[)7 [V7 Submi Sta AEROBIC CELLULOSE DECOMPOSITION BY BACTERIA by William Allen May Jr. AN ABSTRACT Submitted to the College of Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Chemical Engineering 1959 W Wat/l, Hillia sistan an inv of mic dition paper teria Includ compcs consum tion cl PI‘Oduc‘ by the tion C. lulCSe l.g re “33 es Elm] m0 William A. May .14” ABSTRACT Since cellulosic materials have been found to be re- sistant to decomposition in garbage composting processes, an investigation was made of some of the characteristics of microbiological attack on pure cellulose under con- ditions simulating those of a compost mixture. Filter paper was used as a substrate, and a mixed culture of bac- teria obtained from garbage compost was used as inoculum. Included in the factors studied were desired nutrient composition, rate of carbon dioxide production and oxygen consumption, variation of the reSpiratory quotient, varia- tion of pH, characteristics of the microbial population, products of decomposition and rate of nitrogen assimilation by the microorganisms. Work was also done to gain informa- tion concerning the limiting factors for the rate of cel- lulose decomposition. An optimum concentration of kaNOB was found to be l.h per cent by weight of the initial mixture. Magnesium was essential for growth of the microorganisms. The Opti- mum moisture content was from 62 to 70 per cent. The rate of growth of microorganisms was followed by measuring the conversion of nitrate to organic nitrogen in water extracts of samples. The amount of remaining cellu- lose was determined by extracting the samples with water, alcohol and ether to remove the decomposition products. The rate of cellulose oxidation was followed by measuring Willie oxyg e: the d: 17.0 ; total lose . isms ' imum maxim gen p rates initi destr William A. May ABSTRACT oxygen uptake from the exhaust gas that had passed through the decomposing mixture. In a run which lasted eight days, 17.0 per cent of the initial cellulose was oxidized and a total of 3h.3 per cent was destroyed. It was found that the curves for the rate of cellu- lose destruction and for the rate of growth of microorgan- isms were nearly parallel. Both curves reached their max- imum approximately on the fourth day of incubation. The maximum rate of nitrogen assimilation was 5.4 mg of nitro- gen per gram initial cellulose per day, and the maximum rates of cellulose decomposition were 4.6 per cent of the initial cellulose oxidized per day and over 8 per cent destroyed pee day. The curves showed the activity of a decomposing mixture of cellulose to climb rapidly, then level off and decline after four to five days of incubation even though the supply of cellulose had not been exhausted. Experiments concerning the limiting factor for the rate of cellulose decomposition showed that addition of nutrient solution had no stimulating effect during the period of declining activity. An experiment using filter paper discs showed that clumping and restriction of available sufface was not a limiting factor. Submi Sta- AEROBIC CELLULOSE DECOMIOSITION BY BACTERIA by William Allen May Jr. A THESIS Submitted to the College of Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Chemical Engineering 1959 ii ACKNOWLEDGMENT This thesis was only possible with the sincerely appreciated support of an N.I.H. grant. The author wishes to express his sincere appreciation to Dr. Karl L. Schulze of the Department of Sanitary Engineering'and Dr. C. Fred Gurnham for their valuable quidance and assistance in connection with this thesis. ACKNCWI llS'I‘ OF LIST CF SECTION I. II . III. T IV. E TABLE OF CONTENTS ACKNOWLEDGMENT. . . . . . . LIST OF FIGURES . . . . . . LIST OF TABLES. . . . . . . SECTION I. II. INTRODUCTION . . . . . LITERATURE REVIEW . . . A. B, C. D. E. Cellulose Attacking Microorganisms Favorable Conditions and Nutrient Composition. . . . By-products of Decomposition . Mechanism of Attack by Microorganisms Technique of Study j. III. THEORETICAL CONSIDERATIONS. IV. EXPERIMENTAL APPARATUS AND MATERIAL. A. Material. . . . . l. Cellulose . . . 2. Nutrient Solution. 3. Seed Material . . Apparatus . . . . 1. Variation of Nutrient Composition 2. Gas Analysis Apparatus . 3. Apparatus for Filter Paper Sheets iii Page ii vii <3 (n O\ :- 1m 13 13 13 1h 15 15 16 17 iv SECTION Page V. PROCEDURE AND RESULTS. . . . . . . . . 21 A. Variation of Nutrient Composition. . . 21 B. Moisture Variation. . . . . . . . 27 C. Rate of Oxygen Consumption and C02 Production . . . . . . . . . . 29 D. Addition of Nutrient Components at the Point of Maximum Oxygen Consumption . . 34 E. Effect of Increased Surface. . . . I. 39 F. Nitrogen Assimilation and Total Cellulose Destruction. . . . . . . 42 G. Variation of pH. . . . . . . . . 55 H. Variation of ReSpiratory Quotient. . . 56 I. By-products from Decomposition. . . . 59 VL. DISCUSSION . . . . . . . . . . . . 73 VII. CONCLUSIONS . . . . . . . . . . . . 79 BIBLIOGRAPHY . . . . . . . . . . . . . . 80 APPENDIX . . . . . . . . . . . . . . . 83 LIST OF FIGURES Figures 1. Flow Diagram of Apparatus. . . . . . . 2. Plastic Container for Paper Discs . . . . 3. Rate of Weight Loss at 100 Per Cent Humidity 4. Effect of NaN03 Concentration on Weight Loss 5. Effect of NgSOh Concentration on Weight Loss 6. Effect of CaHPOL Concentration on Weight Loss 7. aEffect of KZHPOL Concentration on Weight Loss 8. Effect of FeSOA Concentration on Weight Loss 9. Effect of Moisture Content on Weight Loss . 10. Rate of Oxygen Consumption for Run No. l. . 11. Rate of 002 Production for Run No. 1 . . . 12. Effect of NaNO3 Addition on Oxygen Consumption. 13. Effect of Nutrient Addition on Oxygen Consumption . . . . . . . . . . . 1h. Oxygen Consumption Rate for RunJNo. A. . . 15. C02 Production Rate for Run No. A . . . . 16. Comparison of Runs No. l, 3 and 4 . . . . 17. Total Cellulose Destroyed in Run No. 7 . . 18. Cumulative Nitrogen Conversion . . . . . 19. Oxygen and Nitrogen Consumption Rates. . . 20. Nitrogen Conversion Plotted as a Growth Curse 21. Rate of Weight Loss After Extraction . . . 22. Total Nitrogen Conversion and Cellulose Breakdown . . . . . . . . . . Page 19 20 23 24 24 25 25 26 26 33 33 38 38 Al A1 43 #3 5O 50 53 53 54 Figure 23. 2A. 25. 26. 27. 28. 29. Daily ReSpiratory Quotient. . . . Rate of Cellulose Decomposition by Complete C;Xidation O O I I O O O O 0 Cumulative Cellulose Oxidation . . Rate of Excess 002 Production in Run No. Cumulative 002 Production for Run No. 1 Cumulative Cellulose Oxidized in Run No. Cumulative Cellulose Lost in Run No. 7. vi Page . 58 . 69 16. 17. 18. 19. 20. 21. 22. 23. LIST OF TABLES Nutrient Solution. . . . . . Standard Mixture . . . . . . Variation of Moisture . . . . Data for Runs No. 1 Through 6. . Gas Analysis Data for Run No. 1 . Gas Analysis Data for Runs No. 2 and 3. Gas Analysis Data for Runs No. 5 and 6. Gas Analysis Data for Run No. A . Nitrogen Determination Data . . Nitrogen Determination Calculations. Cumulative Nitrogen Conversion . Gas Analysis Data for Run No. 7 . Nitrogen Conversion and Cellulose loss. Variation of pH . . . . . . Variation of Respiratory Quotient Cellulose Oxidized in Run No. l . Cellulose Oxidized in Runs No. 1 Through Excess 002 in Runs No. 1 Through 6 . 0‘ Material Balance of Weight Loss After Drying. vii Page . 67 Material Balance for Run No. 5 After Extraction. 68 Cellulose Lost in Run No. 7 . . Weight Loss at Standard Composition. . 7O . 7h SECTION I INTRODUCTION The ratio of carbon to nitrogen in the final prod- uct is one criteria for evaluating the quality of com- posted garbage and refuse. Cellulosic material, such as paper, is one of the major components of a typical gar- bage mixture. Since it represents a large per cent of the carbon content, an attempt to decrease the carbon to nitrogen ratio will focus on destruction of cellulose. Finished compost often shows very little attack on the cellulosic components. Wiley and Pearce (1) reported only slight decomposition of cellulosic matter. Gotaas (2) stated that paper in composting material showed little evidence of attack by bacteria. Decomposition took place.after more readily decomposable materials had been utilized, and when conditions favored growth of actino- mycetes and fungi. It was the purpose of this study to investigate some of the characteristics of microbiological attack on pure cellulose. It was hoped that some of the information gained could be applied to obtain increased decomposition of cellulosic materials in the composting process. According to literature, the most common method of studying pure cellulose decomposition has been to suspend the cellulose in liquid medium. In this study it was desired to simulate actual composting conditions. It was decided to use a moisture content in the range of 50 to 75 per cent and a mixed culture of microorganisms. Aerobic conditions were desired, and work was done at room temperature. A gas analysis technique was used to follow the rate of activity. Samples were dried in an oven to determine loss of weight after incubation. In one experiment an extraction technique was used to measure the actual loss of cellulose. Factors investigated during the course of this study include desired nutrient composition, rate of 002 production and oxygen consumption, variation of the respiratory quotient, variation of pH, characteristics of the microbial culture, products of decomposition and rate of nitrogen assimilation by the microorganisms. Work was also done to gain information concerning the limiting factors for the rate of cellulose decomposition. SECTION II LITERATURE REVIEW In the early part of this century, interest in cellulose decomposition by microorganisms was generated by its prominent role in the cycle of carbon transforma- tion in nature. Many investigations were devoted to the type of organisms responsible and the conditions under which they attack cellulose. A more recent stimulus to research in this field resulted from the alarming losses of cellulosic products by bacterial attack in the tropical theaters during World War 11. During 1944 and 1945 teams of mycologists visited various places in the tropical belt and isolated fungi and bacteria from deteriorating cotton fabrics (3). In the last 20 years considerable interest has been directed toward the mechanism of breakdown. A. Cellulose Attacking Microorganisms Hutchinson and Clayton (4) reported isolation of an aerobic organism which showed growth only with cellulose as a source of carbon. Waksman and Skinner (5) studied the types of organisms found in soils capable of decom- posing cellulose. Marsh, Bollenbacker, Butler and Raper (6) gave an extensive report on fungi capable of destroying cellulose. Norman and Fuller (7) reviewed the types of microorganisms capable of decomposing cellulose. Norman and Bartholomew (8) did work with seven different mesOphilic bacteria; and Viljoen, Fred and Peterson (9) studied thermophilic cellulose fermenters. Reese (10) isolated 500 bacteria strains from decom- posing fabrics. He found 8 per cent or 39 of them were capable of destroying cellulose. He divided these into two classes, an aerobic and a second type requiring little or no oxygen, but not responding to differences in oxygen concentration. §;_ngo3g§le Conditions and Nutrient Composition Several nutrient solutions containing no carbon have been developed for enumeration and isolation of cel- lulose decomposing bacteria. Dubos (11) studied effects of nutrient solution composition by immersing strips of filter paper in inoculated solutions and incubating at 28°C. He found an optimum nutrient composition to be: 0.5 grams NaNO3, 1.0 g KZHPO4, 0.5 g MgSOk°7H20, 0.5 g KCl and 0.01 g FeSOh'7H20 dissolved in 1 liter of dis- tilled water. This alkaline solution (pH 7.5) favored growth of bacteria and retarded growth of fungi. He noted that decreasing concentration of NaNO3 decreased the length of the incubation period for the bacteria. Using this nutrient solution, cellulose decomposition could be recorded after 36 to 72 hours. Hutchinson and Clayton (4) used the following mix- ture in their work: 2 g NaNHAHPOh'4H20, l g KHZPOh, 0.1 g Ca012, 0.3 g Mg804°7H20, 0.1 g NaCl and 0.01 g FeCl3 in 1 liter of water. Walker and Warren (12) used the same mixture but found the CaCl2 unnecessary. Perlin, Michaelis and McFarlane (13) worked with an aerobic cellulose decomposing bacterium, Vibrio perimastix. They found that 002 was essential for growth of the bac- teria, but retarded growth at concentrations over 1.2 per cent. Many substances have been found that influence the decomposition of cellulose. For example, Fuller and Norman (14) obtained a utilization of one-third of filter paper suspended in nutrient solution over a fourteen day period. In equal time, cornstalk cellulose was far more extensively decomposed by all organisms tested. They concluded that the presence of xylan in the cellulosan component of this cellulosic material exerted a favorable‘ influence on decomposition. Waksman (15) reported that 80 to 95 per cent mois- ture favored anaerobic decomposition while 50 to 75 per cent favored aerobic cellulose decomposing bacteria. Opti- mum temperature range for aerobic decomposition was 20 to 28°C and 37°C was Optimum for anaerobic decomposition. Reese (10) worked with filter paper suspended in a :nutrient solution. The optimum pH for Sporocytophaga myxococcoides was from 6.5 to 7.5. After three days incu- bation, the optimum NaNOB concentration was from 1 to 3 grams per liter. KCl was found to be toxic at concentra- tions higher than about 0.05 N, but the author suggested that this might have been due to increasing total salt concentration rather than an effect of KCl. MgSOh and iron salts were found to be important to the rate of de- composition. Cu, Zn, Mo and Mn had no stimulating effect on growth. During experimental work to determine the rate of decomposition, Reese used a medium containing 10 ml of 1 M potassium phosphate buffer solution, 1 g NaNOB, 0.5 g MgSOh-7H20, 0.05 g FeSOA'7H20 and 4 g cellulose per liter. A series of 250 ml flasks were prepared and the rate of decomposition was followed by stopping each flask at a different time. He found 50 per cent decomposition of cel- lulose after three days. A maximum decomposition of 80 per cent was reached after six days. Reese suggested that the residue might have been bacterial substance, but no check was made. C;_§y-products of pecomposition Waksman (15) stated that as much as 30 to 40 per cent of cellulose decomposed may be converted into cell material. Heukelekian and Waksman (16) reported only 002 and water as waste products from decomposition of cellu- lose by fungi. Nord and Vitucci (17) reviewed work reporting quantitative determination of products from decomposition of cellulose. Some work was done under aerobic conditions reporting C02, methane and fatty acids including acetic, butyric and valeric; but most work was done in the thermo- philic range and under anaerobic conditions. Products re- ported include 002, hydrogen, methane, ethyl alcohol and higher alcohols and acetic, butyric, valeric, lactic and formic acids. Viljoen, Fred and Peterson (9) reported products from anaerobic organisms which destroyed cellu- lose rapidly at 65°C. The products of fermentation were acetic acid, small amounts of butyric acid, ethyl alcohol, CO2 and hydrogen. The amount of cellulose destroyed in a l to 5 per cent liquid suspension varied from 70 to 95 per cent, of which 50 to 55 Per cent was regained as acetic acid, 5 to 25 per cent as ethyl alcohol and the rest as small amounts of butyric acid, 002, hydrogen and a pigment soluble in ether. I Walker and Warren (12) found that two-thirds of cellulose decomposed under aerobic conditions was account- ed for by 002 evolved. The remaining one-third was in the form of a mucilage substance and small amounts of pigment and other metabolic products. Perlin, Michaelis and NcFarlane (13) reported products similar to those de- scribed by Walker and Warren except that a smaller amount of mucilage was found. D. Mechanism of Attack by Microorganisms Boswell (18) reviewed literature up to 1941 on the mechanism of enzymatic attack on cellulose. He favored evidence that decomposition was facilitated by oxidation resulting in the formation of oxycellulose. Walker and warren (12) suggested that the mucilage substance which they isolated from decomposing cellulose was an oxycellu- lose and an intermediate step in breakdown. Norman and Bartholomew (8) argued that the mucilage was a product of decomposition and should be considered a bacterial gum or polyuronide gum. In 1953, Sin and Reese (19) gave a review of previ- ous work with emphasis on the relationship of organism to the substrate and mechanism of breakdown of the cellulose. They gave evidence for a two stage mechanism of breakdown, and suggested that as a first step natural cellulose is broken down by enzymeiaction to armors easily attacked form. In the second step, the cellulose molecule is con- verted by enzymatic reduction to cellobiose. Evidence was cited that cellulose decomposing organisms assimilated cellobiose rather than glucose. Perlin, Michaelis and McFarlane (13) reported that the oxygen uptake rate of Vibrio perimastix was increased by additions of both glucose and cellobiose. Levinson, Mandela and Reese (20) investigated the enzymatic mechan- ism using paper chromatographic analysis to identify the products of hydrolysis of cellulose. Cellobiose was the principal product of hydrolysis according to their data. They also found that in a rapidly growing culture, the cellobiose was used as rapidly as it was formed. ,E. Technique of Study The most common method of studying pure cellulose decomposition has been to suspend the cellulose in a liquid nutrient medium. Reese (10) suspended ground fil- ter paper in inoculated nutrient solutions. The mixtures were prepared in flasks and agitated with a mechanical shaker during the incubation periods. Remaining cellulose after incubation was determined by filtering and drying. Weight of dried crucibles minus final ash weight was considered to be the weight of remaining cellulose. With this system, he investigated effects of nutrient compo- nents and concentration. Fuller and Norman (14) suspended 3 grams of finely divided cellulosic preparations in 400 ml of nutrient solution, sterilized and inoculated it with pure cultures of bacteria and bubbled sterile, moist air through it. Walker and Warren (12) worked on a large scale to get large quantities of metabolic products. They suspend? ed 20 grams of chopped filter paper in 2 liters of medium and bubbled a slow stream of oxygen through it. In some of their work, the 602 evolved was measured by absorption in baryta bottles followed by titration. The remaining cellulose was measured by filtering the medium through a 10 fine linen cloth. The residue was extracted with hot water, alcohol and ether. The final residue after extrac- tion was considered remaining cellulose. Heukelekian and Waksman (16), working with fungi in liquid, sand and soil mediums, measured remaining cellu- lose by dissolving it in Schweitzer's reagent and precip- itating it with alcohol. They measured C02 evolved by collecting it in Ba(OH)2 similar to the method used by Walker and Warren (12). They also measured nitrogen assimilated by extracting the medium and converting nitro- gen compounds to ammonia by distilling with MgO. The ammonia was collected in standard acid. The measured nitrogen was subtracted from the nitrogen found in an uninoculated control flask to give the nitrogen assimi- lated by the organisms. When nitrates were present, they were converted to ammonia using 3 grams of Devarda alloy (50% Cu, 45% Al and 5% Zn) in alkaline solution. The amount of nitrogen assimilated was shown to be directly related to the amount of cellulose decomposed. Wiley and Pearce (1) used a gas analysis technique to follow the activity of their composting process. They forced air through the composting material and analyzed the exhaust gas for moisture and C02 content. After re- moving moisture with Drierite, they absorbed C02 in Ascarite. In a similar system, Moore (21) used a Beckman magnetic oxygen analyzer to measure oxygen uptake from the exhaust gas of composting garbage. ll - SECTION III THEORETICAL CONSIDERATIONS Wiley and Pearce (l) correlated their gas analysis results from composting garbage with the following rela- tionship for organic matter destroyed: C O + b02 - xCO2 + y/2H20 xHy z This relationship assumed that by-products other than C02 and water were small in comparison to the total amount of material destroyed, and they cautioned that the relation- ship would be expected to change with time. For a pure cellulose substrate, this relationship for complete oxidation per monomer unit of cellulose 'would be: 06H1005 + 602 ' 6C02 + 5H20 The amount of cellulose decomposed by complete oxidation could be calculated by measuring the oxygen consumed. According to this equation, 0.845 grams of cellulose will be oxidized for every gram of oxygen consumed, or for every gram of cellulose oxidized: l g cellulose + 1.185 g 0 = 1.630 g CO 2 + 0.555 g H20 2 12 An indication of the type of microbiological attack taking place is given by the respiratory quotient (R.Q.), computed by dividing the moles of C02 produced by the moles of oxygen consumed. If cellulose is being decomposed by complete oxidation, the moles of oxygen consumed will equal the moles of €02 produced; and the R.Q. will be equal to one. If in addition to oxidation a fermentation reaction takes place, more CO2 is produced than oxygen consumed and the R.Q. will be greater than one. A.Nate W medium. lose. I cut in Study t ed in f experit Phate ‘ Calciu} and ma‘ men t POSsih 13 SECTION IV EXPERIMENTAL APPARATUS AND MATERIAL A. Material 1. Cellulose Whatman No. 1 filter paper was chosen as a working medium. It is almost pure alpha type or long chain cellu- lose. In all experiments except one, the filter paper was cut in small squares varying from 1/8 inch to 3/16 inches. 2. Nutrient Solution A nutrient mixture was chosen on the basis of the study by Dubos (11). It was modified to the mixture list- ed in Table 1 so that the desired low moisture content in experimental work could be obtained. The calcium phos- phate was added to the mixture to give a source of calcium. A grey precipitate, found to be compounds of iron and magnesium phosphate, resulted on mixing the solution. When the nutrient solution was added to the filter paper, the precipitate was kept in suspension as much as possible. 14 TABLE 1 NUTRIENT SOLUTION Component Amount Distilled water 300 m1 K2HP0h-3H20 6 g MgSOh°7H20 0.337 g KCl 0.335 g FeSOA'7H20 0.018 g CaHPOh 0.252 g 3, Sged Material A mixed culture seed was obtained from a sample of finished compost of synthetic garbage containing an ini- tial amount of 35 per cent newspaper on a dry weight basis. The compost, prepared by Moore (21) in his study of aerobic decomposition of organic waste material, had been stored at room temperature for several weeks after completion of the run. It showed more than the usual amount of cellulose breakdown and was therefore used to seed a mixture of 5 grams of filter paper and 15 ml of nutrient solution in a 125 ml Erlenmeyer flask. The mix— ture was kept at room temperature in a desiccator with water in place of desiccant to give 100 per cent humidity. A three da The pape addition fcrmetic mowed t surface miculite surface mica pre Th the decc Paper at SCOpic j teria h; Prom 20; Yeasts ~ large T unmerou evidenc tii‘y tk M328 Celltra. 15 A yellow color appeared on the filter paper after three days. It lost much of its strength after a week. The paper cuts had clumped together slightly with the addition of nutrient but clumped still further due to formation of slime from the organisms. A close inspection showed the yellow discoloration to occur mainly on the surface of the clumps. This led to the addition of ver- miculite to decrease clumping and preserve a greater surface for decomposition. Vermiculite is an expdoded micapreparation and is bacteriologically inert. The culture was maintained by transferring some of the decomposed cellulose into a new mixture of filter paper and nutrient solution about every twenty days. Micro- sc0pic investigation showed that a mixed culture of bac- teria had developed three days after seeding. A few protozoa were present after seven days. In some samples yeasts were found after nine days which developed into a large population by the eleventh day. Protozoa were' numerous in mixtures a month old. Cther than yeasts, no evidence of fungi was found. No attempt was made to iden- tify the components of the mixed bacterial culture. B. Apparatus 1. Variation of Nutrient Composition The equipment used to check optimum nutrient con- centration consisted of a moist chamber made from a desiccator with the desiccant replaced with water. l6 2. Gas Analysis Apparatus An apparatus was set up as shown in Figure l to pump air through a sample of cellulose in a 300 ml Erlenmeyer flask D. A Sigmamotor pump 1, model T68, with a Revco Zero-Fax speed changer, model 142X, was found satisfactory for maintaining low flow rates through the system. The incoming air was purged of CO2 by tube A filled with Sodasorb. The air was then passed through several feet of Tygon tubing submerged in the constant temperature water bath C which was used to hold the samples between 25 and 26°C. The air was humidified in a Fisher-Milligan gas washer bottle B to prevent drying out of the samples. Ascarite was used to collect the CO2 produced by decomposition of the samples. Before the air reached the Ascarite filled tube J, all moisture was removed by passage through a 12 inches long, 5/8 inches inside diam- eter tube E filled with Drierite desiccant. In this way, the increase in weight of the tube filled with Ascarite was due to 002 absorbed. A second tube of Drierite F was included which could be weighed. This gave a check for saturation of the first and larger tube. A Beckman magnetic oxygen analyzer H, model D-2, was used to measure the partial pressure of oxygen in the exhaust gas. A mercury manometer G indicated the reduced pressure in the system at the point of oxygen measurement. 17 The volume of the exhaust gas was measured as it left the system through a Wet-Test gas meter K. 33 Apparatus for Filter Paper Sheets In one experiment, it was desired to decompose the filter paper as whole sheets. The Erlenmeyer sample flask in the previously described apparatus was replaced by a transparent plastic container shown in Figure 2. This container was 4 inches deep and tapered from 10 to 9 inches in width and 13 to 12 inches in length. A hole was drilled in each end of the container and fitted with rubber tubing for air circulation. Four trays were pre- pared as sketched in Figure 2 from 5 mm glass tubes woven in place with 1/16 inch rygon tubing. Spacing between the glass tubes was about 1/8 inch. The trays were separated in the container by 1/2 inch diameter glass tubes running the width of the container at each end of the trays. With this unit, discs of filter paper were spread on the trays. The cover was sealed with stopcock grease so that the container could be submerged under water for temperature control. Figure 1. Flow Diagram of Apparatus A. B. C. D. E. F. G. H. I. J. K. Sodasorb tube Gas washer bottle Water bath Flask containing cellulose preparation Drierite tube Small Drierite tube Manometer OXygen analyzer Pump Ascarite tube Wet-Test gas meter 18 l9 m3L<¢1._.¢zm.~ 92:; MW - D mmalzw EX ml 0 ._.\m.3<1xm pi \ 62.3: 20¢»... ..2\.l\w _ I.\ .i .925: 35o . m) (4L 5;: Es. mi --.. i m>OU WUMJQ KNQ46 «Na 0636ij 4552. 6 «ma no 6: 50+- I 0. N N U R in. 2 R 0 F N m 1%. T C U D m .0 P I... 02 C F .0... 0 E T A R .00 F. R .u. U G F r — IF p b P — O O 0 IO DAYS 34 One to two days of incubation were necessary before activity became measurable. nygen consumption and 002 production indicated rapid growth of microorganisms from the second until the fourth day. This was followed by a leveling off period and a rapid decline of activity after 7 to 10 days. The maximum oxygen consumption rate was 40.8 mg per day per gram initial cellulose, and the max- imum C02 production rate was 69.1 mg per day per gram initial cellulose. D. Addition of Nutrient Components at the Point of Maximum Oxygen Consumption To gain information concerning the reason for the leveling off of activity after four days of incubation, the effect of addition of nutrient components on the rate of oxygen consumption was checked. These additions were made after the leveling off point in the oxygen uptake curves had been reached. Runs No. 2 and 3 were;rufi parallel at a flow rate of 0.4 liters per hour. Remaining oxygen in the exhaust gas decreased to as low as 12 per cent. To check the possibility that activity slowed because of depletion of nitrogen supply, 1.1 grams of NaN03 in 11.3 ml of water were added to sample No. 3 on the seventh day. It is noted that this addition increased the initial moisture content of the mixture from 66 to 70 per cent. 35 The gas analysis data for these runs are listed in Table 6. The oxygen consumption rates are plotted in Figure 12. The maximum oxygen consumption rate was 49.7 mg oxygen per gram initial cellulose per day for run No. 2 and 53.0 mg oxygen per gram initial cellulose per day for run No. 3. Results showed no stimulating effect. Production of 002 and consumption of oxygen in sample No. 3 had been running slightly higher than in sample No. 2. After the addition of NaN03, 002 production and oxygen consumption in sample No. 3 fell below that of sample No. 2. Runs No. 5 and 6 were conducted in parallel at a flow rate of about 0.5 liters per hour. Remaining oxygen in the exhaust gas dropped as low as 14 per cent. 0n the fifth day, 20 m1 of complete nutrient solution were added to sample No. 6 to see if any component supplied in the original nutrient solution acted as a limiting factor. Table 7 lists the gas analysis data for these runs, and the oxygen consumption rates are plotted in Figure 13. The maximum oxygen consumption rate was 46.1 mg oxygen per day per gram initial cellulose for run No. 5 and 57.6 mg oxygen per day per gram initial cellulose for run No; 6. Only a slight increase in oxygen consumption rate was observed after the addition of nutrient solution. Sample No. 5 appeared to lag behind sample No. 6 during the course of the run and did not reach as great a maximum 36 TABLE 6 GAS ANALYSIS DATA FOR RUNS NO. 2 AND 3 Oxygen Consumed, 002 Produced, Oxygen Cons. Rate Days Liters Liters mg/g in.ce11./day No. 2 No. 3 No. 2 No. 3 No. 2 No. 3 0.5 0 0.0015 0.0150 0.0100 0 0.2 1.0 0 0.0045 0.0142 0.0144 0 0.7 1.5 0.0048 0.0076 0.0172 0.0217 0.7 1.1 2.0 0.0212 0.0347 0.0317 0.055 3.2 5.2 2.5 0.054 0.112 0.070 0.143 8.4 16.8 3.0 0.135 0.237 0.166 0.295 24.8 35.6 3.5 0.248 0.338 0.293 0.408 37.2 50.6 4.0 0.312 0.354 0.375 0.418 46.8 53.0 4.5 0.331 0.320 0.376 0.337 49.7 48.0 5.0 0.303 0.297 0.310 0.286 46.5 44.5 5.5 0.257 0.282 0.259 0.273 38.6 42.3 6.0 0.234 0.264 0.238 0.261 35.0 39.6 6.5 0.234 0.260 0.232 0.258 35.0 38.9 7.0 0.229 0.247 0.226 0.247 34.5 37.0* 7.5 0.222 0.239 0.217 0.224 33.4 35.5 8.0 0.217 0.232 0.213 0.226 32.6 34.8 8.5 0.208 0.211 0.200 0.206 31.2 31.7 9.0 0.205 0.191 0.192 0.196 30.7 28.6 9.5 0.190 0.1815 0 180 0.181 28.6 27.1 10.0 0.175 0.1755 0.170 0.175 26.2 26.2 10.5 0.1655 0.1615 0.160 0.162 24.8 24.2 11.0 0.1585 0.150 0.154 0.151 23.8 22.4 11.5 0.149 0.140 0.143 0.139 22.1 21.0 12.0 0.140 0.132 0.138 0.128 21.0 19.8 12.5 0.137 0.119 0.130 0.116 20.5 17.9 13.0 0.131 0.105 0.126 0.108 19.7 15.7 13.5 0.122 0.1005 0.121 0.099 18. 15.1 14.0 0.119 0.093 0.117 0.093 17.9 13.9 37 TABLE 7 GAS ANALYSIS DATA FOR RUNS NO. 5 AND 6 Oxygen Consumed, 002 Produced, Oxygen Cons. Rate Days Liters Liters mg/g in.ce11./day No. 5 No. 6 No. 5 No. 6 No. 5 No. 6 0.5 0.0013 0.0014 0.041 0.0128 0.19 0.22 1.0 0.00815 0.00577 0.0506 0.0156 1.2 0.94 1.5 0.0176 0.0174 0.0566 0.0278 2.65 2.6 2.0 0.0453 0.0638 0.0857 0.0762 6.8 9.6 2.5 0.0986 0.166 0.149 0.190 14.9 25.0 3.0 0.162 0.316 0.239 0.393 24.5 47.6 3.5 0.226 0.382 0.303 0.456 34.1 57.6 4.0 0.294 . 0.336 0.371 0.379 44.4 50.6 4.5 0.306 0.290 0.379 0.316 46.1 43.7 5.0 0.264 0.270 0.297 0.293 40.1 40.6* 5.5 0.216 0.275 0.243 0.319 32.6 41.5 6.0 0.201 0.271 0.221 0.312 30.2 40.8 7.0 0.195 0.217 0.214 0.247 29.3 32.7 7.5 0.199 0.209 0.220 0.244 30.0 31.4 8.0 0.192 0.206 0.209 0.227 28.9 31.0 *Nutrient solution added. **Correction for content of air in the system at the close of the run. 38 3E wrFIGURE I2. EFFficT OF MIND, ADDITION ON OXYGEN CONSUMPTION Q 3‘. 350* x=RUN No.2 3 69 = RUN N0. 3 O 3 I j 40 Lu U 2' 30' Q a w...‘ 0 20 .3 ‘ ‘3‘ '0 z- , m h QN lo. . O . a I ADDITION 0r muo, TO RUN N0. 3 z 1 l L L 1L 00 8 IO I2 “4 “o" 8 8 H6 Oa PER G INITIAL CELL ULOSE PER DAY 0 AYS FIGURE I3. EFFECT OF NUTRIENT ADDITION 0N OXYGEN CONSUMPTION x =RUN No.5 6) =RUN No. 6 r 30+ 20+ I0- I ADDITION OF NUTRIENT To RUN No.6 0 ‘ 1 1 1 1 1 L J O 2 ‘4 b 8 I0 I2 HI DAYS 39 rate. It is noted that sample No. 5 also had a secondary maximum at a corresponding point in its development even though no addition of nutrient solution was made to this sample. E2:§ffect of Increased Surface It was thought that the clumping of the filter paper might be a limiting factor for rate of decomposition. To check this, run No. 4 was made in the plastic container. Since each paper disc was spread individually on the glass grids, practically all of the paper's surface was avail— able for attack by microorganisms and open to the supply of oxygen. The initial dry weight of the 36 discs of filter paper which were spread on the trays was 19.345 grams. The seed material was suSpended in the nutrient solution, and 1.9 ml of this solution were added to each paper disc. A flow rate of 0.3 liters of air per hour was maintained, and remaining oxygen in the exhaust gas decreased to as low as 7 per cent. Table 8 lists the gas analysis data for this run. The maximum oxygen consumption rate was 55.0 mg per day per gram initial cellulose, and the maximum rate of 002 production was 80.3 mg per day per gram initial cellulose. The oxygen consumption rate is plotted in Figure 14, and ‘the 00 production rate is plotted in Figure 15. The flow 2 :rate was increased to 0.4 liters per hour on the eleventh TABLE 8 GAS ANALYSIS DATA FOR RUN N0. 4 4O Oxygen . 002 02 Cons. , C02 Prod. , Days Consumed, Produced, Rate Rate Liters Liters mg/g in.ce11./day 0.5 0.0041 0.0106 0.5 2.0 1.0 0.0088 0.0111 1.2 2.1 1.5 0.0121 0.0139 1.7 2.6 2.0 0.0266 0.0294 3.6 5.5 2.5 0.0813 0.0885 11.0 16.5 3.0 0.1905 0.219 25.8 40.8 3.5 0.308 0.360 41.7 67.0 4.0 0.381 0.432 51.6 80.3 4.5 0.394 0.422 53.5 78.5 5.0 0.392 0.406 53.3 75.5 5.5 0.405 0.405 55.0 75.4 6.0 0.401 0.396 54.5 73.6 6.5 0.397 0.376 54.0 70.0 7.0 0.358 0.340 48.8 63.3 7.5 0.328 0.307 44.6 57.1 8.0 0.297 0.279 40.3 51.9 8.5 0.264 0.249 35.8 46.4 9.0 0.229 0.220 31.0 41.0 9.5 0.202 0.192 27.4 35.7 10.0 0.182 0.171 24.7 31.8 10.5 0.164 0.154 22.3 28.6 11.0 0.1485 0.141 20.2 . 26.2 11.5* 0.153 0.148 20.8 27.5 12.0 0.161 0.159 21.8 29.6 13.8 0.157 0.156 21.4 29.0 13.5** 0.297 0.296 --- --- 14.0 0.145 0.144 19.7 26.8 *Air flow had been increased from 0.3 to 0.4 liters per hour. **Data for a 24 hour period. trl‘ia :dn URPOJNa JJ K J II-(.IP-z. 5 V.— IN P.- .. “V 41 gm FIGURE I4. OXYGEN CONSUMPTION RATE FOR RUN N0, 0, (,0 40 U \ MG 0?. PER G INITIAL CELLULOSE PER DAY 0 n 1 A A 1 IFLO‘W D‘STUBLBED 0 2 '+ 6 8 IO DAYS '2 “5 FIGURE IS. C0,2 PRODUCTION RATE FOR RUN NO. 4 1 IFLOW DISTURBED O 2 4 6 3 I0 DAYS I2 In MG C03 PER G INITIAL CELLULOSE PER DAY 42 day. This change resulted in a disturbance of the measured oxygen uptake and 002 production due to the large volume of the container. For comparison with other data, run No. 4 is plotted along with runs No. 1 and 3 in Figure 16. Run No. 4 appeared to maintain its maximum oxygen uptake rate longer than run No. 3, and both runs reached a higher rate than run No. 1. Runs No. 3 and 4 reached a maximum rate of 53 and 55 compared to a maximum rate of 40.8 mg oxygen per day per gram initial cellulose for run No. 1. Run No. 4 showed a very rapid decline of activity after leveling off. Results indicated that clumping of filter paper was not a limiting factor. F. Nitrogen Assimilation and Total Cellulose Destruction In preliminary work, attempts were made to follow the conversion of NaNO3 to organic nitrogen using the Kjeldahl method of organic nitrogen determination. Curves were obtained for nitrate conversion using this method, but work was hampered by what appeared to be a conversion of the nitrate ion to ammonia in the presence of cellulose during the Kjeldahl procedure. A method was worked out to measure the remaining nitrate rather than organic nitrogen. This method was similar to the procedure described by Heukelekian and Waksman (16). A mixture of Al, Cu and Zn in the presence of NaOH was used to convert the nitrate to ammonia. The 43 FIGURE I6. COMPARISON OF RUNS NO. I, 3 AND II 3£ ‘OI a at" RUN N0. 3 \ Q’ 507 ”If \ RUN N0.” 3.’ 'l \ o ' \ \ S. I” \\ ‘ .1 no» 'I \ .J ' \\ LIJ " \\ U I \\ 4 30»- I’ \ 5 I \\ RUN NO. I t 'I' \\ z x " 20’ I/ \ g ,I \ \ I \ x ’/ \ 8." IO“ I 0' x" £9 2 O I 1 1 A 1 L 1 o u 8 I2 I6 20 2.“ DAYS FIGURE l7. TOTAL CELLULOSE DESTROYED IN RUN No. 7 uoI. I»- 330+ .l m V: O 5‘20~ ‘1 .J U U I-- . zIo Lu U o: LUO _L 1 JL 4 n ‘1 0 2 LI 6 3 I0 DAYS 44 ammonia was collected in standard acid and titrated to determine the amount of nitrate which had been present. The procedure is given in the Appendix. A mixture was prepared using 60 grams of cellulose, 30 grams of vermiculite, 180 m1 of nutrient solution and 1.8 grams of seed material. Samples were taken initially and on the third, fifth and seventh day. These samples were extracted with water, and the extract was analyzed for nitrate. To determine the amount of cellulose destroy- ed, the residue was further extracted with alcohol and ether before drying at 105°C. The dry weight was taken as vermiculite and remaining cellulose. The samples were ashed at 800°C for three hours to determine the amount of vermiculite. Checks of vermiculite alone showed it to be 97.8 per cent ash. Ash from filter paper in the samples was considered negligible and salts from the nutrient had been removed with the water extract. Four samples were taken for each day. Data from these samples are given in Table 9. Since ash content should remain constant, calculations were made using it as a basis. Initially the mixture contained 0.576 grams of nitrogen in the form of nitrate. The initial ash con- tent was 29.3 grams or 10.7 per cent of the initial moist weight of the mixture. This gives 19.7 mg of nitrate per gram ash or 0.21 per cent of the initial moist weight. Initially there should have been 1.97 grams of cellulose per gram of ash. TABLE 9 NITROGEN DETERMINATION DATA 45 Sample Total Dried Wt. Nitrogen as Day No. Moist After Ash, Nitrate, Weight, Extraction, Grams Grams Grams Grams 0 1 7.426 2.418 0.579 0.01425 2 6.187 2.013 0.468 0.0129 3 11.109 3.525 0.806 0.02165 4 7.357 2.387 0.645 0.0131 3 1 3.544 1.197 0.313 0.00464 2 3.261 1.117 0.272 0.00476 3 3.783 1.301 0.314 0.00484 4 5.045 1.652 0.512 0.00725 5 1 4.437 1.342 0.416 0.00138 2 3.374 1.043 0.312 0.00116 3 5.401 1.632 0.527 0.00134 4 5.453 1.609 0.612 0.00231 7 1 4.812 1.360 0.477 0.00071 2 4.180 1.169 0.363 0.00035 3 3.199 0.905 0.294 0.00056 4 3.149 0.834 0.333 0.00064 The actual measurements are listed in Table 10. The per cent nitrogen as nitrate measured initially was slightly low. The initial ash per cent deviated almost 3 per cent from the expected value. This deviation resulted from some of the vermiculite settling to the bottom. Likewise, the calculations tied to the ash have a large deviation from their expected values. The average measured values were taken as the correct initial condition. The per cent of cellulose lost was calculated using TABLE 10 NITROGEN DETERMINATION CALCULATIONS Sample Per Cent Per Cent 0 Nitrate G Cellulose Day No. Nitrate Ash Nitrogen Per G Ash Nitrogen Per 0 Ash Calc. initial value 0.21 10.7 0.0197 1.97 0 1 0.192 7.8 0.0246 3.16 2 0.209 7.6 0.0276 3.29 3 0.195 7.3 0.0269 3.36 4 0.128 8.8 0.0202 2.69 Average 0.194 7.9 0.0248 3.12 3 1 0.131 8.8 0.0148 2.81 2 0.146 8.3 0.0175 3.09 3 0.128 8.3 0.0154 3.13 4 0.144 10.2 0.0142 2.21 Average 0.137 8.9 0.0155 2.81 5 1 0.031 9.4 0.0033 2.21 2 0.034 9.3 0.0037 2.33 3 0.025 9.8 0.0025 2.08 4 0.042 11.2 0.0038 1.61 Average 0.033 9.9 0.0033 2.06 7 1 0.015 9.9 0.0015 1.84 2 0.008 8.7 0.0010 2.21 3 0.018 9.2 0.0019 2.07 4 0.020 10.6 0.0019 1.49 Average 0.015 9.6 0.0016 1.90 47 the data in Table 10. The fraction of cellulose remaining at any time would be the measured cellulose per gram ash divided by the initial cellulose per gram ash. Thus, 9.9 per cent of the initial cellulose had been destroyed by the third day. likewise, 34.0 and 39.1 per cent of the cellulose had been destroyed by the fifth and seventh day. These three values are plotted in Figure 17. Nitrogen converted to an organic form was calculated by subtracting the value of remaining nitrate nitrogen per gram ash from the initial nitrate nitrogen per gram ash. This value was then expressed as mg nitrogen converted per gram initial cellulose. These calculations are tabulated in Table 11. The cumulative nitrogen converted per gram initial cellulose is plotted in Figure 18. Figures 17 and 18 are very similar showing that nitrogen conversion and cellulose destruction were nearly parallel. The maximum possible conversion was 15.2 mg nitrogen per gram initial cellulose as calculated from the initial nitrogen content. To get a rate curve of nitrogen conversion, the cumulative nitrogen conversion curve given in Figure 18 was taken to be correct. Points were taken from it as listed in Table 12 and changed into rates as mg nitrogen converted per gram initial cellulose per day. These rates are plotted in Figure 19. Gas analysis measurements were also made for this run. Results are given in Table 13. Since the weight of the mixture was always changing due to removal of samples, TABLE 11 CUMULATIVE NITROGEN CONVERSION 48 G Nitrate G Nitrogen MG Nitrogen Converted Days Nitrogen Converted Per G Initial Per G Ash Fer G Ash Cellulose 0 0.0248 0 0 3 0.0155 0.0093 5.7 5 0.0033 0.0215 13.2 7 0.0016 0.0232 14.2 TABLE 12 RATE OF NITROGEN CONVERSION (Data taken from Figure 18) MG Nitrogen Converted MG Nitrogen Converted Day Per G Initial Per G Initial Cellulose Cellulose Per Day 005 0.1 “'- l.0 0.4 0.6 1.5 1.0 1.2 2.0 2.0 2.0 2.5 3.5 3.0 3‘0 507* hell, 305 804 50‘.» 4.0 10.8 4.8 4.5 12.3 2.8 5.0 13.2* 1.8 5.5 13.6 0.8 6.0 13.9 0.6 6.5 14.1 0.4 7.0 14.2* 0.2 *Actual measurement TABLE 13 GAS ANALYSIS DATA FOR RUN NO. 7 49 C02 Produced 02 Consumed Oxygen Days Per G Initial Per 0 Initial Consumption Cellulose, Cellulose, Rate, Liters Liters mg/g in.ce11./day 0.5 0.0002 0 0 1.0 0.0003 0.0001 0.12 1.5 0.0008 0.0005 1.68 2.0 0.0017 0.0012 3.6 2.5 0.0034 0.0026 8.4 3.0 0.0073 0.0060 18.95 3.5 0.0141 0.0115 36.4 4.0 0.0199 0.0165 52.0 4.5 0.0212 0.0175 54.4 5.0 0.0173 0.0162 51.1 5.5 0.0164 0.0147 46.3 6.0 0.0147 0.0137 42.9 6.5 0.0141 0.0130 41.0 7.0 0.0137 0.0128 40.3 7.5 0.0136 0.0128 40.3* 8.0 0.0143 0.0135 42.5 8.5 0.0139 0.0132 41.5 9.0 0.0132 0.0119 37.4* 9.5 0.0113 0.0108 34,3 10.0 0.0101 0.0091 28.6** 10.5 0.0083 0.0075 18.0 11.0 0.0075 0.0064 15.4 11.5 0.0066 0.0060 14.4 12.0 0.0062 0.0057 13.7 12.5 0.0060 0.0052 12.5 13.0 0.0057 0.0050 12.0 13.5 0.0054 0.0045 10.8 *Addition of NaN03 **Addition of fresh filter paper I6~ I4" V I2 I0* ”G N: CONVERTED PER G INITIAL CELLULOSE 50 FIGURE I8. CUMULATIVE NITROGEN CONVERSION ______ '_. _. .— _— —- —— -—- INITIAL NITRerN CONTENT 1 IO r a u 2 D AYS m- FIGURE' I7. OXYGEN AND NITROGEN CONSUMPTION RATES I M6 N2. PER G INITIAL CELLULOSE pea DAY OXYGEN CONSUMPTION RATE J. o NITRATE ADDITION l NITRATE ADDITION )f/(w-m- 1 t: 0 CELLULOSE I ADDITION L x» O NITROGEN CONVERSION RATE n3 0 1 O .. I. ,. .. .. I. O p) p 6\ Q) 5 R? E MG oa PER G INITIAL CELLULOSE pan. DAY 51 these measurements were made as grams of 002 produced and liters of oxygen consumed per gram ash and converted to liters per gram initial cellulose. The rate of oxygen consumption was calculated in mg oxygen per gram initial cellulose per day and is plotted in Figure 19. The maximum rate of oxygen consumption was 54.4 mg per day per gram initial cellulose and occurred about 4.5 days after the start of the run. The maximum rate of nitrogen conversion was approximately 5.4 mg nitrogen per gram initial cellu- lose per day and appeared to precede the maximum rate of oxygen uptake by about one day. Since measurements indicated that nearly all the NaN03 had been used, it was thought that nitrate addition might have some effect. After 7.5 days, 0.139 grams of NaN03 in 14 ml of water were added to the mixture. A slight rise in oxygen uptake followed, but this rise corresponded to the normal pattern of other oxygen con- sumption curves. To check this, a second addition of 0.256 grams of NaNO3 in 23 ml of water were added on the ninth day. This had no stimulating effect on the curve. It is noted that the first addition raised the moisture content of the mixture by 2 per cent, and the second addition raised it an additional 4 per cent. 0n the tenth day, 6 grams of fresh filter paper were added. The oxygen con- sumption rate, which had been declining rapidly, leveled off but did not climb. The points of addition are marked on Figure 19. 52 The nitrogen conversion data was taken as a measure of the growth of bacteria in the mixture. The cumulative per cent of the total nitrogen available which had been converted is plotted on a semilogarithmic graph against time in Figure 20. The curve shows a nearly logarithmical rate of conversion followed by a leveling off period. This corresponds to the general behavior of a bacterial growth curve, indicating that nitrogen conversion can be con- sidered a measure of the growth of bacteria in the cellu- lose mixture. According to this curve the maximum growth of the microbial population was reached on the fifth day. This is in agreement with all other data on the activity of the culture including oxygen consumption rates and 002 production rates. If points are taken from the curve for total cellu- lose destruction in Figure 17, a plot of the rate of cel- lulose destruction can be made. This was done to obtain the curve in Figure 21. The maximum rate of cellulose destruction of 8 per cent of the initial cellulose per day appeared to occur about 3.5 to 4 days after the start of the run. This corresponds with the maximum rate of nitrogen conversion by the bacteria as shown in Figure 19, and precedes the maximum rate of oxygen consumption. The total weight of nitrogen converted and weight lost after extraction during run No. 7 are listed in Table 14 and plotted in Figure 22. From these data, grams of cellulose destroyed per gram of nitrogen assimilated PER CENT NITROGEN ' CONVERTED PER CENT \JEIGHT LOSS PER DAY 53 FIGURE 20. NITROGEN CONVERSION PLOTTED AS A GROWTH CURVE mo 90 so 70 60 50 ‘6 N 0 fig 6 <3 1 I ® - ACTUAL MEASUREMENT x = DATA TAKEN FROM FIGURE 18 b DAYS FIGURE ZI. RATE OF WEIGHT LOSS AFTER EXTRACTION T r I. \H' DAYS 54 FIGURE 22.. TOTAL NITROGEN CONVERSION AND CELLULOSE BREAKDOWN .125 TOTAL WEIGHT 0.6 LOST I305 CONVERTED Y Lu > o u: U m 0 20.3 ...I t: 'IIOF '3 r “0.2 <3 t I. Z ‘5 ) W :04 E < < E; a: . L J i 1 J L o 00 I a 3 s 6 7 s 9 o u DAYS 55 are calculated. After a week of incubation, 42 grams of cellulose had been destroyed per gram of nitrogen assim- ilated. TABLE 14 NITROGEN CONVERSION AND CELLULOSE LCSS Nitrogen Total Weight G Weight loss Per Days Converted, Loss, Grams G N2 Converted Grams 3 0.169 5.7 33.8 5 0.478 19.6 41.0 7 0.531 22.5 42.4 0. Variation of pH To check pH it was necessary to have mixtures which could be sampled. Large mixtures were prepared and ana- lyzed in parallel with gas analysis samples. The pH of samples taken from run No. 7 was: also measured. Results are tabulated in Table 15. The pH of the nutrient solution used in each mixture is listed. The pH of these mixtures started II?» about 8.2, which was slightly higher than the pH of the nutrient solution. The pH tended to increase during the run. This would explain the absence of fungi in the mixed culture seed, since a high pH favors aerobic bacteria over fungi. The dip in pH of run No. SA on the third day is the only indication of a pH change at the point of leveling off of 56 activity. The other runs showed no change in pH from the third to the fifth day. TABLE 15 VARIATION OF pH Sample: No. 2A* No. 5A* No. 7 Nutrient solution: 7.8 8.2 7.9 Days 0 8.2 803 709 3 8.6 7.6 8.2 5 8.6 8.0 8.2 7 8.4 8.5 8.8 9 8.6 --- --- 11 8.7 --— --- 14 9.0 --- --- * "A" indicates a large mixture used for sampling and agglyzed in parallel with the designated gas analysis H. Variation of.Respiratory Quotient An indication of the type of microbiological attack taking place is given by the respiratory quotient which is equal to the moles of C02 produced divided by the moles of oxygen consumed. Table 16 lists the average R. Q. for each day during runs No. 1 through 5. The variation is plotted in Figure 23 for runs No. l and 2. The R. Q. started out very high and decreased rapid- Lly. Normally it was nearly one by the fifth or sixth day Iand remained near one for the rest of the run. It dropped 57 slightly below one for the latter part of the run. The high R. Q. value during the first of the run indicated fermentation occurring. TABLE 16 VARIATION OF RESPIRATORY QUOTIENTS 4 No. No. 1 No. 2 No. 3 No. Sample: Day pl. 9 2. 27 1.29 1.19 1.11 1.10 1.09 9 .IM .1 8 235595455h 6 1110999999 lllllOOOOOnU. 6169189600080 4821099900090 0 O O O O O O O O O O O O 101 2111100011 95381985 7769 ”82200999 9999 .1111100000000 .05 5 7628785221332 2023 432210000099999999 00 lellllllanOOnUonwO 123145678901 11 RESPIRATORY QUOTI ENT RESPIRATORY QUOTIENT 58 FIGURE 23. DAILY RESPIRATORY QUOTIENTS RUN NO. I i zIII I 2.2 r 2.0? Li“ I.6* '04P V L2 1 LG 01 1 I . 4 0 u 8 I2. I6 20 24 DAYS RUN No. 2 I 2.0+ u . |.6I- 1.4. 1.2. I.O I- 0.! I2 I6 DAYS FI- co 59 I. By-products from Decomposition The amount of cellulose broken down completely into water and C02 can be calculated from the measured oxygen consumption. For every gram of oxygen consumed, 0.845 grams of cellulose will be oxidized. Likewise, the rate of cellulose oxidation can be calculated from the rate of oxygen consumption. The weight of cellulose decomposed by oxidation each day during run No. l is tabulated in Table 17. The cumulative per cent of cellulose oxidized is also listed. These quantities are plotted in Figures 24 and 25. Figure 24 is essentially the same curve as shown in Figure 10 for oxygen uptake rate since cellulose decomposed by oxidation is a direct conversion from oxygen consumption. These curves show that the rate of cellulose destruction by complete oxidation reached a maximum of 3.4 per cent of initial cellulose per day on the fourth day, then leveled off and declined rapidly after the eleventh day. Oxygen consumption rates for the other runs indicated a higher maximum rate of cellulose oxidation followed by a more rapid decline of activity. A maximum rate of 4.6 per cent of the initial cellulose per day was reached in run No. 4. According to oxygen consumption measurements, a total of 43.5 per cent or 7.90 grams of the initial cellu- lose in run No. l was decomposed by complete oxidation. The actual weight loss of the residue after drying at 105°C was 9.333 grams or 53.5 per cent as plotted in Figure 25. This leaves 1.43 grams or 8.2 per cent of the TABLE 17 CELLULOSE OXIDIZED IN RUN NO. 1 02 Consumed Cellulose Per Cent Cumulative Days Per Day, Oxidized, Per Day Per Cent Liters Grams 1 0.00674 0.00743 0.043 0.043 2 0.03395 0.0375 0.215 0.258 3 0.1794 0.198 1.13 1.39 4 0.461 0.509 2.92 4.32 5 0.531 0.586 3.36 7. 68 6 0.513 0.565 3.24 10. 92 7 0.540 0.596 3.42 14.34 8 0.518 0.572 3.28 17.62 9 0.4725 0.522 3.00 20.62 10 0.440 0.486 2.79 23.41 11 0.461 0.509 2.92 26.33 12 0.4405 0.486 2.79 29.12 13 0.3875 0.428 2.46 31. 58 14 0.347 0.383 2.20 33. 78 15 0.306 0.338 1.94 35 72 16 0.270 0.298 1.71 37. 43 17 0.224 0.248 1.42 38. 85 18 0.1884 0.208 1.19 40.04 19 0.1573 0.174 1.00 41.04 20 0.1366 0.151 0.867 41.91 21 0.1226 0.1355 0.778 42.69 22 0.1139 0.1255 0.720 43.41 23 0.1057 0.1165 0.670 44.08 24 0.0969 0.1070 0.615 44.69 25 0.09365 0.1033 0.594 45.28 61 FIGURE 2“. RATE OF CELLULOSE DECOMPOSITION BY COMPLETE OXIDATION PER CENT CELLULOSE OXIDIZED PER DAY 0 II 8 I2 I6 20 24 DAYS FIGURE 25. CUMULATIVE CELLULOSE OXIDATION FINAL wmur ® L055 8 Us 0 5 PER CENT CELLULOSE OXIDIZED N O 9 DAYS 62 initial weight not accounted for by oxidation of cellu- lose or remaining residue. Table 18 lists per cent of initial cellulose which was oxidized, per cent loss in weight after drying at 105°C and difference between loss of weight from cellulose oxidation and actual loss after drying for all runs made. Sample No. 6 was losthefore the final dry weight was determined. TABLE 18 CELIULOSE OXIDIZED IN RUNS NO. 1 THROUGH 6 ‘ Cellulose Weight Loss Difference Between Run Length, Oxidized, After Drying, Oxidation and Loss No. Days Per Cent Per Cent of Weight, Per Cent NL‘J-‘WV'I NHVJWNL‘ HQOH-INOUI NHDFJOan’ 3 8 4. 7 7 Oxnedwt0ha I-' p I WOONVI Evidence was found from variation of the R. Q. that fermentation occurred during the early part of each run. The relative amount of fermentation going on should be indicated by the amount of excess C02 being produced over that expected from complete oxidation. Figure 26 shows the excess 002 per gram initial cellulose per day produced during run No. 1. The data were computed from Table 5. 63 The rate of excess CO2 production reached a maximum of 15 mg per day per gram initial cellulose on the fourth day. After the tenth day, less moles of CO2 were being pro- duced than moles of oxygen consumed. This was probably due to the fact that during this period not only carbohy- drates were oxidized, but also other components such as proteins and fats, which may have been synthesized by the bacterial culture. Figure 27 shows the relative amount of cumulative excess C02 compared to the total C02 produced during run No. 1. After seven or eight days, the amount of excess 002 had leveled off and became less important to the total amount of decomposition. Data are listed in Table 19 showing the excess 002 at the end of the run for runs No. 1 through 6. The per cent of the initial cellulose weight represented by the amount of excess C02 is also listed. The full weight of the C02 molecule was taken to have come from the cellulose because all oxygen uptake from the air had already been considered in the 002 from oxidation; thus, the excess CO2 must have taken its oxygen content from the cellulose. The ratio of excess 002 to the grams of unaccounted weight loss after drying was calculated and listed in Table 19. The unaccounted weight loss was obtained by subtracting the weight of cellulose oxidized from the actual loss of weight after drying at 105°C. 64 FIGURE 26. RATE OF EXCESS C02 PRODUCTION IN RUN NOJ I6- INITIAL CELLULOSE PER DAY ,s \3 .. U Q C‘J U '11 ‘0 Lu .‘1 m L 9 I2 1: FIGURE 27. CUMULATIVE 003 PRODUCTION FOR RUN N0.I 6‘ 8|- . 3;. ‘2 TOTAL coa a O m 6 D U a a Q 0 e z a “I , I; N 8 e L. E 2‘ a. < I . a I Q, ,3 EXC ESS co, OI— :- 4 L A A L_ 0 H 6 8 I0 l2 DAYS 65 TABLE 19 EXCESS C02 IN RUNS NO. 1 THROUGH 6 Run Length, Excess 002, Per Cent Excess 002 Per No. Days Grams Initial Gram Unaccounted Weight Weight Loss, Grams 1 25 0.215 1.2 0.15 2 14 0.313. 1.8 0.21 3 14 0.415 . 2.4 0.19 4 14 0.490 2.5 0.21 5 8 1.205 7.0 0.68 6 a 0.905 5.3 t--- The results of Table 19 show that the excess 002 is quite significant for runs that lasted only 8 days but is rather small for the runs which lasted longer. If fermentation occurred, it would be expected that products other than CO would be formed that might be 2 volatile at 105°C. Tb check for these products, the vola- tile matter from run No. 5 was condensed and analyzed for carbon. The flask containing the sample was held at 105°C in a small oven, and all volatile matter evolved was condensed in a three neck flask outside of the oven. The noncondensable matter was exhausted through Drierite and Ascarite to collect C02. During the first 24 hours of drying, 0.339 grams of C02 were absorbed in the Ascarite. About 40 ml of color- less liquid had been condensed. A mixture of 25 ml of 0.25 N K2Cr207 solution and 75 m1 of concentrated H2304 66 were added to the condensed material. During the next 12 hours of drying an additional 20 ml of condensate were col- lected. The Ascarite tube absorbed 0.157 grams of 002 during ‘this period which could have come directly from the sample or from oxidation of carbonaceous material in the condensate. After all volatile matter had been collected, the condensate and acid-dichromate mixture was refluxed for three hours. An additional 0.015 grams of C02 were collected while refluxing. A total of 0.511 grams of 002 was collected as volatile products at 105°C, the majority of which evolved directly from the sample as 002. The total weight lost in run No. 5 was 27.3 per cent of the initial weight of cellulose. The cellulose lost by oxidation was 17.0 per cent. The weight of C02 from volatile products during drying represented about 3.0 per cent of the initial weight of cellulose, and therefore only a fraction of the 10.3 per cent difference between weight lost and cellulose oxidized. Gas analysis data for this run showed a total of 1.20 grams of C02 evolved over the amount expected from the com- plete oxidation of cellulose as calculated from oxygen con- sumption. This represents 7.0 per cent of the initial weight of cellulose. As shown in Table 20, cellulose oxidized, excess C02 evolved and 002 collected during drying gave a rough account of the dried weight loss in run No. 5. To determine the nature of the final dried residue of run No. 5, it was extracted with water, alcohol and ether. Extracts with water were done with hot water which cooled in the time necessary for filtration. Aliquot 67 TABLE 20 MATERIAL BALANCE OF WEIGHT LOSS AFTER DRYING Weight, Per Cent Initial Grams Weight of Cellulose Cellulose oxidized 2.94 17.0 Excess 002 . 1.20 7.0 002 as volatile products %f%% ngg Weight loss after drying 4.71 27.3 Difference 0.06 1 0.3 portions of the extracts were gried on a water bath to determine the weight of suspended and dissolved solids. Four 250 ml water extracts contained a total of 0.683 grams of solid material after subtracting the weight of nutrient salts. An alcohol extract of 300 m1 contained 0.096 grams of solid material. An ether extract of 300 ml contained 0.024 grams of solid material. A final cold water extract of 1500 ml contained 0.360 grams of solid material. The complete material balance for run No. 5 is given in Table 21. The residue after extractions was ashed at 800°C for three hours. The initial weight of vermiculite in the sample was 9 grams. The final ash weight was 8.8 grams. This checked exactly with the experimentally determined ash content of 97.8 per cent for vermiculite. 68 TABLE 21 MATERIAL BALANCE FOR RUN NO. 5 AFTER EXTRACTION Weight, Per Cent of Initial Grams Cellulose Weight Initial cellulose l .2 100.00 Cellulose oxidized 2.94 17.04 Excess 002 1.205 6.99 002 from drying 0.511 2.96 Solids in water extracts 1.043 6.05 Solids in alcohol extract 0.096 0.56 Solids in ether extract 0.028 0.1% 5. . Remaining cellulose 11.33 65.68 Total 17.15 99.42 Unaccounted weight 0.10 0.6 Figure 28 shows a plot of cellulose oxidized during run No. 5. The final weight losses before and after ex- traction are shown. The remaining material after extrac- tion was 11.33 grams giving a weight loss of 34.3 per cent. The per cent cellulose lost was calculated for run No. 7 using data in Table 10. The results are listed in Table 22 together with the cellulose oxidized as calculated from the oxygen consumption. These two quantities are plotted in Figure 29. Figure 29 shows that the total cellulose destruction curve began to level off before the cellulose oxidation curve. The curve also demonstrates that the fraction of cellulose oxidized became increasingly larger in time. 69 FIGURE 2?. CUMULATIVE CELLULOSE OXIDIZED IN RUN N0. 5 5 (.9 WEIGHT Loss AFTER EXTRACTION Lu 0 1 ® WEIGHT LOSS AFTER DRYING PER CENT WEIGHT LOST 3 CELLULOSE OXIDIZED IO 0 A L IO 6 DAYS FIGURE 29. CUMULATIVE CELLULOSE LOST IN RUN No.7 40- TOTAL WEIGHT LOST ,_ AFTER EXTRACTION ‘8 .J 4130‘ VI 0 .4 3 320T In 0 CELLULOSE E OXIDIZED Lu . 0 IO 2 In Q. 0 7* J j l 1 A 0 Z '4 IO DAYS 70 TABLE 22 CELLULOSE LOST IN RUN NO. 7 Days Per Cent Cellulose Per Cent Cellulose Lost - Oxidized 3 9.9 1.15 1+ --- [#025 5 34.0 8.0 6 --- 11.1 7 39.1 14.0 8 -“"" 1609 9 —-- 19.8 10 --- 21.8 An obvious by-product from cellulose decomposition is the cell material of the microorganisms. The amount of this material can be estimated from data on nitrogen con- version and approximate nitrogen content of dried bacteria. The "Handbook of Biological Data" (22) lists a range of from 8 to 14 per cent nitrogen content in the dry weight of bacteria. The average is about 10 per cent. In run No. 7, 0.538 grams of nitrate nitrogen had been converted to organic nitrogen by the seventh day. At 10 per cent nitrogen, this represents 5.38 grams of dried bacteria. To estimate the per cent of this weight which came from the nutrient components, the average ash content and nitrogen content of the bacteria were considered. The "Handbook of Biological Data" listed a range of from 4 to 14 per cent ash with an average of about 8 per cent. 71 Combining these two figures gives about 18 per cent of the dry weight of the bacteria due to nutrient salts. This leaves approximately 4.4 grams of weight due to assimilation of cellulose, which represents nearly 7 per cent of the initial cellulose. This is a significant amount and shows that the rapid climb of the total cellu- lose destruction curve could be due largely to the weight assimilated by the organisms. The growth of these micro- organisms would be expected to follow the nitrogen assim- ilation curve shown in Figure 22. The amount of material extracted with water after eight days for run No. 5 appears to be low, since it is only 6 per cent of the initial weight of cellulose and should account for both the weight of bacteria and the weight of by-products formed, such as mucilage, which would not be volatile at 105°C. A microscopic examination of a large mixture ran parallel with run N0. 5 showed growth of yeast and protozoa on the seventh day. It might be that formation of these microorganisms had started in sample N0. 5, and these would not pass through filter paper as easily as bacteria. Also, this sample had been dried before extraction which might have affected it. Of the four samples taken on the seventh day from run No. 7, the water extracts of two were centrifuged for 30 minutes at 3500 revolutions per minute. The substance which settled out was considered mucilage, some of the 72 bacteria and other high molecular weight by-products which had been extracted with water. The supernatant still had a cloudy white color, and a microscopic check showed it to be a suspension of bacteria. The first sample gave 0.046 grams of settled material from 0.875 grams of re- maining cellulose, and the second gave 0.041 grams from 0.796 grams of cellulose. This is 5.26 and 5.15 per cent or an average of 5.2 per cent of the remaining cellulose. This would amount to 1.8 grams based on the remaining weight on the seventh day. On the seventh day an excess of 2.9 grams of 002 had been evolved, and 8.1 grams of cellulose had been oxidized in run No. 7. From nitrogen conversion data, it was estimated that the bacteria had assimilated 4.4 grams of cellulose. If it is assumed that the 1.8 grams of centrifuged material contained a negligible amount of the bacteria, the combined weight of these four quantities is 17.2 grams and accounts for about 30 per cent of the initial cellulose. This is 9 per cent less than the measured loss of weight after extractions. Other losses of weight not considered are solids extracted by alcohol and ether, volatile products present and products which did not settle with centrifuging. 73 SECTION VI DISCUSSION As much as 50 per cent of the initial weight of cel- lulose has been lost over a twenty day period of attack by the cellulose decomposing microorganisms used in this study. By extracting the residue, it was shown that the actual breakdown of cellulose was greater than the weight loss after drying. At the maximum activity during run No. l, 3.5 per cent of the initial weight of cellulose was being oxidized per day. In run No. 4, a maximum rate of 4.6 per cent per day was reached and maintained for two days. Extraction data from run No. 7 indicated a maximum rate of over 8 per cent of the initial cellulose being destroyed per day. Thus, cellulose can be extensively de- composed under conditions simulating the compost process. The variation of NaNO3 concentration from 0.4 to 1.6 weight per cent resulted in a variation in amount of weight loss after drying from 17 to 48 per cent with the maximum occuring at 1.4 per cent NaN03 in the initial mixture. Reese (10) reported an optimum concentration of NaNO3 from 1 to 3 grams per liter in work with cellulose suspended in a liquid medium. The figure reported by Reese is about 0.2 per cent by weight and is not comparable with results of this study. 74 Nitrogen concentration was found to play a role in determining the rate of decomposition as shown in Figure 3. This is in agreement with observations made by Dubos (11); namely, that increasing concentration of NaNO3 increased the lag period before rapid growth of the bacteria. The presence of magnesium was found to be essential for growth of the organisms in this study. Reese (10) re- ported MgSOA to be important to the rate of decomposition in his work. Results for CaHPCh, FeSOh and K2HP04 showed only a small variation of amount of decomposed cellulose over the range of concentrations checked. Since nutrient components were varied from a stand- ard mixture, the curves of Figures 4 through 8 all have a point of identical composition. At these points it would be expected that the final weight loss of material in each experiment would be comparable. The actual amounts are listed in Table 23. TABLE 23 WEIGHT LOSS AT STANDARD COMPOSITION Component Varied Per Cent Weight loss NaNO3 42.5 MgSOh [+3 CaHPOh 47 KZHPOL 36 F6804 40 75 The results cover a range from 36 to 47 per cent or an 11 per cent Spread. This is not interpreted as the magnitude of experimental error for each curve since the data in most cases followed a relatively smooth curve. However, the data were collected over a period of four ' months, during which time the room temperature showed 5 to 10 degree variations; and the mixed culture seed may have changed to some extent. From gas analysis data it was shown that the weight loss of material after drying could be only partially explained by cellulose oxidation as calculated from oxygen consumption. Evidence was found from variation of the R.Q. that fermentation occurred during the early part of each run. It was expected that products volatile at 105°C would have been formed as a result of fermentation, but analysis of volatile matter during drying of run No. 5 showed only about 3 per cent of the initial weight of cellulose repre- sented by the 002 obtained from products evolved during drying. The majority of this C02 was collected directly from the sample rather than by oxidation of volatile products. Thus, it would appear that if many products of fermentation are formed, they must be used up during the course of further activity. The nitrogen conversion data was taken as a measure of the growth of bacteria in the mixture. From the amount of nitrogen converted, it was found that the weight of the bacteria cells formed during the decomposition process was 76 significant. On the seventh day the bacteria represented about 7 per cent of the initial weight of cellulose in run No. 7. This weight was not removed in samples that were dried in an oven. From considerations of fermentation and synthesis of bacterial cell material, it would be expected that the total cellulose destroyed would be greater than the cellu- lose oxidized in the earlier part of the run. During the phase of rapid growth of bacteria in the first 4 to 5 days, a relatively large portion of the cellulose broken down would be converted into cell material and into products of fermentation or of enzymatic degradation. Evidence that this was actually the case is given by the data plotted in Figure 29. The curve for cumulative cellulose destroyed climbed rapidly and leveled off sooner than the cumulative curve of cellulose oxidation. In run No. 7, the maximum rate of cellulose oxidation of 4.0 per cent per day was reached about 4.5 days after the start of the run as shown by the oxygen uptake rate plotted in Figure 19. The maximum rate of total cellulose loss appeared to be about 8 per cent per day and occurred about 3.5 to 4 days after the start of the run as shown in Figure 21. It is noted that the maximum growth of bacteria according to the nitrogen conversion data shown in Figure 19 nearly correSponds with the maximum rate of total cel- lulose destruction. The delayed maximum oxygen uptake rate is interpreted to be a result of its dependence on the 77 total number of bacteria present. The amount of cellulose destroyed per gram of nitro- gen assimilated was listed in Table 14. After a week, 42 grams of cellulose had been destroyed per gram of nitrogen assimilated. Heukelekian and Waksman (16) reported about 30 grams of cellulose decomposed in a liquid medium for every gram of nitrogen assimilated after 24 to 38 days of incubation. Shorter incubation periods gave less cellulose decomposition per gram of nitrogen assimilated. This ratio was higher for experiments done in sand and soil mediums. They reported as much as 43 grams of cellulose decomposed per gram of nitrogen assimilated after 2 weeks incubation in a soil medium. The ratio of total weight lost to grams of nitrogen assimilated found in this study are comparable to the soil medium as reported by Heukelekian and Waksman. The tendency for the ratio to become greater with increas- ing length of incubation was also observed. The maximum rate of cellulose destruction found under the conditions of this study was 8 per cent of the initial cellulose per day. Only limited data have been found in literature of the daily rate of cellulose destruc- tion in other experimental work. Reese (10) gave a curve showing the cumulative amount of cellulose destruction which climbed rapidly and leveled off after five days similar to the curve shown in Figure 17 of this study. The curve shown by Reese reached a maximum of 80 per cent decomposition before leveling off. He feund 75 per cent of 78 the cellulose remaining after 2 days and 50 per cent after 3 days. This would give a rate of 25 per cent of the initial cellulose per day and is three times the rate of maximum decomposition found in this study. Reese worked with bacteria and filter paper in liquid suspension. Attempts were made to determine the reason for lev- eling off of activity after three to four days of incuba— tion. It was found that addition of NaNOB and of the com- plete nutrient solution after reaching the point of maxi- mum activity had no stimulating effect. When clumping of the filter paper was prevented by spreading the paper discs on glass grids, the oxygen uptake rate still leveled off after four days. Addition of fresh filter paper was not found to be stimulating. The pH showed no significant change at the point of maximum activity. Perlin, Michaelis and McFarlane (13) reported con- centrations of 1.2 per cent 002 to petard growth of the organism with which they worked. Although this point was not Specifically checked, no evidence was found in the data of this study that the rate of cellulose decomposition was related to flow rate of concentration of oxygen or 002. However, the range of flow rates used in this study varied only from 0.175 to 0.5 liters per hour. None of these rates kept concentration of €02 below 6 per cent at the point of maximum activity. Low flow rates were necessary to get accurate oxygen consumption measurements. No definite con- clusion could be drawn as to the limiting factor for the rate of cellulose decomposition in this study. 79 SECTION VII CONCLUSIONS 1. Cellulose was extensively attacked under the conditions described in this study. 2. Maximum cellulose destruction occurred from three to five days after mixing the cellulose (filter paper) ‘with nutrient solution and bacterial seed. 3. The maximum rate of nitrogen assimilation by the bacteria occurred at nearly the same time as maximum cellulose destruction and slightly preceded the maximum rate of oxygen consumption. 4. Neither nutrient supply nor available surface appeared to be the limiting factor for the rate of cellu- lose decomposition. 8O BIBLIOGRAPHY Wiley, J. 8., and Pearce, G. W., "A Preliminary Study of High-Rate Composting," Proceedings ASCE, 81: Paper No. 846, Dec. (1955). Gotaas, H. B., "Composting Sanitary Disposal and Reclamation of Organic Wastes, World Health Organization, Geneva, (1956). Siu, R. G. H., "Problems and Speculations on the Decomposition of Cellulose by Fungi," New York Academy of Sciences, Transactions, 17: 38, I 19570 . Hutchinson, H. B., and Clayton, J., "On the Decompo- sition of Cellulose by an Aerobic Organism (Spirochaeta Cytophaga, N. Sp.),?” Journal of Agricultural Science, 9: 143, (1919). Waksman, S. A., and Skinner, C. E., "Microorganisms Concerned in the Decomposition of Cellulose in the Soil," Journal of Bacteriology, 12: 57, 1926 . Marsh, P. B., Bollenbacher, K., Butler, M. L., and Raper, K.B., "The Fungi Concerned in Fiber Deterioration, 11: Their Ability to Decompose Ciéiglose," Textile Research Journal, 19: 462, Norman, A. G., and Fuller, W. H., "Cellulose Decompo- sition by Microorganisms," Advances in Enzym- ology. 2: 293, (1942). Norman, A. G., and Bartholomew, W. V., "The Action of Some Mesophilic Bacteria on Cellulose," Soil Science Society of America, Proceedings, 5: 243, 1940). Viljoen, J. A., Fred, E. B., and Peterson, W. H., "The Fermentation of Cellulose by Thermophilic Bacteria " Journal of Agricultural Science, 16: l, (1926 . 10. ll. 12. l3. 14. 15. 16. 17. 18. 19. 20. 21. 81 Reese, E. T., "On the Effects of Aeration and Nutrition on Cellulose Decomposition by Certain Bacteria," Journal of Bacteriology, Dubos, R. J., "The Decomposition of Cellulose by Aerobic Bacteria," Journal of Bacteriology, 12: 223, (1928). Walker, F., and Warren, F. L., "Decomposition of Cellulose by Cytophaga," Biochemical Journal, 33: 31. (1938). Perlin, A. S., Michaelis, M., and NcFarlane, W. D., "Studies on the Decomposition of Cellulose by Microorganisms," Canadian Journal of Research, 25, Sec. 0: 240, 71947). Fuller, W. H., and Norman, A. S., "Cellulose Decom- position by Aerobic Mesophilic Bacteria from Soil," Journal of Bacteriology, 46: 281, (1943) Waksman, S. A., Soil Microbiology, John Wiley & Sons, Inc., New York, 111, (1942). Heukelekian, H., and Waksman, S. A., "Carbon and Nitrogen Transformation in the Decomposition of Cellulose by Filamentous,Fungi," Journal of Biological Chemistry, 66: 323, (1925). Nord, F. F., and Vitucci, J. 0., "Certain Aspects of the Microbiological Degradation of Cellulose," Advances in Enzymology,_8: 253, (1948). Boswell, J. G., "The Biological Decomposition of Cellulose," New Phytologist, 40: 20, (1941). Siu, R. G. H., and Reese, E. T., "Decomposition of Cellulose by Microorganisms," Botanical Review, _1_2: 377, (1953). Levinson, H. S., Mandels, G. R., and Reese, E. T., "Products of Enzymatic Hydrolysis of Cellulose and Its Derivatives," Archives of Biochemistry, 21: 351, (1951). Moore, A. F., "Oxygen Uptake Rates and ReSpiratory Quotients of an Aerobically Decomposing ' Synthetic—Garbage," Master' 3 thesis, Michigan State University, Department of Civil and Sanitary Engineering, (1958). 82 22. Spector, W. S., Handbook of Biological Data, W. B. Saungers Co., Philadelphia and London, 89, 195 . 83 APPENDIX PROCEDURE USED FOR NITRATE DETERMINATION The weighed sample (from 3 to 10 grams wet weight) was dissolved in 150 ml of distilled water. The suspended matter was filtered out and rinsed with 250 ml of distilled water. (The residue was further extracted with alcohol and ether, then dried and finally ashed at 800°C for three hours.) The water extract containing the nitrate was placed in a Kjeldahl flask. To this was added 75 m1 of saturated NaOH solution and glass beads to prevent bumping. Paraffin wax and paraffin oil were added to prevent frothing. The solution was boiled until BOO ml had been distilled over. (A final portion of this was usually checked to see if ammonia was still being evolved.) The solution was cooled and l g A1, 0.5 g Zn, 1 g Cu and 400 ml of distilled water were added. A blank was always ran and subtracted from final results as a correction for ammonia in the distilled water. 84 The solution was distilled, allowing several hours for reaction, until 300-400 ml of distillate had been collected in a measured amount of standard H2304 solution. The acid solution was titrated with standard NaOH to find the amount of acid used by the ammonia. The amount of nitrogen as nitrate was calculated as follows: g N2 = 1.4 X N Acid X m1 acid used up by the NH3 1ij my 1111 mm 303