World Journal of Microbiology & Biotechnology 13, 519525

Microbial activities during composting of pulp and paper-mill primary solids

C.F. Atkinson,* D.D. Jones and J.J. Gauthier
In this study, bench-scale aerobic reactors were used to monitor microbial activities and determine the degradability of pulp and paper-mill primary solids. Over the 43-day composting period, the level of microorganisms detected increased four-fold. However, based on amounts of CO2 produced, the increase expected was 19-fold, suggesting lysis of some cells in the population. The composition of the microbial population, determined through species identication by the BIOLOG method, and by spot plating, changed throughout the composting process with Cellulomonas spp. not appearing until day 22. Chemical analyses suggested a fall of 33% in the cellulose content, but no other statistically signicant chemical changes were observed. Key words: BIOLOG, degradability, enzymatic activity, microorganisms. Most pulp and paper-mills use primary clariers and some form of secondary treatment before releasing mill efuent into receiving waters. In 1988 treatment of efuent generated 2.3 million tons of dewatered sludge in the USA (Amberg 1988). Composting can be used to stabilize many solid wastes. Although a number of studies have documented composting of pulp and paper-mill sludges, few have specically examined primary sludge. In others, experimental details were omitted (e.g. Smyser 1982; Carter 1983). Valente et al. (1987) composted primary sludge and reported signicant cellulose degradation, but no distinction was made between cellulose degradation in the primary sludge and in the wood chips used as a bulking agent. Campbell et al. (1995) composted primary sludge mixed with tailings, wood ash, and cattle paunch. Although the C:N ratio was reduced from > 270:1 to 1467:1 and thermophilic temperatures were maintained for approximately 80 days, the compost was found to be immature. Successful composting of a solid waste is dependent on microorganisms producing specic enzymes to convert complex molecules into energy, cellular constituents, and metabolic endproducts (Golueke 1987; Senior & Balba 1990). Golueke (1987) suggested that determining the roles of microorganisms involved in composting could be used for process improvement. Most studies that characterize microbial populations associated with pulp and paper-mill wastes concentrate on microorganisms responsible for degrading one specic class of compounds or utilize isolated microorganisms (e.g., Fulthorpe et al. 1993). However, microbial consortia are known to degrade complex molecules more readily than microorganisms in isolation. The purpose of the present study was to monitor changes in activities of microbial consortia during composting of pulp and paper-mill primary solids using bench-scale reactors. Inorganic bulking agents (clay and vermiculite) were used to allow determination of the degradability of only the primary solids. Changes in the composition of the culturable microbial population were followed by use of a spot-plating technique and the BIOLOG system. Total microbial densities were monitored using epiuorescent microscopy. Fibrous components were measured at the beginning (day 1) and end (day 43) of composting to determine changes in recalcitrant compounds (i.e., lignin, cellulose, and hemicellulose).

Materials and Methods

C.F. Atkinson was, and D.D. Jones and J.J. Gauthier are with the Department of Biology, University of Alabama at Birmingham, 1300 University Boulevard, Birmingham, AL 35294, USA. C.F. Atkinson is now with NASA, Mail Code JJ-G, Kennedy Space Center, FL 32899, USA; fax: 407 853-4165. *Corresponding author.

Compost Mix and Processing Freshly collected, belt-ltered pulp and paper-mi ll prima ry solids (20% dry solids) were mixed with ground clay and vermiculite to adjust the dry solids content to 40% and supple-

1997 Rapid Science Publishers

World Journal of Microbiology & Biotechnology, Vol 13, 1997

C.F. Atkinson et al.

mented to adjust the C:N ratio to approx imately 40:1. The supplem ent contained a total of 0.99 l water, 69 g potassium phosphat e, 14.89 ml trace minerals (Greer et al. 1990), 94.3 g ammonium sulphate, 14.9 g magnesium sulphate, 8.0 g vitamin supplem ent (DVM, Inc.), and 7.0 g lactose. The mixture was placed into duplicate bench-sc ale reactors which have been described previously (Atkinson et al. 1996b). Two separate reactors were loaded with a mixture of unsuppl emented prima ry solids, clay, and vermiculite. The reactors were operated in a vertical position, rotated by 180 three times a week, and the inlet and outlet air supplies reversed to redistribute moisture throughout the reactors. After 21 days of operation the reactors were unloaded (on day 22), additional supplements [0.97 l water, 67 g potassium phosphate, 83.5 g ammonium sulphate, 8.5 g vitamin supplem ent (DVM, Inc.), and 7.5 g lactose] were added and the reactors were reloaded. Samples were removed periodically and processed as described previously (Atkinson et al. 1996b); on day 22 they were remov ed before the moisture content was adjusted. Biodegradability When compost ing small amounts of organic material, the amount of material lost is insufcient to be determin ed accurately by mass balance. Therefo re, biodegr adability of the primary solids was determin ed based on the relationship between the respirato ry quotient (RQ), g of substrate oxidized, and g of CO2 produced (Atkinson et al. 1996c). Microbial Assays Serially diluted samples were spread in duplicate onto tryptic soy agar (TSA) plates and incuba ted at 23 C (mesophil es) and at 55 C (thermoph iles). Individual colony types on the spread plates were isolated and prepared according to manufact urer's instructions for use in the BIOLO G microbi al identication system (Gram-pos itive and Gram- negative). Unknown microorganisms with a similarity index 0.500 are considered positive identi cations. Those with a similarity index < 0.500 are considered to be only metabolica lly similar to microorgani sms listed in the database. Three 1-ml portions of a serially diluted sample were stained with the uorochro me 4,6-diami dino-2-phen ylindole (DAPI) (0.1 mg/ml, 30 min contact time) (Porter & Feig 1980). A Leitz Diaplan microscope equipped with a 100-watt mercury bulb, a 340380 nm excitation lter (Leitz BP 340-380) with a 430 nm barrier/s uppression lter (Leitz LP 430) was used for examination of DAPI-s tained cells. A spot-plate technique was utilized to moni tor activities of the microbial commun ity on various macro molecules. This technique involved placing 10 ll of each serial dilution onto a minimal medium (Greer et al. 1990). Also, 1% (w/v) casein, 1% (w/v) a-cellulose, 1% (w/v) starch (soluble potato), Gram-ne gative cell walls (Pseudomonas aeruginosa), and Gram-positive cell walls (Bacillus subtilis) (Atkinson et al. 1996b) were supplemented with 0.025% yeast extract for spot-pla te counts. Levels of fungi were estimated by spot-plati ng on Sabour aud's agar. All plates were inoculat ed in duplicate and incubated at 23 C and at 55 C to enumer ate mesophiles and thermophile s, respectively. Plates were examined after 4 days of incubat ion. Level of growth/g dry solids (ds) was estimated by determin ing the most diluted sample from which microbial growth was observed. Potential enzymatic activity was based on the most diluted sample in which clearing of the medium around microorgani sms was observed. Only growth on plates that exceeded growth on a minimal medium with no added carbon source was considered positive. The sensitivity of this approach is 10-fold changes. Chemical Analyses The bre-content of compost samples was determin ed by the method of Goering & VanSoest (1970) which separates components into neutral- or acid- deterg ent-soluble fractions, cellulose, lignin, and ash. Hemicellulose content was estimated as the difference between neutral- and acid-detergent bres. Statistical Analyses Differences between means were statistically analyzed by AN OVA and/or t-tests using Microso ft Excel Version 5.0 after determining that appropria te statistical requiremen ts had been met. All a values were set at 0.05.

Results
Metabolic Activity and Biodegradability Metabolic activity, as measured by an increase in temperature and by CO2 production, did not increase signicantly in the reactors loaded with the unsupplemented compost mixture. Therefore, only data from the supplemented compost mixture are presented. The temperature in the reactors increased slowly to the thermophilic range up to day 13 (around 325 h), but then steadily decreased to the mesophilic range (Figure 1A). After the mix was re-hydrated, re-supplemented, and re-mixed on day 22, temperatures again reached the thermophilic range at about 850 h (day 35), then steadily declined. Mean CO2 (n = 2) produced from the mix was 303 g (SD = 64.4) before day 22 and 250 g (SD = 34.3) after day 22, a total of 553.3 g over the 43-day composting period (Figure 1B). Rates of CO2 production from the mix were between 0.5 and 0.8 g/h before day 22 and 1.0 g/h after day 22 (Figure 1C). Total biodegradability for the mix was calculated to be 0.22 (Table 1) (Atkinson et al. 1996c). Total Microbial Levels and Spot-Plating Counts of intact cells stained with DAPI showed a signicant increase in microbial density (p < 0.001) over the composting period (Table 2). The overall increase was about four-fold. On Gram-negative and Gram-positive cell-wall media, mesophiles were observed at relatively high levels (Table 3). Thermophiles were initially present at lower levels, increased until about day 22, then remained at relatively constant densities. Exoenzymes were observed at dilutions similar to growth on most sampling days. Protein and starch are readily degradable by numerous microorganisms. These media supported mesophilic growth and observable exoenzyme production at high dilutions throughout the experiment (Table 3). Ther-

Microbial activity in paper-mill compost

Table 1. Mean biodegradability and paper- mill primary solids. of volatile solids (n = 2) in pulp

Days 122 Total VS* (kg) RQ F CO2 produced (kg) Substrate lost (kg)G Biodegradability coefcient 1.48 0.92 0.30 0.17 0.11

Days 2243 1.27 0.92 0.25 0.14 0.11

* VS, volatile solids. F RQ, respira tory quotient calcu lated as moles CO2 prod uced/moles O2 consumed. G Substrate lost, grams CO2 produced/[(1.42 ) RQ)/0.247] (Atkinson et al. 1996c). Biodegradability coefcient, grams substrate lost/total grams VS.

Table 2. Mean cou nts of microorg anisms stained with DAPI (n = 4, 10 elds per lter)/g dry solids from composted pulp and paper- mill primary solids. Samples were remov ed on day 22 before supplementatio n. Day 1 8 15 22 29 36 43 * SD, standard deviation. DAPI counts 4.77 1.25 9.05 1.18 2.14 1.92 2.14 10 10 10 109 10 10 10 10 1010 1010
9 SD *

Figure 1. Representati ve proles of pulp and paper-mill primary solids during 43 days of compo sting. (A) Temperature ( C); (B) Total CO 2 prod uced (g). (C) Rate of carbon dioxide production (g/h).

6.70 2.05 1.94 2.56 3.56 3.29 2.30

10 10 9 10 9 9 10 9 10 10 9 10 9

mophilic growth and exoenzyme production, however, increased steadily (104105-fold) up to day 22. On cellulose plates observable mesophilic growth initially increased 104-fold between days 1 and 8, but then decreased to a relatively constant level similar to the

initial level (Table 3). Thermophiles were observed at their highest level on day 22. With the exception of day 8, exoenzymes were produced at levels similar to those of mesophilic and thermophilic growth.

Table 3. Level of microbial growth (log 10) / microorgan isms clearing medium (log 10) from pulp and paper-mill primary solids detect able on spot-plates after 4 days inc ubation at 23 or 55 C. Values repre sent the mean of three replicates and have been normalized to growth on minimal medium alo ne. Temper ature 23 C Day 1 8 15 22 29 36 43 1 8 15 22 29 36 43
4

Gram-negative 9/9 10/10 8/8 8/8 9/9 10/9 10/10 6/6 6/6 8/7 11/10 10/10 10/9 9/9

Gram-pos itive 9/9 10/10 9/9 8/8 10/10 10/9 9/9 7/7 8/8 8/8 10/10 9/9 9/9 9/9

Casein 10/10 9/9 9/8 8/8 8/8 11/10 9/9 5/ND 8/7 8/8 10/10 9/9 10/9 9/9

Starch 9/9 10/10 8/8 9/8 9/9 10/9 9/9 5/5 6/6 8/8 9/9 8/8 10/9 9/9

Cellulose 7/7 11/6 6/6 8/7 9/9 8/8 8/8 5/5 7/6 8/8 9/9 10/8 10/10 8/8

55 C

ND,

below limit of detection (10 ).

Table 4. Level of fungi (log 10) detect able on spot-plates incubated at 23 and 55 C from pulp and paper-mill primary solids. Day 1 8 15 22 29 36 43
ND,

Gram ) 5/ND 5/ND 7/ND ND/ND 7/ND 8/ND ND/ND

Gram +
ND/ND

Casein
ND/ND

Starch
ND/ND

Cellulose 5/ND ND/ND 6/6 7/ND 6/7 5/6 ND/ND

Sabouraud's 9/5 9/7 7/8 10/9 8/7 9/8 9/7

5/ND 8/ND ND/ND 7/ND 7/ND ND/ND

7/ND 8/ND ND/ND ND/5 7/ND ND/ND

4

7/ND 7/ND ND/9 7/7 8/7 6/ND

Bacillus coagulans, Xanthomonas campestris) (Table 6). Some organisms appeared most frequently in early to middle samples (e.g. Pseudomonas paucimobilis), whereas others appeared in middle to late samples (e.g. Acinetobacter johnsonii). Cellulomonas sp. were not detected in the early stages of composting, but appeared in mid to late samples. Chemical Analyses Fibrous components of the compost were analysed in the starting mix (day 1) and when the experiment was terminated (day 43) (Table 7). Dry weight of the compost mix decreased signicantly (t = 6.20, df = 2, p < 0.05). The unusually high ash content is a result of the bulking agents used. Cellulose content decreased signicantly (t = 12.63, df = 2, p < 0.05) by about 33%. No other statistically signicant changes were noted.

below limit of detection (10 ). Valu es (presented as level at 23 C/level at 55 C) represent the mean of three replica tes and have been normalized to growth on minimal medium alon e.

On each sampling day fungal growth on Sabouraud's agar indicated that high levels (up to 1010 propagules/g ds) were present in the compost (Table 4). However, on macromolecular substrates growth by mesophilic and thermophilic fungi was observed in only 22 and seven of 35 subsamplings, respectively. BIOLOG Identications To further investigate changes in subpopulations during composting, a representative of each visually different colony type on TSA spread plates was subcultured for identication by BIOLOG. A total of 497 isolates were selected for identication. Depending on the sampling day, between 51 and 100% of the microorganisms growing on spread plates could be subcultured (Table 5). In most of the samples (six out of seven), the majority of organisms were Gram-negative. Examination of the BIOLOG data showed that some organisms (or at least organisms that were metabolically similar to the named organisms) were present at varying frequencies throughout the composting process (e.g.

Discussion
Metabolic Activity Increases in temperature reect active decomposition of organic matter by microorganisms (Richard & Zimmerman 1995). Wastes of high lignocellulose content are known to degrade slowly, the rate being dependent on environmental factors (N'Dayegamiye & Isfan 1991), but can be composted successfully with proper aeration, moisture, and nutrients (Campbell et al. 1990). Thus, in the present bench-scale study, CO2 production and increases in temperature (Figure 1) suggest that nutrient levels were adequate and indicate degradation of organic substrates in the compost mix. Biodegradability coefcients in the present study reect that decomposition was low. Metabolic activity in the compost mix was stimulated by nutrients and further enhanced when the compost was re-mixed and additional nutrients were supplied on day 22. This indicates that physical and/or nutritional conditions within the

Table 5. Results of BIOLOG analysis of colonies isolated from spread plates of pulp and paper- mill solids. Day Total colonies* % Total no F regrowt h 6 0 49 14 7 21 15 % Total colonies Gram-po sitive 5 36 9 19 32 54 34 % Total colonies Gram-negative 89 64 42 67 61 26 51 % Grampositive G (23 C) 5 36 4 19 25 40 31 % Grampositive G (55 C) <1 <1 5 <1 7 14 4 % Gramnegative (23 C) 85 63 22 26 35 12 34 % Gramnegative (55 C) 4 1 20 41 25 14 17

1 8 15 22 29 36 43

4.13 1.34 1.01 1.07 2.92 2.39 1.48

10 1010 109 1010 1010 1010 1010

* Total colonies, mean (n = 8) colonies growing on TSA spread plates. F % Total no regrowth, % total colonies that could not be recultured after initial spread plate counts. G % Gram-positive (23 C) and (55 C), % Gram-positive organisms classed as mesophiles and thermophile s, respectively. % Gram-n egative (23 C) and (55 C), % Gram-negative orga nisms classed as mesophiles and thermo philes, respectively.

Table 6. Partial listing of microorganisms Organism Acinet obacter johnsonii Alterom onas haloplanktis Bacillus coag ulans Bacillus insolitus Brucella abortus Cellulomo nas cartae Cellulomo nas cella sea Corynebacter ium bovis Klebsiella pneumoniae Klebsiella terrigena Pseudomon as mendocina Pseudomon as paucimobilis Xanth omonas campestris
ND,

from BIOLO G study. Day 8

ND /ND ND /ND ND /ND

Day 1
ND /ND

Day 15
ND /ND ND /ND

Day 22
ND /ND ND /ND ND /ND

Day 29
ND /ND

Day 36 2/<1
ND /ND

Day 43 <1/<1 2/1 <1/ ND 1/ND 28/ND 1/ND ND/ND ND/ND ND/<1 <1/<1 ND/ND ND/ND <1/ ND

1/ ND ND /ND ND /ND 23/ND ND /ND ND /ND ND /<1 20/ND ND /ND ND /ND <1/14 ND /ND

<1/ ND 1/1 ND /ND ND /ND ND /8 2/ND <1/<1 5/10 11/<1 ND /3

<1/ ND 4/ND <1/ ND ND /ND ND /ND ND /ND ND /ND ND /ND ND /ND ND /ND 2/ND

3/ND 14/ND <1/ ND <1/<1 ND /ND ND /ND ND /ND ND /ND ND /ND 3/5

2/3 2/ND <1/ ND ND /20 6/ND ND /ND ND /ND ND /<1 <1/ ND 2/ND 1/ND ND /3

<1/ ND 14/ND <1/ ND 5/ND 16/ND ND /ND ND /<1 ND /ND ND /ND ND /ND <1/ ND

not detected. The rst number in each column represents the perce nt of all microo rganisms named that sampling day that were identied (Similarity Index > 0.5) as the named microo rganism. The second number in each column represents the percent of all microo rganisms named that sampling day that were metabolically (SI <0.5) similar to the named microorgani sm.

mix change during composting and slow the composting process. Further degradation of the compost may have been possible with subsequent re-supplementation, rehydration, and re-mixing. The moisture content of the compost mix was initially 60%. By day 22 (when the reactors were unloaded, remixed, and re-hydrated), moisture content had decreased by about 10%. Between days 22 and 43, moisture content decreased by about 2%. If the composted sludge were to be hauled for land application or other agricultural use, a substantial decrease in moisture content would reduce hauling costs as has been noted by Campbell & Tripepi (1992). Based on the results of the present study, transportation cost reductions would be unlikely to offset the capital investment required for invessel composting of this solid waste. Levels of Microorganisms Many studies involving pulp and paper-mill solid wastes do not measure levels of microorganisms (Carter 1983;

Valente et al. 1987; Diehn & Zuercher 1990; Campbell et al. 1995). Because composting is dependent on the activities of microorganisms, measuring levels of microorganisms or functional community changes should be an integral part of monitoring (Insam et al. 1996). Enumeration of microorganisms by serial dilution and spread-plating on agar media is known to underestimate the total level of microorganisms in environmental samples (Chung & Neethling 1988; Palmisano et al. 1993). Levels of microorganisms in the present study were determined independently of growth requirements by staining samples with DAPI and enumerating cells (Table 2). Increases were about four-fold. However, based on amounts of CO2 produced, the average respiratory coefcient of the material oxidized, and an assumed yield coefcient of 0.5, the number of cells detectable should have increased 19-fold. This may be explained if production of a portion of the CO2 were due to lysis of some of the cells in the population as has been reported in other solid wastes (Atkinson et al. 1996a, 1996b). Spot-Plating Shifts in subpopulations and potential metabolic activities within the overall microbial population have been demonstrated in composted oxidation-ditch sludge, poultry litter, municipal solid waste (Atkinson et al. 1996a, 1996b, 1996c, respectively), and sawdust (Atkinson et al. 1996a, 1996b). To investigate such shifts in composted primary solids, we used the spot-plating method described previously (Atkinson et al. 1996b) which permits growth of microbial consortia rather than isolated microorganisms. Enzymatic activities of microorganisms are known to differ under experimental and natural conditions (Faure & Deschamps 1991). Thus,

Table 7. Fibrous solids. Component Dry solids Ash Cellulose Lignin Hemicellul ose therF

components

of pulp and paper-mill

primary

Initial kg 6.2 3.4 0.9 0.5 0.2 1.1

Final kg 5.9 3.4 0.6 0.5 0.3 1.1

D )0.3* )0.3* +0.1

* Indicates statistically signicant change (t-test). Valu es repres ent the mean of three replicates. F Other, components not listed.

exoenzyme production on agar plates does not demonstrate production of enzymes in the compost itself, but does indicate that microorganisms with the potential to produce specic exoenzymes are present. Microbial growth and exoenzyme production on cellwall media show that there is the potential for degradation of microbial biomass throughout the composting process. This potential has been reported in oxidationditch sludge and poultry litter composts (Atkinson et al. 1996a, 1996b, respectively). Subpopulations capable of degrading microbial cell walls may increase or decrease in response to changing environmental conditions as a means of recycling nutrients (Golueke 1987). Cellulose-degrading exoenzymes were produced on solid media throughout composting, but production increased as composting progressed. Others have reported little cellulose degradation during the early days of composting (Faure & Deschamps 1990). Thus, it would be expected that microorganisms producing cellulosedegrading exoenzymes would increase later in composting. Low levels of fungi observed on spot plates indicate that fungi are not able to compete effectively with bacteria on agar media containing the various macromolecules. In composted simulated municipal solid waste, Palmisano et al. (1993) concluded that fungi did not appear to be dominant members of the microbial community. N'Dayegamiye & Isfan (1991) also found that fungal levels were lower than other microbial groups (bacteria and actinomycetes) in wood shavings, sawdust, and peat moss composts. BIOLOG When environmental samples are plated onto TSA, it is not uncommon to observe colonies that cannot be transferred to fresh TSA agar. This inability to transfer may be due to growth factor requirements or to physical conditions not available in the medium (e.g., proximity to other cells). The changing percentage of cells incapable of regrowth (Table 5) supports the concept of shifts in subpopulations. Identications in the BIOLOG system are based on metabolic patterns in 96-well plates containing dehydrated substrates. In a study of pulp and paper-mill wastewater, Fulthorpe et al. (1993) reported that a majority of microorganisms isolated did not match the BIOLOG database. This was also our experience. For example, on every sampling day an organism was named by BIOLOG as Brucella abortus biovar 2. When this microorganism was identied by gas chromatographic analysis of the fatty acid prole of the microorganism, the microorganism almost always identied as Xanthomonas maltophila (Atkinson, data not shown).

One objective of composting is to `sanitize' solid wastes, that is to reduce levels of pathogenic microorganisms, larvae, and weed seeds (Finstein & Morris 1975), which is accomplished by maintenance of thermophilic temperatures for a specic period of time. Klebsiella pneumoniae, a human pathogen, and other Klebsiella species were isolated frequently from primary solids compost. Klebsiella species have also been isolated from pulp and paper-mill machinery, clariers, and efuent (Va a ta nen & Nimel a 1983; Fulthorpe et al. 1993). Va a ta nen and Nimel a isolated 134 strains of Klebsiella from a paper mill in Finland and found that elevated temperature (above 55 to 60 C) signicantly reduced levels. In our study, thermophilic temperatures were maintained for only a short period of time (Figure 1A). Therefore, `sanitization' of pulp and paper-mill primary solids might require cocomposting with a waste likely to generate sufcient metabolic heat (e.g., municipal solid waste or sewage sludge) to destroy pathogenic micro- organisms. In a study of bark composts, Campbell et al. (1990) found that the majority of culturable microorganisms were Gram-negative thermophiles. In our study most of the total isolates were Gram-negative mesophiles; in only three samples were thermophiles present in higher proportions (Table 5). The difference in results may be due to the relatively short period of time that thermophilic temperatures were maintained in the compost (Figure 1A). The composition of the microbial population changed during composting as was indicated by changes in identications suggested by the BIOLOG system. Some organisms were present throughout composting (e.g., Klebsiella sp.) while other organisms appeared only at certain times (e.g., Cellulomonas). These results agree with those of others who have reported microbial succession during composting (Faure & Deschamps 1990; Palmisano et al. 1993). Chemical Analyses Degradation of primary solids during composting was considerably lower than municipal solid wastes (Atkinson et al. 1996c). The difference may be related to the form of cellulose in the wastes. Cellulose in municipal solid waste is more puried (e.g., paper) than cellulose in primary solids (lignocellulose) and may, therefore, be easier for the microbial population to degrade. Although not statistically signicant, the apparent change in hemicellulose content (+0.1 kg) likely reects degradation of larger macromolecules into forms extractable in the hemicellulosic fraction. A similar increase was also observed in composted municipal solid waste (Atkinson et al. 1996c).

Conclusions
Biodegradability of pulp and paper-mill primary solids was low in aerobic bench-scale reactors, suggesting that in-vessel systems may not be appropriate for composting this type of waste. Organic material in the compost mix was degraded with production of energy and microbial biomass. Subpopulations of microorganisms increased and decreased continuously as was observed on spot plates, by changes in Gram reactions, regrowth capability, by BIOLOG identications, and by counting DAPIstained cells. The potential for microbial cell wall degradation, as well as readily degradable macromolecules like starch and casein, exists throughout the composting period.

Acknowledgem ents
The authors would like to express their sincere appreciation to Gloria Robinson, John Bentley, Stepanie McElwain, and Dennis Gentle for technical assistance. Bob Wilson provided helpful advice. This research was supported in part by PWT Waste Solutions, Birmingham, Alabama, and the Department of Biology, University of Alabama at Birmingham, Birmingham, Alabama.

References
Amberg, H. 1988 Sludge dewatering and disposal in the United States pulp and paper industr y. NCASI Technical Bulletin No. 552. Atkinson, C.F., Jones, D.D. & Gauthier, J.J. 1996a Biodegra dabilities and microbial activities during compost ing of oxidation ditch sludge. Compost Science and Utilization 4(1), 84 96. Atkinson, C.F., Jones, D.D. & Gauthier, J.J. 1996b Biodegra dability and microbi al activities during composting of poult ry litter. Poultry Science 75, 608617. Atkinson, C.F., Jones, D.D. & Gauthie r, J.J. 1996c Biodegra dabilities and microbi al activities during composting of municipal solid waste in bench-sc ale reactors. Compost Science and Utilization, 4(4), 1423. Campbell, A.G. & Tripepi, R.R. 1992 Logyard residues: products, markets, and research needs. Forest Products Journal 42(9), 60 64. Campbell, C.D., Darbyshi re, J.F. & Anderso n, J.G. 1990 The compost ing of tree bark in small reactors self-heating experimen ts. Biological Wastes 31, 145161. Campbell, A.G., Zhang, X. & Tripepi, R.R. 1995 Composti ng and evaluating a pulp and paper sludge for use as a soil amend ment/mulch. Compost Science and Utilization 3(1), 84 95.

Carter, C.N. 1983 Composting disposes of sludge, yields byproduct at Glatfelte r. Pulp Paper 57(3), 102104. Chung, Y.-C. & Neethling, J.B. 1988 ATP as a measure of anaerobic sludge digest er activity. Journal Water Pollution Control Federation 60, 107112. Diehn, K. & Zuercher, B. 1990 A waste management program for paper mill sludge high in ash. Tappi Journal 73(4), 8186. Faure, D. & Deschamps, A.M. 1990 Physico- chemical and microbiolog ical aspects in compos ting of grape pulps. Biological Wastes 34, 251258. Faure, D. & Deschamps, A.M. 1991 The effect of bacterial inoculation on the initiation of compos ting of grape pulps. Bioresource Technology 37, 235238. Finstein, M.S. & Morris, M.L. 1975 Microbiolo gy of municipal solid waste compos ting. Advances in Applied Microbiology 19, 113151. Fulthorp e, R.R. Liss, S.N. & Allen, D.G. 1993 Characteriza tion of bacteria isolated from a bleached kraft pulp mill wastewa ter treatment system. Canadian Journal of Microbiology 39, 1324. Goering, H.K. & van Soest, P.J. 1970 Forage ber analyses. In USDA Agricultural Handbook 379. pp. 120 Washingt on, DC: USDA. Golueke, C.G. 1987 Biological Reclamation of Solid Wastes. Emmaus, PA: Rodale Press. Greer, C.W., Hawari, J. & Samson, R. 1990 Inuence of environmen tal factors on 2,4-dichl orophenox yacetic acid degradation by Pseudomonas cepacia isolated from peat. Archives of Microbiology 154, 317322. Insam, H., Amor, K., Renner, M. & Crepaz, C. 1996 Changes in functional abilities of the microbi al community during composting of manure. Microbial Ecology 31, 7787. N'Dayegami ye, A. & Isfan, D. 1991 Chemical and biological changes in compost of wood shavin gs, sawdust and peat moss. Canadian Journal of Soil Science 71, 475484. Palmisan o, A.C., Marusc ik, D.A., Ritchie, C.J., Schwab, B.S., Harper, S.R. & Rapapor t, R.A. 1993 A novel bioreactor simulating composting of municipal solid waste. Journal of Microbiological Methods 18, 99117. Porter, K.G. & Feig, Y.S. 1980 The use of DAPI for identifying and counting aquat ic microor a. Limnology and Oceanology 25, 943948. Richard, D. & Zimmerman, R. 1995 Respiration rate reheati ng potential: a comparison of measur es of compost stability. Compost Science and Utilization 3(2), 7479. Senior, E. & Balba, M.T.M. 1990 Refuse Decompositi on. In Microbiology of Landll Sites, ed Senior, E. pp. 1757. Boca Raton: CRC Press, Inc. Smyser, S. 1982 Compost paying its way for paper producer. BioCycle 23, 2526. Va a ta nen, P. & Niemel a , S.I. 1983 Factors regulating the density of bacteria in process waters of a paper mill. Journal of Applied Bacteriology 54, 367371. Valente, C.A., Vaz, A.R. & de Carvalh o, A.P. 1987 Compo sting pulp mill sludge. BioCycle 28, 4649.

(Received in revised form 24 September 1996; accepted 11 October 1996)