TH -‘Q'u —v—-———- ,_ - SW. @1233? {KNEE} RES if“? TEE .. t” it?! ' 5‘»:- g as" 5,2,5 a yr BSCMEOSEEEC‘N ui cilia} \ ‘ 51.“:2‘9. fliests Sow {he Dfiqi'm 3‘? M. 5. f‘fiECI-il GAB} STATE UEEVERS‘LTY ‘Jilci: arc. giQeyes i??? WWW!WW]IWH'IWIUIWWII 3 1293 01747 7021 ; ”LIBRA R y '4 Michigan State University (3?”‘3 ‘E‘: ‘ .a' ‘l ‘ L ”ma AUG. 2 5 2004 ABSTRACT THE INTERRELATIONSHIPS OF SOIL ANIMALS AND SOIL MICROORGANISMS IN THE DECOMPOSITION OF ORGANIC MATTER By Victor G. Reyes The interrelationships of soil animals and soil microorganisms in degrading uniformly labeled 14C-yeast was.studied using a radioisotopic method. The gut of the animal used, Tracheonisous rathkei (Brandt), was examined for its ultrastructure and microbial population. After 28 days, the animal~microbial system (a) respired 56.4 percent of the labeled food while only 33.8 and 50.8 was respired in the animal (b) and microorganism (c) system,respectively. The animals were responsible for the high initial degradation rate of the food while the soil micro- organisms degraded the feces. wood fed animals were more efficient utilizers of the yeast than yeast fed animals as indicated by higher assimilation, 44.4% vs. 37.8%, of the,origina1 food and lower fecal excretion, 28.2% vs. 37.4%, of the original food. These differences suggest a possible difference in the microbial population associated with the animal gut. The feces from yeast fed animals decomposed in.the soil at a faster rate than the feces from.wood fed animals. This indicates. that the feces from the former were less resistant than the feces from the.1atter. Pure yeast decomposed-rapidly in the soil but the rate' declined abruptly after three days. The feces decomposed at a slower Victor G. Reyes rate but for a longer period. The feces degraded faster in the soil of higher moisture level than at the lower moisture level. Moisture did not affect.the decomposition rate of pure 14C-yeast. Bristle-like protrusions extending from the chitinous wall lining of the hindgut was observed. At least six microscopically distinct types of bacteria and two kinds of fungi were isolated from the gut. The estimated microbial population was 3 to 7 x106 bacteria/gut. The enrichment for cellulolytic microorganisms from the gut was successful based on growth and the disappearance of ground filter paper. The radioisotopic method employed for following the_fate.of decomposing organic material had reproducible results and showed a relatively high recovery of the labels. THE INTERRELATIONSHIPS OF SOIL ANIMALS‘ AND SOIL MICROORGANISMS IN THE DECOMPOSITION OF ORGANIC MATTER By 1! :1 I ~ ‘ 5-. Victor GL Reyes A THESIS Submittsdxto Michigan State University. in partial fulfillment of the requirements for the degree of. MASTER OF SCIENCE Department of Crop and Soil Sciences- 1971 Dedications, To my.parents, brothers, and sisters whose memory gave me the inspiration to finish this little piece of work To Dr. Thomas S. C. Wang whose wisdom enlightened me 11 ACKNOWLEDGEMENTS To Dr. J. M. Tiedje, my heartfelt gratitude is due for his encourage- ment, patience and invaluable guidance. To Dr. A. R. Wolcott, the author is indebted for his constructive criticisms of the manuscript. To Dr. B. G. Ellis, Dr. J. W. Butcher, and Dr. M. J. Klug, special‘ thanks is also due for their helpful-suggestions. To the_staff of the Soil Biology laboratory of the Entomology Department, their contributions to the selection and rearing of the soil animals are well appreciated. The use of the equipment of Dr. D. Penner is also appreciated. The financial assistance from the,Michigan State Pesticide Project and Grant No. 00801-06 Environmental Protection Agency, previously. Grant No. ROlSD-OOZZB-OJ, Food and Drug Administration, is acknowledged. iii TABLE OF INTRODUCTION . . .'. . . .-. . . .'. MATERIALS.AND METHODS. . . . .'. . . Soil animal . . . . . . . . Preparation of the substrate. ‘Metabolism experiment_. . . . Radioactivity determination . Gut enamination . . . . . .-. “SULTS. O O O O O O '0 O O O r. O O 0 Soil arthropod-microorganism vs. arthropod.or CONTENTS" . micro- organism system 0 Se 0 e e e e e e Se e e e 'e e >e e 'e Yeast fed vs. wood fed Isopods. . . . . . . . °,° .- Biodegradability of feces vs. yeast in soil at two m 18 ture levels e e e e e e e Gut examination . . . . . .'. DISCUSSION e 'e e e e e e e e e e ‘e e REFERENCES e e e e e e le e e e ‘e e e iv Page 12 . 12- 13 16 18 20‘ 24 LIST OF TABLES Table Page 1 The fate of uniformly labeled 14C-.-yeast28 days after feeding to woodlice or acted upon by soil microorganisms. . . 12 2 Distribution of:S40slabel after one and two days:follow- ing feeding of woodlice with uniformly labeled yeast for one day 0 O O O “O O O O .0 O O I I. O O O ‘0 O O O .0 O O O O O Q 16 Figure LIST OF FIGURES A schematic diagram for growing uniformly labeled 14C- yeast 0 I O O O O O O I O O I .0 O ‘0 O O O I O ‘0 O O O O O O A-side.view of the screwecap.inCubation jar (50 x 53 mm) showing the various components. . . . .'. ._. . . -.- . . . A side view of the modified oxygen combustion flask showing various parts. Rubber hose is connected to.an.adapter for evacuation and.oxygen flushing. .The basket holds the wrapped sample during ignition. .Rubber.capped glass tubing accepts the CO2 trap after combustion. . . . , . . . Percent decomposition per day of l4C-yeast- in system: (a) animal-microorganism; (b) animal, and (c) micro- orgmismO O O O O O O O O O O O I I O O O O I O O O O O O O Progressive.decomposition.of.14C-yeast in system:,.(a) animal~microorganism; (b) animal; and (c) microorganism . . Percent decomposition per day in the soil of pure 140- yeast. and feces from yeast feeding and wood feeding wood- lice fed.with 14C~yeast . . . . . . . . . . . . . . . . . . An electron micrograph of a thin cross section of hindgut. epithelium showing chitinous intima (i) and bristle (b), 5625 Xe O O O O O C O C O C O O 0 O O l. O O O O O O O I O ,. vi Page 10- 14 15 .17 -19_ INTRODUCTION The balance of our dynamic terrestrial ecosystem depends on nutrient recycling and rate of energy flow mediated by its biotic components. Traditionally, soil scientists have assumed that the major agents of degradation of organic materials are-microorganisms. Recently, the role of soil animals in decay has been studied intensively and results sug- gested that they are important in decomposition (Bocock, 1964; Clarke, 1965; Torne, 1967). On the contrary, however, there is evidence showing that soil fauna are insignificant in the metabolism of plant residues in, the soil (Bleak, 1970; Curry, 1969; Zachariae, 1963). This inconsistency.is due to the lack of concerted efforts among microbiologists, zoologists, and soil scientists. As van der Drift (1970) and Coupland et al. (1969) stressed, workers in different disciplines of soil biology must coordinate their research to understand the_complex interactions of the diverse groups of micro- and macroorganisms which drive the biological decomposition processes. In addition, the methods that have been used to study them were as varied'and numerous as the number of investigators. Heath et al. (1964) recognized this variety and the limitations of the methods used. It is therefore the purpose of this paper to demonstrate that an integrated approach is possible: that soil microorganisms and soil animals,-as they interact, are efficient decomposers. Since currently used biocides reach the soils directly or indirectly and may indiscrimi~ nateLyinhibit beneficial populatiOns (Edwards, 1969), it is evident 1 2 that an understanding of their interrelationships is necessary. It is also the purpose of this paper to introduce a radioisotopic method that is.sensitive and quantitative for assessing the biological activity in. soils. MATERIALS AND METHODS Soil animal Tracheoniscus rathkei (Brandt), commonly known as woodlice, was used in this study. It is a terrestrial Isopod and one of the most active soil arthropods (van-der Drift, 1962; Kuhnelt, 1961; Macfadyen, 1962). It thrives mainly on forest litter and wood but can live on almost any kind of organic material as long as the relatiVe humidity is high. The choice was.based on its availability, easy maintenance in the laboratory, ready collection of feces, and ability to withstand low oxygen and.wide fluc- tuation of temperatures. They were collected-from campus woodlots and reared in the laboratory on a diet of either Baker's yeast or wood-accord- ing to the method of Butcher et a2. (1969). In case of mass rearing, 10 x 19.5 cm plastic boxes were used instead of rearing jars. Preparation 2£_the substrate Uniformly labeled 14C-yeast was used to represent plant residues at various degrees of decomposition because it was a suitable food for the‘ soil animals and easy to prepare. In addition, previous workers have~ suggested that the fungal mycelium and bacterial cells associated with decomposing organic residues are the major source of nutrition of grazing animals (Cmelik and Douglas, 1970; Cragg, 1961; Macfadyen, 1962; Minderman and Daniels, 1966).. The yeast-was prepared by aerobically growing Saccharomyces oerevisiae for 24 hr in 200 m1 of medium (pH 5.5) as modified from.Olson and Johnson. 4 (1949). It consisted of KH2P04, 0.8 gm; NHANOB’ 0.1 gm; Mg804-7H20, 40.0 mg; CaC12°2H20, 4.0 mg; Fe013°6H-0, 2.0 ppm; CuSO4-5H20, 0.10 ppm; H3BO3, 1.0 ppm; MnSO4°4H20, 1.75 ppm; Zn50407H20, 1.0 ppm; glucose, 2.0%; yeast extract,0.015% biotin, 0.16 ppm; thiamin, 0.10 ppm; and pyridoxin, 0.10 ppm.. This medium was derived to give optimal yield while minimizing the quantity of unlabeled carbon materials added. To supply the needed label, 0.5 mC of uniformly labeled 14C-glucose,(Tracerlab) was added (2.78 x 10-3 mM) in the form of 50 ml-aqueous solution (180 mC/mM), rins- ing the containing vials with 50 m1 of the salt solution used. The medium was then inoculated with 1 ml of the yeast in the logarithmic phase grow- ing in 2% glucose-yeast,extract-mineral salt medium. A 500 ml-flask containing the above medium was attached to_a rotary shaker operating at. 125 rpm. This, together with the removal of CO from the perfusing 2 atmosphere,was necessary to insure aerobic growth for high yield of cells. To aerate the flask, compressed air that had passed through drierite (W. A. Drierite Co.) and ascarite (Arthur H. Thomas Co.) to remove 002 was bubbled into the medium (Figure 1). The respired CO2 was trapped from the exhaust by a series of three gas scrubbing towers containing 1 M NaOH. ‘After incubation, the trap contained 4.35% of the original label. The cells were harvested-in the late log phase at an 0.D. of 0.75 by centrifuging the suspension in 250 ml-polypropylene bottles at 5000 rpm for 25 min~in a refrigerated centrifuge (Sorvall) set at 4° C. After discarding the supernatant liquid and including the washings from the. grower flask, the cells were washed by resuspending them twice in 250 ml' of refrigerated distilled water. The washed yeast cells were carefully pipetted out on a preweighed ovendry filter paper (Whatman No. 42), with two extra filter papers fitted to a watch glass to absorb excess water. The rate of flow was slower than .ummmmnoqa moaonma hHfiHOMfias mafiaoum Mom amuwmaw oflumawsom < .H shaman momz 2 H £ua3 moan» Honommua m Jenam manuaso . o>Hm> ,5 oufiumomny a oufiuofiun was I LIIH wommoumaou , AV AIII. Houawmwmn nouuou 1a maaumum 6 the rate of water absorption to retain the cells in_a smallest possible area thus facilitating the separation of the yeast cells from the filter paper after oven drying for 12 hr at 60° C and weighing.“ The caked dry yeast was ground into a fine powder in an agate mortar and placed in.a vial, and stored in a colored bottle containing drierite as desiccant, overlaid with cotton at 4° C. The total yield of labeled yeast cells was 0.69 g which contained 8.35% of the original label and had a specific activity of 0.06 uC/mg. Metabolism_gxperiment All experiments were done in the rearing jar (Figure 2) inside a temperature controlled walk-in incubator (Chicago Electrical and Surgical Co.).' The cap liner was repasted with rubber cement to prevent-separav tion after prolonged tight usage. The temperature was maintained at 20 1'2° C'and the relative humidity was kept high by placing a pan of water in the incubator. The incubator was kept dark. The above rearing jars used to determine the organic matter breakdown contained the following components: (a) three IsOpods and soil containing the.natural microbial population; (b) three Isopods and charcoal—plaster of Paris; and (c) soil containing the natural microbial population. The soil was freshly obtained from the animal collection site; 25 g per jar was used. The labeled yeast was fed or added at the rate of 2.5 mg (0.15_uC) per jar and placed on a glass cover slip in the treatments con- taining animals (a and b) or spread on the soil surface where animals' were absent (c). This quantity of yeast was previously determined as the amount which three animals would totally consume within 24 hr. Non- labeled Baker's yeast was supplied to the animals one day after the initial feeding and at every other inspection thereafter. Inspections "'i‘===5"'7 I Cap I fill ’ Wire support I CO2 trap . Cover 311p --Charcoal-p1aster of paris or soil Figure 2. A side view of the screw-cap incubation jar (50 x 53 mm.) showing the various components. 8 occurred after 1 to 7, 9, 12, 15, 18, 23, and 28 days; during these periods, respired 14CO2 was measured while the feces were collected after 3, 7, 23, and 28 days. The feces in the first treatment (a) were notr collected but allowed to be metabolized by soil microorganiSms. The animals were_anesthetized with chloroform in a cotton ball after 28 days and collected. The feces and the animals were picked using disposable needles in a 1 cc syringe (Tbmac) and wrapped in glacine weighing paper. Both were immediately.dried (60° C) and kept in a freezer to minimize decomposition prior to radicactivity determination. Each treatment was replicated six times. Yeast-fed and wood-fed animals were compared in an experiment similar to treatment (b) above but lasting only for 48 hr. All of the animals used were reared for two months in the laboratory. Respired 14CO2 and feces were collected and measured after 24 and 48 hr, while the animals were collected after 48 hr. Onethalf-of the first fecal collection was. used.for radioactivity assay while the other half was saved for biode— gradability determination below. The experiment was replicated eight-times. The biodegradability of the above labeled feces and the labeled yeast was compared in a soil containing the natural microbial population (similar, to c). The soil was held at two moisture levels, 16.4% which was near field capacity (17.4% measured at 1/3 atm for 48 hr) and 11.4%. The.higher‘ moisture level was that of the freshly collected soil (October) while the other was air dried to the precalculated weight. Respired 14CO2 was measured daily for the first 10 days and then at 12, 14, and 16 days. The experiment was replicated eight times. Radioactivity,determination Respired CO was trapped in 2 m1 of 1 M NaOH contained in a 2 ml- 2 disposable polystyrene beaker inside the jar as shown in Figure 2. The beaker and contents were both transferred directly to a glass scintilla- tion vial. Samples of yeast, feces, and animals were first combusted to C02 using a combustion flask (Figure 3) as modified from Dobbs (1963). The sample to be burned was wrapped in a black paper (Arthur H. Thomas Co.) and oven dried before combustion. Ignition was initiated by an Infra Red igniter (Arthur H. Thomas Co.). After combustion, 20 m1 of l M NaOH was injected into the flask, swirled for 15 sec and allowed to stand for 2 hr. A one milliliter aliquot was pipetted into a glass scintillation vial. A mixture (1:1 v/v) of Bray's scintillator (Bray, 1960) and Cab-O-Sil (New-England Nuclear) was added to the sample in the scintillation vial to a total volume of 20 ml; The mixture was vigorously shaken, equilibrated to scintillation counter temperature for 30 min, and counted using a Packard Linuid Scintillation Spectrometer, model 3310. A11 counts were. corrected for quenching by internal standardization and for machine efficiency. The NaOH as C02 trap was used throughout because it showed little quenching, was cheap, stable and did not affect the animals. Con- trary to previous claims, NaOH did not produce extra background count due to spontaneous breakdown of the scintillators. Gut examination The gut was fixed according to normal procedure using buffered 6.25% glutaraldehyde (pH 7.2) at 4° C and postfixed with buffered 1% osmium tetroxide (pH 7.2); and the thin section double stained with uranyl nitrate and lead acetate. A Philips electron microscope model EM 100 was used for examination. 10 rubber capped glass tubing rubber stopper JL-J rubber hose Nichrome wire support and basket l-liter filter flask Figure 3. A side view of the modified oxygen combustion flask showing various parts. Rubber hose is connected to an adapter for evacuation and oxygen flushing. The basket holds the wrapped sample during ignition. Rubber capped glass tubing accepts the CO2 trap after combustion. ll Microorganisms in the gut of yeast feeding and wood feeding animals were_isolated in YEA (yeast extract agar) and HIA (heart infusion agar). The gut was removed by pulling away from each other the head and the last two to three body segments with sterile fine—tipped forceps. This was done in a sterile disposable petri-dish. Immediately after removal an insect stuffing pin which was held in glass tubing and ethanol-flame sterilized was inserted into the still moist foregut and stabbed four to five times in the agar plates. This was repeated two times, each pref ceded by sterilization. Each stabbing was alternated between YEA and. HIA. The same procedure for the hindgut immediately followed to avoid rapid drying of the gut after removal. As a control after each gut had been sampled, the pin.was resterilized as before and stabbed into agar. To estimate the relative counts of bacteria in the gut, the guts of five previously yeast fed IsOpods (12 hr) and five starved Isopods were aseptically crushed with glass beads in 0.9 ml distilled water and made to 1.0 ml with standardized 1.95 u—polyvinyl-toluene beads. (Diagnostic Products). The number was calculated from the ratio of bacteria and beads under the phase microsc0pe and from the known concen- tration of the initial bead suspension (4.6 x 108 beads/m1). Each treat- ment was duplicated. Each replicate was sampled and counted twice. Cellulose decomposers were enriched (Aaronson, 1970) from 0.5 g of feces and five crushed guts of IsOpods one week after collection of the animals. Each treatment was duplicated and as a control, the enrich- ment medium was not inoculated. RESULTS Soil arthropodemicroorganism gs. arthroEOdIg£_microo£g§nism system The combined activity of soil arthropod and microorganisms (system a) was more efficient in degrading uniformly labeled 14C-yeastthan either the arthropods (b) or microorganisms (c) as shown in Table 1. After 28 days, the animal~microbia1 system respired 56.4% of the labeled food while only 33.8% and 50.8% was respired in the animal and microorganism system, respectively. Table 1. The fate of uniformly labeled 14C-yeast 28 days after feeding to woodlice or acted upon by soil microorganisms Percent of the original food Degradation system respiration a] feces b] assimilation 3] total a. soil arthropod- microorganisms 56.4 17.1 73.5 b. animals 33.8 29.8 14.9' 78.5 c. microorganisms 50.8 a/differences significant at 5% level, T-test b/difference between two computed values of decomposed feces insignificant at 5% level, T-test Ejdifference insignificant at 5% level, T-test The feces accounted for 29.8% of the label. Calculated from the table, two values of fecal decomposition may be ascribed to the soil 12 l3 microorganisms: directly as the difference in respiration of treatment (a) and treatment (b), 22.6%; and indirectly as the difference of the label in the undecomposed feces (difference between total recovered label in system (a) and system (b), 5.0%) with the label present in the feces, 24.8%. This suggests that at least 75.8% or three-quarters of the fecal. material was converted to CO2 by microorganisms. The animal retained an average 15.8% of the labeled material originally fed._ It is assumed that the bulk of the labeled atoms are in stable body components as shown by the low rate of respiration (Figure 4) on the 28th day. All the systems were respiring almost at the same slow rate on the last sampling as seen in the merging of the curves. Clearly, the presence of the animals boosted the degradation of the yeast on the first day while the microorganisms took 3 days to reach their maximum decomposition rate. When the same data are presented in a cumulat- ive manner (Figure 5), it was evident that decomposition was approaching a constant rate and that no further degradation could result in one system overtaking the other. Unavoidable losses or errors resulting from inspections, adsorption to subatratum, combustion, counting, and the length of time involved are assumed to account for the recovery of only 78.5% of the label originally. introduced. Yeast fed :2, wood fed IsOpods Table 2 shows that the respiration of yeast and wood fed animals after two days accounted for 9,6: and 11.7% of the total initial material, respectively. The difference was not statistically significant at the 5% level, T-test.‘ In the same order as above, the percent of original material present in the feces was‘37.4% and 28.2%while assimilation took 14 .amanmwuoouofia on mom “HmaHnm Anv mamfiomwuoouowalamaaom Amv "amumhm ca unmohluqa mo esp use :ofiuflmomaooov unmouom .e muowfim MN ON mH OH m n 1 q 1 d + d u! [1 u M 1 d d amfiommnoouoaz To :35 Ella amfinmwuoouofialamafic< AQFIIIIAQV L 0H I93°3 ;o nuaOJad 89 Dirt VI Asp 18d zoo 15 .amaomwuoowoaa on mom mamafiom Anv mamaomwuoouofialamafiam Amv "acumen as ammoelo mo oofiufimoeaooov oPHmmonmoum .m madman amanmwuoouofialamafia< «H mm om ma - 0H m 41 ddqqdfi4l14444uldfidqda \ k»\ in b ad > o\ b» and Ar .- b u ed .57 my .. .o 4‘ _u —u 0. AV -— n— 0. 4V 4 o. 4 .0 o 4 0. 4 AV coauamomaoomw Hmoom ou mom ROWIIIIMDV amfiamwuoouofiz AVIIIIIAU SEE 0H ON on O \1’ on on xenon go auaoled SB GOVT 371 Z 16 Table 2. Distribution of 14C'label after one and two days following feeding of woodlice with uniformly labeled yeast for one day Percent of the original food Laboratory C927 feces animal total food 1 da a] 2 da b] 1 da 2] 2 da d!" 2 da*'g/ 2 da* f] Yeast 5.8 3.8 33.9 3.5 37.8 84.8 WOod 6.5 5.2 23.9‘ 4.3 44.4 84.3 * after 2 days a], 2], g], and.fjnot significant at 5% level, T-test E] and Ejsignificant at 5% level, T-test 37,8zand 44.4%, respectively. In_both cases the difference between the animals was statistically significant at the 5% level. The difference in feces and assimilation between yeast and wood fed animals is possibly due to a difference in the microbial population associated with the animal gut as conditioned by the previous type of feeding. WOod fed animals were more efficient utilizers of the yeast substrate as indicated by higher assimilation and lower fecal excretion. The lesser efficiency of yeast fed animals is indicated by the opposite of the above.‘ The maximum nine- fold decrease in radioactivity of the feces from.the first to second day and the high assimilation compared to what had been respired indicates that the potential ability of these animals to utilize decomposable organic materials is high. Biodegradability gf_feces gs, yeast in_soil §£_two moisture levels The percent decomposition per day of the labeled feces and yeast at two moisture.levels (16.4% and 11.4%, respectively) are shown in Figure 6. Yeast, which is more nutritious and containing more digestible materials 17 C>——