Changes in microbial population numbers during the composting of pine bark

Bioresource Technology39 (1992) 85-92

Changes in Microbial Population Numbers During the Composting of Pine Bark C. L. Davis Department of Zoology, University of Cape Town, Private Bag, Rondebosch 7700, Republic of South Africa

S. A. Hinch, a C. J. D o n k i n a & P. J. G e r m i s h u i z e n b ~Department of Microbiology and Plant Pathology, bDepartment of Horticultural Science, University of Natal, PO Box 375, Pietermaritzburg 3200, Republic of South Africa (Received 20 November 1990; revised version received 9 January 1991; accepted 13 January 1991 )

Abstract

Microorganisms associated with the composting of pine bark were enumerated by plating. Microbial numbers increased concurrently with temperature when composting of the bark was initiated by nitrogen addition, and remained high during stabilisation of the compost. The numbers of bacterial colony forming units (CFU) were higher than fungal CFU throughout the composting process, of both mesophilic and thermophilic microorganisms (grown at 30 and 60°C, respectively). Urea and chicken litter additions to bark resulted in greater bacterial numbers than nitrate addition. Mesophiles and thermophiles were fairly evenly distributed throughout the compost heaps. It is suggested that many of the microorganisms bringing about pine bark composting are thermotolerant. They may not only be able to survive a wide range of temperatures, but also to grow actively at temperatures betwen 25 and 60°C. Key words: Composting, pine bark, urea-, chicken litter-nitrogen-addition, microbial population changes, thermotolerance. INTRODUCTION The disposal of various solid wastes is an everincreasing problem, both financially and environmentally. Composting is used as a treatment method for some wastes, and the process yields a stable, profitable product. The term composting

has been defined by Crawford (1983) as 'the incomplete, artificially acccelerated, decomposition of heterogenous organic matter by a mixed microbial population in a warm, moist environment'. Studies have been carried out on the microorganisms involved in the composting of a number of solid wastes, for example municipal sewage sludge (McKinley & Vestal, 1985) and wheat straw (Chang & Hudson, 1967; Fermor et al., 1979). Pine bark is an organic waste generated in large amounts by the timber industry. This waste has been found, after composting, to be suitable for use as a growing medium for vegetable, citrus and forestry seedlings, and other plants. Pine-bark compost has good physical properties, and has the added advantage of being suppressive to numerous plant diseases (Hoitink & Fahy, 1986). As a result, the composting of pine bark is practised commercially on a large scale in some countries. A typical composting cycle usually includes the following processes. The coarsely milled ('hogged') bark is piled into heaps of approximately 500 m 3, and left to 'age'. Thereafter water and nitrogen (which is usually in the form of urea) are added to initiate composting. Temperatures within the heaps rise dramatically, with peak temperatures sometimes as high as 80°C. Subsequently the heaps are watered and 'turned' or mixed weekly for aeration, for a period of about three months as composting continues (stabilisation). The finished product is then screened to obtain particles of a uniform size, and then distributed.

85 Bioresource Technology 0960-8524/92/S03.50 © 1991 Elsevier Science Publishers Ltd, England. Printed in Great Britain

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C. L. Davis, S. A. Hinch, C. J. Donkin, P. J. Germishuizen

In South Africa, problems were initially experienced with the product, as some batches of compost would grow excellent seedlings, while others would give poorer results. Much research has been carried out to determine the physical and chemical changes that occur in the bark during composting (Bunt, 1988), but little is known about the microbiology of the process. As Crawford's (1983) definition of composting indicates, microbes are the biological catalysts in the process. Thus a study was undertaken with the following aims: to develop a method for monitoring the changes in the number of microorganisms during composting; to determine what happens to numbers of mesophiles and thermophiles during a composting; to examine the effect of various nitrogen sources on composting and microbial numbers; and to determine whether aging of the raw bark has an effect on composting. The results presented in this paper demonstrate the vital role microorganisms play in the composting of pine bark.

METHODS Composting facilities Composting of pine bark was performed at a commercial factory in Natal, South Africa. Compost stacks were maintained in the open, and thus exposed to the prevailing weather conditions. Large commercial heaps had the dimensions 50 m × 6 m at the base of the stack and were up to 3 m high.

Composting A mixture of the bark of three pine species was used routinely for composting: Pinus patula, P. elliottii and P. taeda. Composting was performed as described in the Introduction, with 4 kg urea per m 3 bark being used to initiate composting in the commercial heaps. Water was used to run off at the beginning, and aeration was achieved by turning the heaps weekly with a payloader followed by further water addition. At the end of composting, the product was sieved through a 10 nun mesh screen. Temperatures of the heaps were monitored during the process using thermometers inserted at different heights in the heaps and read after 5 min.

Compost samples At sampling times a composite sample from approximately six sites within a heap was assem-

bled, mixed and sieved through a 4 mm mesh screen. The bark was transported to the laboratory and tested within 4 h.

Isolation media Tryptic soy agar (TSA) was made by Dffco (Detroit, Michigan, USA). Peptone agar (PA) contained 0"5% peptone (Difco), 0.1% yeast extract (Difco) and 1.5% agar in distilled water. Cycloheximide (50 mg/litre final concentration), where used, was added to TSA and PA after they were autoclaved. Peptone dextrose agar (PDA) and Rose Bengal agar (RBA) were made as described in Booth (1971), and streptomycin (50 mg/litre) and aureomycin (35 mg/litre final concentration), respectively were added to them after autoclaving.

Standard isolation procedure Ten grams (wet weight) of a compost sample was shaken at 28°C for 30 min in 50 ml of sterile distilled water on a rotary shaker set at 150 rpm. Thereafter the bark chips in the sample were removed by coarse filtering through muslin. Dilutions of the filtrate were made in distilled water and three appropriate dilutions were plated. TSA was used for enumerating and isolating mesophiles, and the plates were incubated at 30 or 37°C for 2 days and at 25°C for a further 3 days before the colonies were counted. Thermophilic microorganisms were grown on PA at 60°C for 18-20 h and counted.

Other isolation procedures Experiments were performed to check the effect of different variables in the isolation procedure. The number of bacteria in the filtrate after various time periods of shaking was monitored. Phosphate buffer (Na2HPO 4, 2.1 g/litre; KH2PO4, 1-09 g/iitre) with 150 rnM NaC1 (pH 7.0), was tested as the suspending medium and diluent. Finally, three 'extraction' methods were compared (in each case using 10 g bark in 50 ml of water): (a) shaking at 150 rpm for 30 min; (b) homogenisation of bark in water in a commercial food blender set at the highest speed, followed by shaking as in (a); and (c) sonication for three bursts of 1 min on ice, followed by shaking as in (a).

Anaerobic growth PA plates were incubated anaerobically in a Gaspak jar at 60°C for 1 week and counted. TSA plates were incubated in an anaerobic glove box (Forma Scientific, Marietta, Ohio), with the incubator set at 35°C.

Microbial numbers during the composting of pine bark

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RESULTS

Isolation procedures TSA and PA were compared as growth media for mesophiles and thermophiles, and gave similar results for the two kinds of organisms. TSA was chosen for growing mesophiles (Strom, 1985), and PA for thermophiles. The latter choice was made as there were fewer spreading colonies on this medium, although the total number of colonies remained the same. Phosphate buffer and water as suspending and diluting medium, respectively, gave similar results; thus water was routinely used. Three methods of 'removing' microbes from the bark for enumeration were compared. The method adopted as standard (that is, shaking in water for 30 min) yielded 6.0 x 1 0 9 mesophilic C F U per g of a test compost sample, and 2.9 x 108 thermophiles. The other methods of homogenisation and sonication gave 2.7 x 1 0 9 and 2.5 x 1 0 9 mesophiles, and 3.0 x 108 and 3-7 x 108 thermophiles per g compost, respectively. A 30 min period of shaking was chosen after shaking times of 25 s, 5 min, 10 min and 30 min were tested. The results were 9.5 x 107, 2.8 x 108, 3.0 x 108 and 3.7 x 108 mesophiles per g compost for these time periods, respectively.

9

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0

I 40

20

I 60

I 80

I 100

DAYS

Fig. 1. Numbers of mesophiles (e) and thermophiles (o) present during composting of bark in a commercial-scale heap. Day 0 indicates the time of urea addition.

~

.ERMOP.I'ES

Typical commercial compost heap The numbers of mesophilic and thermophilic C F U present during the composting process are shown in Fig. 1. Mesophiles outnumbered the thermophiles at all times, and both groups underwent a striking increase in numbers, concommitant with the increase in heap temperature, on the addition of urea to the heap. Mesophile and thermophile numbers stabilised after about 3 weeks.

IO n

~E 0 0

\ ii

Types of microflora A n attempt was made to quantify the fungal propagules present during composting, under conditions where bacterial growth was inhibited on the plates (Fig. 2). The selective media used for fungi included PDA with streptomycin, and RBA with aureomycin. Despite the antibacterial agents, bacterial or yeast colonies arose on some of the plates, but in greatly reduced numbers. Mesophilic and thermophihc fungal C F U were found at all stages of composting, but in lower numbers than bacteria growing on plates with the fungal inhibitor. PDA and R B A allowed growth of similar numbers of fungal CFU. It was found that if

0

2

4

6

0

2

4

6

WEEKS

Fig. 2. Numbers of microorganismsin bark during 7 weeks of composting, with mesophiles and thermophiles shown separately. Bacteria were grown on TS (e) and TS+cycloheximide (o). Fungi were cultured on PDA+streptomycin ( • ) and RBA+ aureomycin ( zx). TS and PA were also incubated anaerobically (n) at 35 and 60°C respectively.

88

C L. Davis, S. A. Hinch, C J. Donkin, P. J. Germ&hu&en MESOPHI LES

7 T H E R M O P H I L ~

ku) 0

lm

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Fig. 3. Profile of compost heap showing sample sites referred to in Table 1. The gap in the middle shows where the heap was dug out to obtain the internal samples C, D and E.

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Table 1. Distribution

of mesophilic bacteria in a pine bark compost heap

Sample sitea

A B C D E F

Temperatureof site (°C) 25 55 57 58 61 64

and

thermophilic

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10 20 COMPOSTING

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I O

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(DAYS)

Fig. 4.

Log CFU/g compost Mesophiles

Thermophiles

9.38 8"87 7"18 6"87 8"60 8"97

7.92 7-38 7-81 6"76 7"92 6"76

aSample sites A-F were as indicated in Fig. 3.

cycloheximide was included in TSA to exclude fungal growth, the bacterial counts were similar to those on unamended media. The number of microbes able to grow anaerobically was also monitored during this trial. This group of microbes was increasing in number towards the end of the period of monitoring.

Distribution of mesophiles and thermophiles in a commercial heap The occurrence of mesophilic microorganisms throughout the composting period was surprising. Therefore the distribution of both mesophiles and thermophiles in a heap was investigated. A commercial heap was sampled at various sites after the temperatures were taken (Fig. 3 and Table 1). The highest and lowest values for both groups were not associated with the coolest and warmest sites within the heap. Effect of urea concentration Experimental heaps were set up with varying concentrations of urea. One batch of pine bark was

Numbers of bacteria in bark heaps treated wtih different urea concentrations: 0 (o), 4 ( A ) and 8 (zx) kg urea per m 3 bark, respectively. Bark was aged for 2 weeks, divided into 3 heaps and urea added at day 0.

aged for two weeks before it was split up and urea added to initiate composting. Figure 4 shows that 4 or 8 kg urea per m 3 bark caused similar increases in mesophiles and thermophiles, respectively. However, if no urea was added, microbial numbers remained low, particularly the thermophilic counts. These results were reflected in the temperatures recorded for the heaps, as the 4 and 8 kg per m 3 heaps became far hotter (peak temperatures 71 and 76°C, respectively), indicating intense microbial activity, compared with 55°C peak temperature in the heap with no added urea. The addition of 2 and 6 kg urea per m 3 bark also caused heaps to heat up and microbial numbers to increase above the corresponding values recorded for the u n a m e n d e d heap (data not shown). Use of different nitrogen sources Figures 5 and 6 show the results of trials carried out utilising urea and other nitrogen sources to initiate composting. W h e n K N O 3 or Ca(NO3) 2 were used as an alternative to urea (Fig. 5), microbial numbers did not increase to the same extent as in standard heaps. This was particularly noticeable in the thermophilic populations. The temperatures of the nitrate-treated heaps were also lower, as the peak temperatures of the K N O 3 and Ca(NO3) 2 heaps were 65 and 63°C, respectively, compared with 80°C for the urea-treated heap. In a second set of trials (Fig. 6), the effect of chicken litter, and a combination of urea and nit-

89

Microbial numbers during the composting of pine bark THERMOPHILES

MESOPHILES

I

MESOPHILES

THERMOPHILES

8

0

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0

I 0

I 10

I 20

COMPOSTING

I

30

~

TIME

I O

I 10

I 20

I 30

(DAYS)

Fig. 5. Numbers of mesophilic and thermophilic bacteria associated with bark treated with 4 kg urea per m 3 (O), 15.4 kg K N O 3 per m 3 ( • ) and 12'9 kg Ca(NO3) 2 per m 3 (zx) (approximately 2 kg nitrogen per m3).

rate were compared with the urea treatment. These two treatments had approximately the same effect on the microbial populations as urea.

Effect of different aging periods For a period of 5 months from December 1987 to May 1988 pine bark chips were set aside monthly to age. Numbers of mesophiles and thermophiles in uncomposted, unaged bark were constant in the months tested. Thereafter the resulting five heaps and an unaged heap were composted concurrently. Microbial numbers in both groups showed little variation in numbers during the aging period of all heaps. However, after urea was added to each heap, differences became apparent (Fig. 7). Although the mesophile numbers in all heaps did not vary greatly for three weeks after urea addition, bark heaps that had been aged for longer periods (3 and 5 months) contained fewer thermophiles after 1 week of composting than the heap aged for 1 month. The unaged 'fresh' bark showed the greatest increase in the number of thermophiles of all the heaps.

0

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0

2

4

6

WEEKS

Fig. 6. Numbers of mesophilic and thermophilic bacteria associated with bark treated with 4.4 kg urea per m 3 (e) and 2.2 kg urea + 7-7 kg K N O 3 per m 3 ( zx ). Chicken litter ( • ) was added at the rate of 1:7 (v/v) to bark.

lESOPHILES

10

o9

THERMOPHILES

f

8

7

o • 0

DISCUSSION I

0

Composting processes are generally developed empirically, and the biological component is often regarded as a simple chemical reaction, rather than the complex set of interactions that actually occur. Key factors that affect the rate of compost-

I

1

I

2

I

3

0

1

2

3

WEEKS

Fig. 7. Numbers of bacteria associated with bark during composting of heaps aged for different periods of time. Composting was initiated by urea addition at week 0, in heaps that had been aged prior to this for 0 (o), 1 (o), 3 ( • ) and 5 (zx) months, respectively.

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C. L. Davis, S. A. Hinch, C. J. Donkin, P. J. Germishuizen

ing, such as temperature, pH, moisture and nutrient availability, are all factors that profoundly influence microbial growth and activity. It is generally accepted that composting occurs in three stages: first, the stage when readily degradable organic compounds are utilised; second, the thermophilic stage where temperatures rise; and third, the stabilisation period when temperatures drop. The 'self-heating' that takes place is due to heat liberation from microbial metabolic activity (Finstein & Morris, 1975). The optimal moisture content for most composting processes is 50-70% w/w (Crawford, 1983), and the oxygen concentration should be maintained at greater than 0.1%, preferably 5-12% (Hoitink & Poole, 1980). The pH of milled pine bark is 3.7-3.8 (Ogden et al., 1987), but of mature bark compost is 6.4-7.2 (Hoitink & Poole, 1980). Another important characteristic of milled pine bark is a total nitrogen concentration of approximately 0.28% (Ogden et al., 1987). The bark has a C:N ratio of 100-120:1, but after composting this falls to 30-35:1 (Riegel, 1980). The results of the selective plating for bacteria and fungi (Fig. 2) showed that at all stages of composting, bacterial CFU outnumbered fungal propagules. Thus the subsequent work focused on bacteria (including actinomycetes). Earlier workers (Chang & Hudson, 1967) showed that mesophilic and thermophilic fungi were killed off as the temperature of wheat-straw compost rose to a maximum. These workers also found that thermophilic bacterial and actinomycete CFU outnumbered the mesophilic populations during the latter part of composting. Subsequently, Fermor et al. (1979) studied bacterial and fungal numbers in wheat straw with amendments, and found mesophilic and thermophilic bacterial numbers to be similar after about 50 h of composting, and low numbers of mesophilic fungi present throughout a 200 h composting process. Thus the findings in this study are different from both of these. Although the relative contributions to cornposting activity by bacteria and fungi are difficult to determine, methods such as electron microscope visualisation of the bark chips (Donkin, unpublished) showed the importance of bacteria in the composting process, particularly in the initial stages. It has also been stated (Finstein & Morris, 1975) that bacterial metabolism is responsible for the dramatic temperature increases that occur in composting. Fungi may well be important in the later stages of composting.

Actinomycetes are also problematic to enumerate using standard techniques, as they are often adherent to their substrate. Their characteristic powdery colonies were noted on plates incubated at 60°C rather than 30°C throughout these trials. Estimates of microbial numbers obtained by plating environmental samples are always an underestimation, perhaps more so in the case of actinomycetes. In order to carry out a number of trials simultaneously, small trial heaps were used. Maximum microbial numbers in the large commercial heaps were similar to those of the smaller trial heaps. However, there was variability in the patterns of increases and decreases in numbers from trial to trial (compare urea trials in Figs 5 and 6). Nevertheless, similar trends were found when trials were repeated. Composite samples from a number of sites within each heap were used in order to ease the problem of patchy distribution of microorganisms within a heap. Various definitions of the terms mesophile and thermophile have been proposed. According to Atlas and Bartha (1987), microorganisms are classified as mesophiles and thermophiles if their optimal growth temperatures are moderate (25-40°C) or high (40-60°C), respectively. However, Brock and Madigan (1988) have defined the terms more simply as 'an organism living in the temperature range around that of warm blooded animals' (mesophile) and 'an organism living at a high temperature' (thermophile). In this study, mesophiles and thermophiles were considered to be the organisms growing on plates incubated at 30 and 60°C, respectively. A profile of mesophile and thermophile distributions in the heaps was undertaken (Fig. 3), in order to see if the outer, cooler insulating shell of the compost heap contained mainly mesophiles, and the inner, hotter portion the thermophiles. It appeared that there was a fairly even distribution of the two groups of microbes within the heap. Waste substrates for composting that have high C:N ratios generally require the addition of a nitrogen source to initiate the intense microbial activity associated with composting. Urea may be used, for example, with wheat straw (Gupta et al., 1987), and in this case, pine bark. Other organic wastes may be added, for example chicken and piggery manure, and soy waste (Verdonck & Pennick, 1985). ff a suitable nitrogen source is not added, the high temperatures associated with composting do not occur, as shown in this study and that of Indbar et al. (1988). Although the dif-

Microbial numbers during the composting of pine bark

ferent concentrations of urea tested in this study allowed similar composting of the trial heaps to occur (shown by the increases in microbial numbers), the heaps to which 2 and 8 kg per m 3 bark were added had lower and higher peak temperatures than the 4 kg per m 3 heap. Thus urea concentration may have affected the rate of composting. Nitrates are used as fertilisers when growing seedlings or plants in bark compost or barkamended media. Thus the use of nitrate as a nitrogen source in the composting process was investigated, with little success (Fig. 5). Hoitink and Kuter (1986) have advocated the use of ammonium nitrogen or poultry manure, rather than nitrates, and attributed the good effects of the former two to the way they increase the pH of the compost. Heaps of bark to which nitrates were added were more acidic (data not shown) and a higher proportion of fungal C F U were noted on plates from these heaps. The lower pH generated in nitrate-treated heaps would also discourage actinomycete growth (Lacey, 1973). The presence of nitrate itself is not inhibitory, as shown in Fig. 6, where a heap treated with equal quantities of ammonium and nitrate composted as well as a standard urea heap. The reason for Ca(NO3) 2 being more inhibitory to composfing than K N O 3 is not known, but could be due to the greater acidifying effect of Ca(NO3) 2. Chicken litter appeared to be excellent as a nitrogen source for cocomposting with pine bark. When bark arrives at the factory for composting, it does not absorb water well, and is reddish in colour. During the period of aging (before the addition of the nitrogen source) when the bark chips remain undisturbed in a heap, some changes occur, and the bark becomes darker and more wettable. The temperatures rise above the ambient, and the heaps may become slightly anaerobic. This may be the reason for the relatively smaller increase in microbial numbers in well-aged (3 and 5 months) bark compared to the newer bark (0 and 1 month of aging), as shown in Fig. 7. Although numbers of mesophiles and thermophiles were similar for all heaps at the start of composting, there were possibly subtle changes in the microbial species making up the populations during the aging period, or changes due to degradation of bark constituents. Hoifink and Fahy (1986) have commented on 'sour composting', noting that anaerobic decompositions liberate less heat and generate different end products from their aerobic equivalents. From a corn-

91

merical bark composting point of view, a period of one or two months of aging seems advisable, to allow the bark to become more wettable, but not longer than that, to prevent undesirable changes in the microbial populations present in the bark. Microbial numbers are not necessarily a good indicator of compost quality, and plant or seedling growth in the finished product is the ultimate test. Pine bark compost has the advantage over some other gowing media of being suppressive to many plant pathogens. This is probably a result of the periods of self-heating during the composting process being inhibitory to the survival of the pathogens, as well as antagonism from saprophytic compost microorganisms. The ability of the microflora to detoxify or degrade phytotoxic compounds such as resins, phenols and tannins also merits investigation. The microbes in a pine-bark compost heap thus play a vital role in transforming what would be a waste problem into a useful product.

ACKNOWLEDGEMENTS This research was funded by the Organic Wastes Programme of the CSIR, Pretoria, Republic of South Africa, with project leader Prof. I. E. Smith. The assistance of M. Sherrif in sample collection is gratefully acknowledged.

REFERENCES

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Hoitink, H. A. & Fahy, P. C. (1986). Basis for the control of soilborne plant pathogens with composts. Ann. Rev. Path., 24,93-114. Hoitink, H. A. J. & Kuter, G. A. (1986). Effects of composts in growth media on soilborne pathogens. In The Role of Organic Matter in Modern Agriculture, ed. Y. Chen & Y. Avnimelech. Martinus Nijhof, The Hague. Hoitink, H. A. J. & Poole, H. A. (1980). Factors affecting quality of composts for utilisation in container media. Hort. Sci., 15, 171-3. Indbar, Y., Chen, Y. & Hadar, Y. (1988). Composting of agricultural wastes for their use as container media: Simulation of the composting process. Biol. Wastes,26, 247-59. Lacey, J. (1973). Actinomycetes in soils, composts and fodders. In Actinomycetales: Characteristics and Practical

Importance, ed. G. Skykes & E A. Skinner. Academic Press, London, pp. 231-51. McKinley, V. L. & Vestal, J. R. (1985). Physical and chemical correlates of microbial activity and biomass in composting municipal sewage sludge. AppL Environ. MicrobioL, 50, 1395-403. Ogden, R. J., Porkorny, E A., Mills, H. A. & Dunavent, M. G. (1987). Elemental status of pine bark-based potting media. Hort. Rev., 9, 102-31. Riegel, E (1980). Composting and agricultural use of bark and sludge. Das Papier, 34, V92-V98. Strom, E E (1985). Effect of temperature on bacterial species diversity in thermophilic solid-waste composting. AppL Environ. MicrobioL, 50, 899-905. Verdonck, O. & Pennick, R. (1985). Composting of bark with soy scrap sludge. Acta Hort., 172, 183-7.