 5.01 1.95 X (Biomass. ug C/mL) 43B 191 216 315 327 Aluminum Stressed Soils umol exch. Al/g soil 1 9 .2 2.85 2.96 5.44. u (nmol C/g soil/h) 1277 755 845 602 939 K (umol C/g soil) 14.0 1.90 11.1 1.46 7.00 X (Biomass. ug C/mL) 321 284 284 167 216 Monod model equation: dX/dt = uX a u-(S/(K+S))ox S = umol substrate C/mL 45 apart along the transect. The carbon dioxide evolution activities for the non-Al stressed soils exhibited maximum velocities ranging from 490 to 3077 nmol C/g Soil/h (Fig. 6a) with the previously mentioned carbon amendments. Carbon dioxide evolution rates for the Al stressed soil gave a max- imum velocity range of 602 to 1277 nmol C/g Soil/h (Fig. 6b). Aside from the differences in magnitude. the low-Al stressed soils demonstr- ated as a group a more rapid increase to the maximal velocity than did the Al stressed soil series (Fig. 6a and 6b). Amendments of Fésand C substrate to the low-Al stressed soils were seen to slighty increase the carbon dioxide evolution rates (Fig. 7a). When similar F8“ and C substrate amendments were made to the A1 stressed soil series. activities not unlike that demonstrated by B.megaterium (B-12) (Fig. 38). were observed (Fig. 7b). Biomass estimates were assayed by the method of Anderson and Domsch (1978) for both group of soils. The biomass estimates were conducted with glucose-only. glucose-FeH . and glucose-Mg. Figures Ba and 8b show the results for the glucose only amend- ments plotted against the exchangable Al concentrations for both groups of soils. No trend can be seen in the low-Al stressed soils relating exchangeable Al to biomass carbon (Fig. Ba). For the Al stressed soil group. a generally decreasing trend for biomass carbon against increasing exchangeable A1 can be seen (Fig. Sb). When the same biomass estimates are made in the presence of Fe . significant changes in the biomass estimates occur relative to the glucose only estimates. The low-Al stressed soils all exhibited increases in biomass carbon compared to the glucose only estimates L111 Al SOIL HICRUBIFL ACTIVITY A # 46 25% i 88m ”9: 15m ___‘ W 81 3) 1m 8 a . 01(19 8 0 10 0.94 g 9% 8 F319 8.0? a E! 13 0.11 e 0.01 0.1 1.0 1.3 D nsm 0.15 mol Fe(III)/3 soil mol 91/9 5011 Fig. 7a. Carbon dioxide evolution rates from Fe(III) amended low aluminum soils. Carbon substrate used was glucose at 8 umol C/g soil. Al-STRESSED SOIL HICROBIN. ACT .5: 2. '8' W 91 3) - IA 1.19 3 ’ n ma 2.22 g , [1 NA 2.85 E) WA 2.96 a 0.01 0.1 1.0 1.3 [I m 5.44 mol Fe(III)/3:011 (.01 81/3 soil Fig. 7b. Carbon dioxide evolution rates from Fe(III) amended aluminum stressed soils. Carbon substrate used was glucose at 8 umol C/g soil. 47 L011 F11. 3011. 3101-1038 C 0.04 0.07 0.11 0.15 mol exch. All soil Fig. Ba. The relationship between soil biomass C and exchangeable soil Al for the low aluminum soils. Al STRESSEII SOIL RICH-133 C i_. _l l—1 umol biomass C/g soil 3 1.19 2.22 8.85 2.96 5.44 mol exch. avg soil Fig. 8b. The relationship between soil biomass C and exchangeable soil Al for the aluminum stessed soils. 48 (Fig. 9a). With the Al stressed soils plus Fe amendment. all soils except IIIA demonstrated decreases in biomass carbon relative to the glucose only estimates (Fig. Sb and 9b). Significant differences were determined for the glucose and glucose9Fe biomass estimates using the F test. The F test was conducted at (P < 0.05) and (P < 0.01) levels of confidence. The depressed biomass carbon estimates for the Al stressed soils amended with Fe’3(Fig. 9b) would appear to support the previous hypo- thesis regarding iron induced Al stress. A third treatment involving Mg amendments to the two groups of soils were conducted (Fig. 108 and 10b). Past investigators (Keyser and Munns. 1979a; Keyser and Munns. 1979b) have examined the effects of Pq . Mn". and Ca amendments on microbial activity under Al stress. These investigators found no significant releif from Al stress by these amendments. Examination of Al stressed soils often demonstrate low available Mg concentrations. In the low Al stress soils. random changes in active biomass C were observed (Fig. 88 and 108). For the Al stressed soil amended with Mg. only the sample with 2.85 umol exch- angeable Al/g Soil exhibited a significant decrease in biomass C. while the other demonstrated no significant difference from the glu- cose only treatments (Fig. Sb and 10b). Hargrove and Thomas (1981) have demonstrated that increasing soil organic matter can relieve the phytotoxic effects of Al in Al stressed soils. They showed that increasing soil organic matter from 1 to 2 percent could increase the dry weight yield of barley by 10 to 15 percent. Figures 11a and 11b demonstrate the relationship between biomass C and soil organic matter for the two groups of soils. In 49 (.014 Al SCIL+Fe 31011033 C 50 OlflOSS 0.04 0.07 0.11 0.15 mol exch. 91/ soil Fig. 9a. The relationship between biomass C and exchangeable Al for the Fe(III) amended low aluminum soils. Al STRESSEII SOIUFe 3101883 C 80 H '9 a S O 1.19 2.22 2.85 2.96 5.44 uolexch. Al! soil Fig. 9b. The relationship between biomass C and exchangeable Al for the Fe(III) amended aluminum stressed soils. 50 L011 AI 3131ng 81011033 C 0.04 0.07 0.11 0.15 mol exch. All 5011 Fig. 108. The relationship between biomass C and exchangeable Al for the Mg amended low aluminum soils. AL smsszn 301L019 moms c 30 umol biomass C/g soil 1.19 2.22 2.85 2.96 5.44 mol exch. (ll/3 soil Fig. 10b. The relationship between biomass C and exchangeable Al for the Mg amended aluminum stressed soils. 51 contrast to Hargrove and Thomas (1981) the data in Figures 118 and 11b exhibit no apparent trend relating biomass C and soil organic matter for these soils. The amendment of the two groups of soils with Fen‘demonstrated two interesting trends. The low Al stress soils all exhibited slight increases in active biomass C over the glucose only amendments (Fig. 118 and 128). This increase was generally expected. However. Fe‘3 amendments to the Al stressed soil group were associated with a sign- ificant (P < 0.05) positive correlation (0.879) between biomass C and soil organic matter (Fig. 12b). varying greatly from the glucose only control (Fig. 11b). The association between biomass C and soil organic matter in soils amended with Fe” suggests a participation by organic matter in reducing the effects of Fe induced Al stress on microbial activity. Most likely. the effect of soil organic matter in reducing Fe induced Al stress is the reduction of Fe.3 activity through various ionic associations. Assuming Fe"3 binding by soil organic matter. it seems fair to suggest that the data in Fig. 12b support the Fe induced Al stress model described earlier. A significant correlation between ex- changeable Al and soil organic matter was not found for either group of soils. However. a negative correlation of (-)0.875 was found be- tween exchangeable Al and soil organic matter for the Al stressed soils. The Mg amended treatments exhibited no significant correlation between biomass C and soil organic matter (Fig. 13a and 13b). In the Al stressed soil. the active biomass C did give a significant (P < 0.05) correlation with soil pH (r=0.899). L011 AI SOIL 310N033 C 783 816 041 992 1725 no! soil 0/ soil Fig 11:. The relationship between biomass C and soil organic C for the low aluminum soils. AI STRESSED SOIL RIMS C i_r Fl umol biomass C/g soil 8 m 783 841 841 867 ueol soil org. C13 soil Fig. llb. The relationship between biomass C and soil organic C for the aluminum stressed soils. 53 L011 AI SUIU-Fe 311311053 C 783 016 041 992 1725 molsoil 0/ soil Fig. lZa. The relationship between biomass C and soil organic C for the Fe(III) amended low aluminum soils. SOIUFe RIMS C 700 783 041 841 067 mol soil CI soil Fig l2 b. The relationship between biomass C and soil organic C for the Fe(III) amended aluminum stressed soils. 54 1.011 m 501ng 01011833 1: 783 816 041 992 1725 (.01 soil El 3011 Fig 13a. The relationship between biomass C and soil organic C for the Mg amended low aluminum soils. AL 51m 801m, mm c 30 , d 25 .3 .__i " 20 3’ iv 11 1 F— ; 1a .2 .9 3 0 3 no 783 841 041 067 mol soil 0P3. 179 soil Fig l3b. The relationship between biomass C and soil organic C for the Mg amended aluminum stressed soils. 55 ENZYME ASSAYS: Enzyme assays were conducted to help further describe the) biological-biochemical parameters of biological processes under Al stress in nature. For reasons of aluminum soil chemistry. all enzyme assays were conducted without pH buffers. As most enzyme buffering regimes occur at pH > 5.5. effects caused by soluble aluminum species would be negated. ' Pyrophosphatase: Pyrophosphatase was the first enzyme activity assayed. Pyrophosphatase mediates the hydrolysis of pyrophosphate to two ortho- phosphates: 9.0, + Hlo—-—)2HPO; The maJor interest in pyrophosphatase activity lays in the applicat- ion of pyrophosphate as a fertilizer for agricultural soils. Pyrophosphatase activity has been correlated primarily with soil organic matter. This enzyme has generally exhibited an optimum activ— ity at pH 8.0 (Tabatabai. 1982). The substrate used for this assay was sodium pyrophosphate. The pyrophosphatase assayed in low Al stressed soil showed no significant correlation with any of the factors measured in this study However. in the Al stressed soils. pyrophosphatase activity was significantly (P < 0.05) correlated with the glucose only biomass C estimate (r= +0.887). No relationship was found to exist between pyro— phosphatase activity and exchangeable Al for either group of soils (Fig. 14a and 14b). Phosphatase: The phosphatase assayed was the monoesterase type. presumably an acid phosphatase. This enzyme catalyses the reaction: 56 3011. WTASE ACT. 0 0.04 0.07 0.11 0.15 mol exch. 5011 Fig 148. The relationship between pyrophosphatase activity and exchangeable soil aluminum for the low aluminum soils. Al SOIL PWMSE ACT. .__. g 4 nmol P/g soil/h I" J 1.19 2.22 2.85 2.96 5.44 . mol exch. (ll/g soil Fig. 14b. The relationship between pyrophosphatase activity and exchangeable soil aluminum for the aluminum stressed soils. 57 R-PO+ + HzO-—>R-OH + HPo; A variety of inhibitors including heavy metals will non-competitively inhibit phosphatase. Orthophosphate will competitively inhibit phos- phatase activity also (Tabatabai. 1982). The substrate used in this assay was p-nitrophenol phosphate. In the low A1 stressed soils studied. a highly significant (P < 0.05) negative correlation (r= -0.943) existed between phosphatase activity and the orthophosphate concentrations for these soils. The soil which exhibited no phosphatase activity had recently been amended with a phosphate fertilizer prior to sampling. This soil had the highest orthophosphate concentration of all the soils examined. This lack of activity might suggest that the competitive inhibition cited above be invoked to explain the absense of activity. Also. a signif- icant (P < 0.01) negative correlation (r= -0.990) between phosphatase activity and potassium. and a significant (P < 0.05) positive correla- tion (r= +0.919) between K and Po; were found in this study. The Al stressed soil exhibited no significant correlations be- tween phosphatase activity and the other soil factors measured. except dehydrogenase activity (r=0.S77). Results of the phophatase assays for both groups of soils can be seen plotted against exchangable Al concentrations in Fig. 158 and 15b. Sulfatase: The sulfatase assay conducted was for the arylsulfatase type. breaking the O-S bond in the following reaction: FPO-803+ H‘O—bR-OH + H”+ so; Sulfatase activity has been shown to be correlated with soil organic matter content. Sulfatases are competitively inhibited by Moo . AsO . 58 SOIL Pl-IJSPI-HTASE ACT. C g 0.04 0.07 0.11 0.15 mol exch. All soil Fig. 15a. The relationship between phosphatase activity and exchangeable soil aluminum for . the low aluminum soils. 1.19 2.22 2.85 2.96 5.44 mol exch. All soil Fig. l5b. The relationship between phosphatase activity and exchangeable soil aluminum for the aluminum stressed soils. 59 and PO4(Tabatabai. 1982). The substrate used in this assay was p- nitrophenol sulfate. Figures 16a and 16b show sulfatase activity plotted against exchangable Al concentrations for low Al and Al stres- sed soils. respectively. with the low Al stressed soils. no significant correlation was found between sulfatase activity and any of the other variables measured. The soil exhibiting no sulfatase activity is the same soil which exhibited no phosphatase activity. Competitive inhibition is suggested for the same reasons given for phosphatases lack of activ- ity. However. in the Al stressed soils. sulfatase activity was significantly (P < 0.05) correlated with the exchangable Al and Mg. r=(+)0.906 and r=(+)0.929. respectively. An explanation which might help to understand this association lay in the aluminum-phosphate soil chemistry. In this study. sulfa- tase activity shows a fairly high (though not significant) negative correlation with both phosphatase activity (r= -0.S47) and soil ortho- phosphate concentration (r= -0.858). It is possible that the Al under acid soil conditions removes phosphate from the soil solution. This situation would relieve any competitive inhibition from P04 as cited above. The ability of Al under acid conditions to precipitate soluble orthophosphate is well documented (Lindsay. 1979). Also. the fixation of orthophosphates in soils by Al(C)H); has been demonstrated by Sims and Ellis (1983). These types of mechanisms are suggested as an explanation for the significant positive correlation between sulfatase activity and A1. No beneficial effects have been cited in the literature for ei- 60 SOIL SULFATASE ACT. 12000 .C .— 3.10000 '6 '0 0000 33’ 5 6m 0 ‘8 4000 O i. 3 2000 C '3 0 g 0 0.04 0.07 0.11 0.15 mol exch. Alla soil Fig. l6a. The relationship between sulfatase activity and exchangeable soil aluminum for the low aluminum soils. Al SOIL TASE C E 1.19 2.22 2.05 2.96 5.44 mol exch. All 5011 Fig. 16b. The relationship between sulfatase activity and exchangeable soil aluminum for the aluminum stressed soils. SOIL Dfl-fi'IiROEiASE ACT. 0.04 0.07 0.11 0.15 mol exch. 111/ soil Fig. 178. The relationship between dehydrogenase activity and exchangeable soil aluminum for the low aluminum soils. Al SOIL W ACT. 1.19 2.22 2.85 2.96 5.44 ueol exch. All soil Fig. 17b. The relationship between dehydrogenase activity and exchangeable soil aluminum for the aluminum stressed soils. 62 ther Al or Mg directly related to sulfatase activity. Dehydrogenase: Dehydrogenase activity is described by the general reaction: XHz + A-—4X + AHz Here. XH is an organic substrate. and A is a hydrogen-electron acceptor (Tabatabai. 1982). Dehydrogenase has been correlated with C01 evolution rates. proteolytic activity. and nitrification activity in soils examined by SkuJins (1973). Triphenyl tetrazolium chloride was used as the hydrogen-electron acceptor. The low Al stressed soils exhibited significant (P < 0.05) posi- tive correlations for dehydrogenase activity with Mn concentrations (r= 0.955). soil organic C (r= 0.950). and biomass C (r= 0.926). The Al stressed soils exhibited a significant positive correla- tion for both dehydrogenase activity with soil pH (r= 0.983). and phosphatase activity (r= 0.877). It would be interesting to speculate on the association between phophatase and dehydrogenase activity. The available phosphorous in Al stressed soils is likely limited by the ~aluminum species. The only phosphorous to become available might be organic-P cleaved by phosphatase. No such association was found be- tween dehydrogenase and phosphatase activity in low Al stressed soils. Figure 17a and 17b show dehydrogenase activity plotted against exchangable Al for low Al and Al stressed soils. respectively. SyMMARY AND CONCEQSIONS: Data obtained from the artificial soil studies suggest a general reduction in the maximum velocities (89 due to Al stress. This effect is much more pronounced with B. megaterium (B-12) than with Rhizobium 63 see. (I-110). The data from the Fefland Fe"3 chelate studies demonstrate a defi- nite interaction involving FeI‘and Al under Fe induced A1 stress conditions. The effect of Fé‘uas to intensify the Al stress effect on microbial activity. The Al-biomass association data presented suggests a definite increase in the amount of Al associated with microbial biomass in the presences of Fe.3 . Surface interactions (precipitation or cation exch.) could be invoked to explain the Fe induced Al stress effects. However. exchange phenomena (Féadisplacing Al) is not supported by the Al-biomass association data which show increasing biomass Al in the presence of Fe" . Precipitation due to Fe"3 -Al interactions as the stress mechanism is not supported by data from the growth study. The last two treatments for both organisms in the growth study differ only in the reversal of the order of addition of Al and Fe»1 . Though pos- sible. it does not seem probable that the order of addition would eff- ect precipitation if it were the stress mechanism in this system. Yet marked differences in the stress response do occur relative to this reversal. The Al stressed soils exhibited a generally hyperbolic response to increasing carbon substrate concentration as with the low Al stres- sed soils. The Fefsamended Al stressed soils. however. exhibited a dynamic character. similar to the Fe induced Al stress seen with the artificial soil system. Under Fe induced Al stress. the initial drop in CO; evolution activity occurred after the lowest Fegconcentration and leveled off for higher concentrations for the artificial soil system. The initial drop in activity was less pronounced with the 64 non-chelated Fefsamendements. The A1 stressed soil used in this study was a fine-loamy. mixed. mesic. Typic Hapludalf. Soils exhibiting classical A1 stress problems are tropical and subtropical Oxisols. Oxisols characteristically are acid soils (pH 3.5 to 5.5). and are high in aluminum and iron oxides. Also. being acid in nature. one would be led to suspect these soils to have relative high concentrations of soluble iron and aluminum. Lindsay (1979) indicated that soils at pH 4.6 should yield soluble Fe+3 activity of approximately 103' 0 when in equilibrium with soil-Fe. Aluminum activity for Alfl’in equilibrium with gibbsite should be on the order of'10'“ M, (Lindsay. 1979). These activities are not unlike the expected activities from concentrations used in this study. The conclusion drawn firom the data presented is that in the pres- ence of Fe?’. an increase in biomass associated Al occurs for l; mggatgrium (B-12) and Rhizobium see. (I-110). Also an intensifica- tion of Al stress occurs in the presence of Féafor these two test org- anisms. Therefore. it is suggested that the most likely mechanism for Fe induced Al stress is the import of Al intracellularly in association with Fe9'. This effect might either be direct as in dir- ect competition with Feaassimilation or indirect being assimilated in association with other nutrients with their assimilation being accelerated by FeI3’s effect on metabolism. The final assessment of the effects of Fe on Al stress in microorganisms will have to include a more detailed examination of the specific chemical and growth kinet- ics of biological Fe-Al interactions. 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