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Microbial composition of poultry excreta
Biological Wastes 33 (1990) 95-105
Microbial Composition of Poultry Excreta R. Nodar, M. J. Acea* & T. Carballas Instituto de lnvestigaciones Agrobiol6gicas de Galicia (CSIC), Apartado de Correos 122, 15080 Santiago de Compostela, Spain (Received 26 October 1989; accepted 22 November 1989)
ABSTRACT Total microbial populations, bacteria, actinomycetes, fungi and algae of three samples of poultry excreta were studied. The physiological groups involved in C. N and S ~3'cles have been determined. The results were compared with the common numbers in soils and organic wastes. Poultry excreta had a high density of micro-organisms. Bacteria, most of them being strict or facultative anaerobes, predominated. Actinomycetes and fungi were also in relatively high density. Fungi were mainly as propagules and their mycelium was nearly indetectable. Algae were in low density. A small percentage of aerobic' bacteria was acidophilic or acid-tolerant, a lesser number was spore-forming, and cyanobacteria were not detected. Most of the microbial population had proteolytic, ammonificant, anaerobic cel[ulolytic, denitrO~'cant and anaerobic nitrogen-fixing capacities, followed by amylolytics, pectolytics, sulphate reducers and anaerobic mineralizers of sulphur; whereas micro-organisms favoured by aerobic conditions as aerobic' cellulolytic, aerobic .fi'ee-living nitrogen fixers, ammonium oxidizers, nitrite oxidizers and sulphur oxidizers were in low densities, and sulphide oxidizers were not detected.
INTRODUCTION The worldwide increase in organic wastes production has intensified and diversified the investigations into their value as soil fertilizers, energetic and nutritive substrates or environmental contaminants (FAO, 1983; Leir6s * To whom correspondence should be addressed. 95 Biological Wastes 0269-7483/90/$03"50 © 1990 Elsevier Science Publishers Ltd, England. Printed in Great Britain
96
R. Nodar, M. J. Acea, T. Carballas
et al., 1984; E1-Ashry et al., 1987; Tate III, 1987; Chen et al., 1988; DiazFierros et al., 1988). These compounds are ecosystems with special
characteristics; their microbial diversity makes their study interesting for its own sake; thus, an increasing number of investigators have given their efforts to the knowledge of the biotic properties of organic wastes (Riviere et al., 1974; Finstein & Morris, 1975; Acea & Carballas, 1983, 1988a, b; Corominas et al., 1987; De Bertoldi et al., 1987; Diaz-Ravifia et al., 1989). The most rational means of disposal of large quantities of manure generated by the poultry industry seems to be its application to soil. The potential of such materials to change soil conditions or to increase plant production has been extensively studied (Tinsley & Nowakowski, 1959a, b, c; Perkins et al., 1964; Perkins & Parker, 1971; Hafez, 1974; Lau & Wu, 1987). Because microbiota plays a unique role in the energy circulation through the ecosystems and in the cycles of C, N, P and S (Alexander, 1967; Dommergues, 1974; Atlas & Bartha, 1981) organic wastes mineralization by micro-organisms is essential, and a decisive factor in the soil fertility. In spite of this, research on microbial properties of poultry wastes is scarce and incomplete. The aim of the work described in this paper was to study the microbial populations of poultry faeces from the point of view of their participation in the nutrient cycles and in the soil fertility, as well as to compare their densities with common numbers in soils and organic wastes.
METHODS The poultry excreta used in this study, named P1, P2 and P3, were taken in January, May and July (1989) from a poultry farm in La Corufia (north-west Spain). In each case, a 3-kg sample of poultry excreta was obtained with thirty 100-g subsamples of fresh fowl droppings from different points of the room where about 6000 animals were stocked. Samples were homogenized prior to use.
Physical and chemical characteristics The methods described by Guitifin and Carballas (1976) were used to study the following characteristics of poultry manures. Dry matter was determined by oven-drying the samples at 110°C. Texture was determined with a set of sieves and a cellulose filter with 1.5/~m pore size; per cent of particle sizes were expressed as samples dried at 60°C. pH in distilled water or KC1 1N (1:2-5 poultry/water or KC1) were measured with a glass
97
Microbial composition of poultry excreta TABLE 1 Main Characteristics of the Poultry Excreta
pH
Dry matter
Ash
Total C
P1 P2 P3
7'70 7'68 7.72
(%) 14'66 13"85 8"66
(%) 31'30 30"80 33"15
(%) 44-70 45"20 46"90
Material P1 P2 P3
Total 4'40 4"41 4-45
3"63 3"62 3"67
Material PI P2 P3
CaO 9"76 9"73 9'82
MgO 0"76 0.73 0.82
Material P1 P2 P3
>2 3"3 3"5 4'0
2-1 6'17 6"15 6"13
Mater&P
Nitrogen (%) Organic Ammoniacal 0"70 0-69 0"74
Available (%) K20 2.77 2.74 2.83
Na20 0"98 0"96 1.03
C/N I0 10 11 Nitric 0"07 O"10 0-04 Total P (%) P205 2'56 2.53 2-62
Particle size (mm) (%) lab5 9-32 9-24 9"22
0"5 1"5 × 10- 3 < 1"5 × 10- 3 45"91 35-17 45'86 35"10 45"60 35"09
"Samples of excreta, see Methods.
electrode. Ash was determined by combustion at 400°C. Available cations were extracted with 0"5N acetic acid; Ca and Mg were measured by atomic absorption spectrophotometry and K and Na by flame photometry. Total Kjeldahl N was determined by digestion and steam distillation, and ammonium-N and nitrate-N by steam distillation with MgO and Devarda's alloy (Bremner & Keeney, 1965). Total C was determined by combustion in a Carmhograph 12 (H. Wosthoff, oHG, Bochum, FRG). Total P was determined by ignition and extraction with 1N H z S O 4 (Saunders & Williams, 1955), and measured colorimetrically (Murphy & Riley, 1962). Main physical and chemical characteristics of poultry faeces are listed in Table 1. Microbial population A 20 g subsample of poultry faeces was diluted ten-fold in sterile water. Five Petri dishes containing solid media or five test-tubes containing liquid media were inoculated from each dilunon. The methods and media used for the culture and counts of the viable microbial population, bacteria, aerobic
98
R. Nodar, M. J. Acea, T. Carballas
spore-forming bacteria, actinomycetes, algae, nitrifiers, denitrifiers and aerobic free-living nitrogen-fixers (azotobacters) of the poultry manures were those described by different authors in Black and Evans (1965). Acidophile bacteria were assessed by plating at pH 4. The cyanobacteria were grown in standard mineral medium with omission of a combined nitrogen source (Rippka et al., 1979). Fungi were cultured on Czapek-Dox medium. For estimation of length of fungal hyphae, the membrane filter technique (Sundman & Silvela, 1978) combined with a method of estimating the total length of hyphae (Newman, 1966) was followed. The growth of the nitrobacter group was detected using diphenylamine-sulphuric acid reagent. Anaerobic nitrogen fixers (clostridia), proteolytics, ammonifiers, aerobic and anaerobic cellulolytics, amylolytics, pectolytics, sulphate reducers, sulphide oxidizers, elementary sulphur oxidizers and anaerobic mineralizers of organic sulphur were studied using the methods described by Pochon and Tardieux (1962). Bacteria, acidophiles and aerobic spore-forming bacteria, actinomycetes and fungi, were determined after 7-10 days of incubation by colony counts in agar plates. The other microbiological groups were incubated in liquid media in test-tubes for 21 days at 28°C, except algae and cyanobacteria which were cultured for six weeks in a sunlight camera (about 500 Ix), with periodic cycles of light and dark both of 8 h. Their number was estimated from a most-probable-number table. The density of microorganisms was related to dry weight (110°C) of poultry excreta.
RESULTS As Table 1 shows, in poultry droppings small-size particles predominate, a great percentage of them being equal to, or smaller than, 0.5 mm. The three samples had a basic pH and 80-90% humidity. The percentages of C, N and P were relatively high, and 82% of the nitrogen was organic. Inorganic nitrogen was mostly ammoniacal. Also, the available cations content was high, especially in Ca and K. The numbers of viable micro-organisms in poultry excreta (Fig. 1) were about 108 per gram dry excreta (d.e.). Bacteria predominated over the other groups. Aerobic bacteria represented a small proportion of total bacteria with values of 1 0 6 colony-forming units (CFU) per gram d.e. Twelve per cent of aerobic bacteria were acidophiles or acid-tolerant, a smaller proportion (5"6%) were spore-forming and cyanobacteria were not detected. Actinomycetes reached densities of 105 C F U per gram d.e. and fungi of 104 propagules per gram d.e. Fungal mycelium was present, but it was too short (less than 13 cm per gram d.e.) to be measured by the method used. Algal populations showed densities about 1 0 2 algae per gram d.e.
d
a
m
~2
~^
~^ ,^
.^
;:4i '::¢i:
,+
,÷
'3
Microbial population of three samples of poultry excreta. ?M, Total microbial population: AB, aerobic bacteria; AeB, acidophilic .erobic bacteria; SB, aerobic spore-forming bacteria; ACT, actinomycetes: F, fungi; A, algae; CB, cyanobacteria.
:ig. 1.
I.
i
~'1
P2
P3
Fig. 2. Microbial population of three samples of poultry excreta. ~,C, Aerobic cellulolytics; ANC, anaerobic cellulolytics; AML, amylolytics: PEC, pectolytics.
r,. .o
!;
Pl
P2
.| P3
~ig. 3. Microbial population of three samples of poultry excreta. ~.Z,Azotobacter; CL, Clostridium; PR, proteolytics; AM, ammonifiers; AO, ammonium-oxidizers; NO, nitrite-oxidizers; DN, denitrifiers.
:D
t. =
I AZ - - CL
)I
P2
P3
rig. 4. Microbial population of three samples of poultry excreta. )SM, Anaerobic organic sulphur-oxidizers; SR, sulphate-reducers; ESO, elementary sulphur-oxidizers; SUO, sulphide-oxidizers.
C~
~2
7 5
c~
! -
?
Microbial composition of poultry excreta
101
With regard to the micro-organisms involved in the carbon cycle (Fig. 2) the majority were anaerobic cellulolytics, with densities about 106 per gram d.c., followed by amylolytics and pectolytics (about 105 per gram d.e.), and aerobic cellulolytics had lowest densities (102 per gram d.e.). Among the micro-organisms related to the nitrogen cycle (Fig. 3) the proteolytics and the ammonificants were the most abundant, with densities of 10a per gram d.e. The densities of anaerobic free-living nitrogen-fixers (Clostridium) and denitrifiers, both with values about 105 per gram d.e., were also relatively high, whereas, the numbers of ammonium oxidizers (about 104 per gram d.e.), nitrite oxidizers (103 per gram d.e.) and aerobic free-living nitrogen-fixers (Azotobacter) (102 per gram d.e.) were relatively low. Finally, of the micro-organisms associated with the sulphur cycle (Fig. 4), the sulphate reducers and the anaerobic organic sulphur mineralizers showed densities of 104 per gram d.e., the elementary sulphur oxidizers were one order of magnitude lower and the sulphide oxidizers were not detected in any of the three samples studied.
DISCUSSION There were no significant differences among the three samples of poultry excreta studied and they showed similar microbiological, chemical and physical characteristics. The high number of total viable micro-organisms reached densities close to the upper values given for many soils (Alexander, 1967; Berthelin & Toutain, 1979) and was comparable to the populations of composted urban refuses and cattle slurry (Acea & Carballas, 1983; DiazRavifia et al., 1989). However, these densities were much more than a logarithmic order higher than in pig slurry (Rivi6re et al., 1974); these results were in agreement with the fact that chemical and physical properties of fowl faeces were between the limits needed for microbial proliferation (Pochon & Barjac, 1958; Dommergues & Mangenot, 1970) and resembled the ones corresponding to a soil with high chemical fertility (Duchaufour, 1987), but flooded. Bacteria predominate over the other taxonomic groups, as had been found in most soils (Lynch, 1979; Atlas & Bartha, 1981) and organic wastes (Acea & Carballas, 1983, 1988a). However, only a small proportion of bacteria were aerobes. Probably, anaerobic conditions are due to the high water content of faeces and promoted by the elevated microbial metabolic activity common in substrates with high macro- and micro-nutrient content. High anaerobic populations were also found in cattle and pig slurries (Rivi+re et al., 1974; Acea & Carballas, 1983). A small percentage of aerobic bacteria were acidophilics or acid-tolerant and were aerobic spore-forming, and no cyanobacteria were found. The densities of actinomycetes, fungal
102
R. Nodar, M. J. Acea, T. Carballas
units and algae were three, four and six orders of lower magnitude, respectively, than bacterial density. Actinomycetes represented a percentage of the total microbial population lower than those given for most soils (Kfister, 1967; Acea & Carballas, 1986) and some manures (Allievi et al., 1987); also, there was a lower actinomycetes content than in other organic wastes (Diaz-Ravifia et al., 1989). The number of fungal propagules was lower than in soils (Dommergues & Mangenot, 1970) and manures. However, it may be pointed out that the ratio of propagules to mycelia was relatively high because fungal mycelium was almost undetectable. Therefore, poultry excreta showed negative conditions for fungal development, especially if compared with soils (West, 1988). Also unfavoured were autotrophic micro-organisms such as cyanobacteria and algae, less competitive than heterotrophic groups in materials with high organic-matter content (Dommergues & Mangenot, 1970). However, algae abundance was similar to some soils and cattle slurry (Acea & Carballas, 1983, 1986) and higher than in composted urban refuses (Diaz-Ravifia et al., 1989). The largest groups, by far, were those that carry out the breakdown of proteins and the ammonifying organisms; similar predominance was found in many soils and manures (Alexander, 1967; Acea & Carballas, 1983, 1986). By contrast, nitrifiers were scarce. These results are in accordance with the fact that almost all the inorganic nitrogen in poultry excreta is ammonium. This gap between the two steps of nitrogen mineralization may be explained for two reasons: on the one hand, it may be due to the biochemical heterogeneity and ubiquity of micro-organisms bringing about proteolysis and nitrogen ammonification, these two processes occurring in both aerobic and anaerobic environments; on the other hand, since oxygen is an obligate requirement for all species concerned with nitrification, where the O 2 supply is inadequate for both ammonium and nitrate oxidizers, there will be little ammonium oxidation. Denitrifiers were in relatively high densities, probably favoured by anaerobic conditions and a substrate rich in organic materials (Sprent, 1987; Groffman et aL, 1988). The same reasons could have made the density of anaerobic free-living nitrogen-fixers three orders of magnitude higher than the aerobic ones. Except for aerobic cellulolytics, all the studied physiological groups involved in the carbon cycle were present in relatively high densities in poultry excreta, in accordance with the high carbon content of this waste, although anaerobic cellulolytics clearly predominated over the other carbon cycle micro-organisms: results opposite to those found in soils, cattle slurry and composted urban residues (Alexander, 1967; Acea & Carballas, 1983, 1986; Diaz-Ravifia et al., 1989). The populations of amylolytics, pectolytics and aerobic cellulolytics were lower than in soils and other wastes. By
Microbial composition of poultry excreta
103
contrast, anaerobic cellulolytics were found in higher numbers than in soils and in composted urban residues (Acea & Carballas, 1986; Diaz-Ravifia et al., 1989). The anaerobic groups involved in the sulphur cycle clearly predominated over the aerobes. Sulphate reducers and anaerobic mineralizers of organic sulphur were in higher densities than in soils, and in similar number to other wastes (Acea & Caballas, 1986; Diaz-Ravifia et al., 1989). Nevertheless, the sulphate reducers were in lower densities than in cattle and pig slurries (Rivi6re et al., 1974; Acea & Carballas, 1983). The elementary sulphur oxidizers were present in poultry wastes in similar densities to those in soils and cattle slurry, and higher than in urban refuse compost (Acea & Carballas, 1983, 1986; Diaz-Ravifia, 1989). In conclusion, the distribution of the different taxonomic and physiological groups of micro-organisms showed that most of the organic matter of poultry faeces will tend to be incompletely oxidized by anaerobic organisms, and intermediate metabolic products will be produced in higher quantities than in well-aerated soils or wastes.
A C K N O W L E D G E M ENTS The authors thank Mrs Blanca Arnaiz for technical assistance and Dr E. Eiroa for drawing the figures for this paper.
REFERENCES Acea, M. J. & Carballas, T. (1983). Caracterizaci6n de la poblaci6n microbiana de un purin de vacuno. An. Edafol. Agrobiol., 42, 149-59. Acea, M. J. & Carballas, T. (1986). Estudio de la poblaci6n microbiana de diversos tipos de suelos de zona hfimeda (N. O. de Espafia). An. Edafol. Agrobiol., 45, 381-98. Acea, M. J. & Carballas, T. (1988a). The influence of cattle slurry on soil microbial population and nitrogen cycle microorganisms. Biol. Wastes, 23, 229-41. Acea, M. J. & Carballas, T. (1988b). Effects of cattle-slurry treatment on the microorganisms of the carbon- and sulphur-cycles in the soil. Biol. Wastes, 24, 251-8. Alexander, M. (1967). Introduction to Soil Microbiology. John Wiley, New York. Allievi, L., Citterio, B. & Ferrari, A. (1987). Vermicomposting of rabbit manure: Modification of microflora. In Compost: Production, Quality and Use, ed. M. De Bertoldi, M. P. Ferranti, P. L'Hermite & F. Zucconi. Elsevier Applied Science, London, pp. 115-26. Atlas, R. M. & Bartha, R. (1981). Microbial Ecology: Fundamentals and Applications. Addison-Wesley Publishing Company, London.
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Berthelin, J. & Toutain, F. (1979). Biologie des sols. In Pbdologie 2. Constituans et Propi~t~s du Sol, ed. M. Bonneau & B. Souchier. Masson, Paris, pp. 123-53. Black, C. A. & Evans, D. D. (eds) (1965). Methods of Soil,4nalysis. Part 2. Chemical and Microbiological Properties. American Society of Agronomy, Madison, WI. Bremner, J. M. & Keeney, D. R. (1965). Steam distillation methods for determination of ammonium, nitrate and nitrite. Anal Chim. ,4cta, 32, 485-95. Chen, T. H., Day, D. L. & Steinberg, M. P. (1988). Methane production from fresh versus dry dairy manure. Biol. Wastes, 24, 297-306. Corominas, S., Perestelo, F., Perez, M. L. & Falcon, M. A. (1987). Microorganisms and environmental factors in composting of agricultural waste of the Canary Islands. In Compost: Production, Quality and Use, ed. M. De Bertoldi, M. P. Ferranti, P. L'Hermite & F. Zucconi. Elsevier Applied Science, London, pp. 127-34. De Bertoldi, M., Ferranti, M. P., L'Hermite, P. & Zucconi, F. (eds) (1987). Compost: Production, Quality and Use. Elsevier Applied Science, London. Diaz-Fierros, F., Villar, M. C., Carballas, M., Leiros, M. C., Carballas, T. & Cabaneiro, A. (1988). Effect of cattle slurry fractions on nitrogen mineralization in soil. J. ,4gric. Sci. Camb., 110, 491-7. Diaz-Ravifia, M., Acea, M. J. & Carballas, T. (1989). Microbiological characterization of four composted urban refuses. Biol. Wastes, 30, 89-100. Dommergues, J. R. (1974). Role des microorganismes dans la biosphere. Comision francaise pour L'Unesco. Unesco, Paris. Dommergues, Y. & Mangenot, T. F. (1970). Ecologie Microbienne du Sol. Masson, Paris. Duchaufour, P. (1987). Manual de Edafologia. Masson, Barcelona. EI-Ashry, M. A., Khattab, H. M., E1-Serafy, A., Solimam, H. & Abd Elmoula, S. M. (1987). Nutritive value of poultry wastes for sheep. An. Wastes, 19, 287-98. Fao (1983). Animal waste utilization. Selected Scientific papers from the Fourth Consultation, Budapest. Swedish National Board of Agriculture. Finstein, M. S. & Morris, M. L. (1975). Microbiology of municipal solid wastes composting. Adv. Appl. Microbiol., 19, 113-51. Groffman, P. M., Tiedje, J. M., Robertson, G. P. & Christensen, S. (1988). Denitrification at different temporal and geographical scales: Proximal and distal controls. In Advances in Nitrogen Cycling in Agricultural Ecosystems, ed. J. R. Wilson. CAB International, Wallingford, UK, pp. 174-92. Guitian, F. & Carballas, T. (1976). Tkcnicas de Anhlisis de Suelos. Pico Sacro, Santiago de Compostela, Spain. Hafez, A. A. R. (1974). Comparative changes in soil-physical properties induced by admixtures of manures from various domestic animals. Soil Sci., 118, 53-9. Kiister, E. (1967). The actinomycetes. In Soil Biology, ed. A. Burges & F. Raw. Academic Press, London, pp. 111-27. Lau, D. C. W. & Wu, M. M. W. (1987). Manure composting as an option for utilization and management of animal waste. Resour. Conserv., 13, 145-56. Leir6s, M. C., Villar, M. C., Cabaneiro, A., Carballas, T., Diaz-Fierros, F., Gil, F. & Gomez, C. (1984). Caracterizaci6n y valor fertilizante de los purines de vacuno en Galicia. An. Edafol. Agrobiol., 42, 753-68. Lynch, J. M. (1979). The terrestrial environment. In Microbial Ecology: .4 conceptual approach, ed. J. M. Lynch & N. J. Poole. Blackwell Scientific Publications, Oxford, pp. 67-91.
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Murphy, J. & Riley, J. P. (1962). A modified single solution method for the determination of phosphate in natural waters. Anal Chim. Acta., 27, 31-6. Newman, L. (1966). A method of estimating the total length of root in a sample. J. Appl. Ecol., 3, 139-45. Perkins, H. F. & Parker, M. B. (1971). Chemical composition of broiler and hen manures. University of Georgia College of Agriculture, Athens, GA. Perkins, H. F., Parker, M. B. & Walker, M. L. (1964). Chicken manure: Its production, composition and use as fertilizer. University of Georgia College of Agriculture, Athens, GA. Pochon, J. & Barjac, H. (1958). Traitk de Microbiologie des Sols. Applications Agronomiques. Dunod, Paris. Pochon, J. & Tardieux, P. (1962). Techniques d'Analyse en Microbiologie du Sol. La Tourelle, St Mand6, France. Rippka, R. M., Deruelles, J., Waterbury, J. B., Herman, M. & Stainer, R. Y. (1979). Generic assignment, strain histories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol., 111, 1-61. Rivi6re, J., Subtil, J. C. & Catroux, G. (1974). Etude de revolution physicochimique et microbiologique du lisier de porcs pendant le stockage anfierobie. Ann. Agron., 25, 383-401. Saunders, W. M. H. & Williams, E. G. (1955). Observations on the determination of total organic phosphorus in soils. J. Soil Sci., 6, 254-67. Sprent, J. I. (1987). The Ecology of the Nitrogen Cycle. Cambridge University Press, Cambridge, UK. Sundman, V. & Silvela, S. (1978). A comment on the membrane filter technique for estimation of length of fungal hyphae in soil. Soil Biol. Biochem., 10, 399-401. Tate III, R. L. (1987). Ecosystem management and soil organic matter nivel. In Soil Organic Matter. John Wiley, New York, 13, pp. 260-80. Tinsley, J. & Nowakowski, T. Z. (1959a). The composition and manurial value of poultry excreta, straw-droppings compost and deep litter. I. Introduction, experimentals, methods of sampling and analysis. J. Sci. Food Agric., 10, 145-50. Tinsley, J. & Nowakowski, T. Z. (1959b). The composition and manurial value of poultry excreta, straw-droppings compost and deep litter. II. Experimental studies on composts. J. Sci. Food Agric., 10, 150-67. Tinsley, J. &. Nowakowski, T. Z. (1959c). The composition and manurial value of poultry excreta, straw-dropping compost and deep litter. III. Experimental studies on deep litter. J. Sci. Food Agric., 10, 224-32. West, A. W. (1988). Specimen preparation, stain type, and extraction and observation procedures as factors in the estimation of soil mycelial lengths and volumes by light microscopy. Biol. Fertil. Soils, 7, 88-94.
Microbial Composition of Poultry Excreta R. Nodar, M. J. Acea* & T. Carballas Instituto de lnvestigaciones Agrobiol6gicas de Galicia (CSIC), Apartado de Correos 122, 15080 Santiago de Compostela, Spain (Received 26 October 1989; accepted 22 November 1989)
ABSTRACT Total microbial populations, bacteria, actinomycetes, fungi and algae of three samples of poultry excreta were studied. The physiological groups involved in C. N and S ~3'cles have been determined. The results were compared with the common numbers in soils and organic wastes. Poultry excreta had a high density of micro-organisms. Bacteria, most of them being strict or facultative anaerobes, predominated. Actinomycetes and fungi were also in relatively high density. Fungi were mainly as propagules and their mycelium was nearly indetectable. Algae were in low density. A small percentage of aerobic' bacteria was acidophilic or acid-tolerant, a lesser number was spore-forming, and cyanobacteria were not detected. Most of the microbial population had proteolytic, ammonificant, anaerobic cel[ulolytic, denitrO~'cant and anaerobic nitrogen-fixing capacities, followed by amylolytics, pectolytics, sulphate reducers and anaerobic mineralizers of sulphur; whereas micro-organisms favoured by aerobic conditions as aerobic' cellulolytic, aerobic .fi'ee-living nitrogen fixers, ammonium oxidizers, nitrite oxidizers and sulphur oxidizers were in low densities, and sulphide oxidizers were not detected.
INTRODUCTION The worldwide increase in organic wastes production has intensified and diversified the investigations into their value as soil fertilizers, energetic and nutritive substrates or environmental contaminants (FAO, 1983; Leir6s * To whom correspondence should be addressed. 95 Biological Wastes 0269-7483/90/$03"50 © 1990 Elsevier Science Publishers Ltd, England. Printed in Great Britain
96
R. Nodar, M. J. Acea, T. Carballas
et al., 1984; E1-Ashry et al., 1987; Tate III, 1987; Chen et al., 1988; DiazFierros et al., 1988). These compounds are ecosystems with special
characteristics; their microbial diversity makes their study interesting for its own sake; thus, an increasing number of investigators have given their efforts to the knowledge of the biotic properties of organic wastes (Riviere et al., 1974; Finstein & Morris, 1975; Acea & Carballas, 1983, 1988a, b; Corominas et al., 1987; De Bertoldi et al., 1987; Diaz-Ravifia et al., 1989). The most rational means of disposal of large quantities of manure generated by the poultry industry seems to be its application to soil. The potential of such materials to change soil conditions or to increase plant production has been extensively studied (Tinsley & Nowakowski, 1959a, b, c; Perkins et al., 1964; Perkins & Parker, 1971; Hafez, 1974; Lau & Wu, 1987). Because microbiota plays a unique role in the energy circulation through the ecosystems and in the cycles of C, N, P and S (Alexander, 1967; Dommergues, 1974; Atlas & Bartha, 1981) organic wastes mineralization by micro-organisms is essential, and a decisive factor in the soil fertility. In spite of this, research on microbial properties of poultry wastes is scarce and incomplete. The aim of the work described in this paper was to study the microbial populations of poultry faeces from the point of view of their participation in the nutrient cycles and in the soil fertility, as well as to compare their densities with common numbers in soils and organic wastes.
METHODS The poultry excreta used in this study, named P1, P2 and P3, were taken in January, May and July (1989) from a poultry farm in La Corufia (north-west Spain). In each case, a 3-kg sample of poultry excreta was obtained with thirty 100-g subsamples of fresh fowl droppings from different points of the room where about 6000 animals were stocked. Samples were homogenized prior to use.
Physical and chemical characteristics The methods described by Guitifin and Carballas (1976) were used to study the following characteristics of poultry manures. Dry matter was determined by oven-drying the samples at 110°C. Texture was determined with a set of sieves and a cellulose filter with 1.5/~m pore size; per cent of particle sizes were expressed as samples dried at 60°C. pH in distilled water or KC1 1N (1:2-5 poultry/water or KC1) were measured with a glass
97
Microbial composition of poultry excreta TABLE 1 Main Characteristics of the Poultry Excreta
pH
Dry matter
Ash
Total C
P1 P2 P3
7'70 7'68 7.72
(%) 14'66 13"85 8"66
(%) 31'30 30"80 33"15
(%) 44-70 45"20 46"90
Material P1 P2 P3
Total 4'40 4"41 4-45
3"63 3"62 3"67
Material PI P2 P3
CaO 9"76 9"73 9'82
MgO 0"76 0.73 0.82
Material P1 P2 P3
>2 3"3 3"5 4'0
2-1 6'17 6"15 6"13
Mater&P
Nitrogen (%) Organic Ammoniacal 0"70 0-69 0"74
Available (%) K20 2.77 2.74 2.83
Na20 0"98 0"96 1.03
C/N I0 10 11 Nitric 0"07 O"10 0-04 Total P (%) P205 2'56 2.53 2-62
Particle size (mm) (%) lab5 9-32 9-24 9"22
0"5 1"5 × 10- 3 < 1"5 × 10- 3 45"91 35-17 45'86 35"10 45"60 35"09
"Samples of excreta, see Methods.
electrode. Ash was determined by combustion at 400°C. Available cations were extracted with 0"5N acetic acid; Ca and Mg were measured by atomic absorption spectrophotometry and K and Na by flame photometry. Total Kjeldahl N was determined by digestion and steam distillation, and ammonium-N and nitrate-N by steam distillation with MgO and Devarda's alloy (Bremner & Keeney, 1965). Total C was determined by combustion in a Carmhograph 12 (H. Wosthoff, oHG, Bochum, FRG). Total P was determined by ignition and extraction with 1N H z S O 4 (Saunders & Williams, 1955), and measured colorimetrically (Murphy & Riley, 1962). Main physical and chemical characteristics of poultry faeces are listed in Table 1. Microbial population A 20 g subsample of poultry faeces was diluted ten-fold in sterile water. Five Petri dishes containing solid media or five test-tubes containing liquid media were inoculated from each dilunon. The methods and media used for the culture and counts of the viable microbial population, bacteria, aerobic
98
R. Nodar, M. J. Acea, T. Carballas
spore-forming bacteria, actinomycetes, algae, nitrifiers, denitrifiers and aerobic free-living nitrogen-fixers (azotobacters) of the poultry manures were those described by different authors in Black and Evans (1965). Acidophile bacteria were assessed by plating at pH 4. The cyanobacteria were grown in standard mineral medium with omission of a combined nitrogen source (Rippka et al., 1979). Fungi were cultured on Czapek-Dox medium. For estimation of length of fungal hyphae, the membrane filter technique (Sundman & Silvela, 1978) combined with a method of estimating the total length of hyphae (Newman, 1966) was followed. The growth of the nitrobacter group was detected using diphenylamine-sulphuric acid reagent. Anaerobic nitrogen fixers (clostridia), proteolytics, ammonifiers, aerobic and anaerobic cellulolytics, amylolytics, pectolytics, sulphate reducers, sulphide oxidizers, elementary sulphur oxidizers and anaerobic mineralizers of organic sulphur were studied using the methods described by Pochon and Tardieux (1962). Bacteria, acidophiles and aerobic spore-forming bacteria, actinomycetes and fungi, were determined after 7-10 days of incubation by colony counts in agar plates. The other microbiological groups were incubated in liquid media in test-tubes for 21 days at 28°C, except algae and cyanobacteria which were cultured for six weeks in a sunlight camera (about 500 Ix), with periodic cycles of light and dark both of 8 h. Their number was estimated from a most-probable-number table. The density of microorganisms was related to dry weight (110°C) of poultry excreta.
RESULTS As Table 1 shows, in poultry droppings small-size particles predominate, a great percentage of them being equal to, or smaller than, 0.5 mm. The three samples had a basic pH and 80-90% humidity. The percentages of C, N and P were relatively high, and 82% of the nitrogen was organic. Inorganic nitrogen was mostly ammoniacal. Also, the available cations content was high, especially in Ca and K. The numbers of viable micro-organisms in poultry excreta (Fig. 1) were about 108 per gram dry excreta (d.e.). Bacteria predominated over the other groups. Aerobic bacteria represented a small proportion of total bacteria with values of 1 0 6 colony-forming units (CFU) per gram d.e. Twelve per cent of aerobic bacteria were acidophiles or acid-tolerant, a smaller proportion (5"6%) were spore-forming and cyanobacteria were not detected. Actinomycetes reached densities of 105 C F U per gram d.e. and fungi of 104 propagules per gram d.e. Fungal mycelium was present, but it was too short (less than 13 cm per gram d.e.) to be measured by the method used. Algal populations showed densities about 1 0 2 algae per gram d.e.
d
a
m
~2
~^
~^ ,^
.^
;:4i '::¢i:
,+
,÷
'3
Microbial population of three samples of poultry excreta. ?M, Total microbial population: AB, aerobic bacteria; AeB, acidophilic .erobic bacteria; SB, aerobic spore-forming bacteria; ACT, actinomycetes: F, fungi; A, algae; CB, cyanobacteria.
:ig. 1.
I.
i
~'1
P2
P3
Fig. 2. Microbial population of three samples of poultry excreta. ~,C, Aerobic cellulolytics; ANC, anaerobic cellulolytics; AML, amylolytics: PEC, pectolytics.
r,. .o
!;
Pl
P2
.| P3
~ig. 3. Microbial population of three samples of poultry excreta. ~.Z,Azotobacter; CL, Clostridium; PR, proteolytics; AM, ammonifiers; AO, ammonium-oxidizers; NO, nitrite-oxidizers; DN, denitrifiers.
:D
t. =
I AZ - - CL
)I
P2
P3
rig. 4. Microbial population of three samples of poultry excreta. )SM, Anaerobic organic sulphur-oxidizers; SR, sulphate-reducers; ESO, elementary sulphur-oxidizers; SUO, sulphide-oxidizers.
C~
~2
7 5
c~
! -
?
Microbial composition of poultry excreta
101
With regard to the micro-organisms involved in the carbon cycle (Fig. 2) the majority were anaerobic cellulolytics, with densities about 106 per gram d.c., followed by amylolytics and pectolytics (about 105 per gram d.e.), and aerobic cellulolytics had lowest densities (102 per gram d.e.). Among the micro-organisms related to the nitrogen cycle (Fig. 3) the proteolytics and the ammonificants were the most abundant, with densities of 10a per gram d.e. The densities of anaerobic free-living nitrogen-fixers (Clostridium) and denitrifiers, both with values about 105 per gram d.e., were also relatively high, whereas, the numbers of ammonium oxidizers (about 104 per gram d.e.), nitrite oxidizers (103 per gram d.e.) and aerobic free-living nitrogen-fixers (Azotobacter) (102 per gram d.e.) were relatively low. Finally, of the micro-organisms associated with the sulphur cycle (Fig. 4), the sulphate reducers and the anaerobic organic sulphur mineralizers showed densities of 104 per gram d.e., the elementary sulphur oxidizers were one order of magnitude lower and the sulphide oxidizers were not detected in any of the three samples studied.
DISCUSSION There were no significant differences among the three samples of poultry excreta studied and they showed similar microbiological, chemical and physical characteristics. The high number of total viable micro-organisms reached densities close to the upper values given for many soils (Alexander, 1967; Berthelin & Toutain, 1979) and was comparable to the populations of composted urban refuses and cattle slurry (Acea & Carballas, 1983; DiazRavifia et al., 1989). However, these densities were much more than a logarithmic order higher than in pig slurry (Rivi6re et al., 1974); these results were in agreement with the fact that chemical and physical properties of fowl faeces were between the limits needed for microbial proliferation (Pochon & Barjac, 1958; Dommergues & Mangenot, 1970) and resembled the ones corresponding to a soil with high chemical fertility (Duchaufour, 1987), but flooded. Bacteria predominate over the other taxonomic groups, as had been found in most soils (Lynch, 1979; Atlas & Bartha, 1981) and organic wastes (Acea & Carballas, 1983, 1988a). However, only a small proportion of bacteria were aerobes. Probably, anaerobic conditions are due to the high water content of faeces and promoted by the elevated microbial metabolic activity common in substrates with high macro- and micro-nutrient content. High anaerobic populations were also found in cattle and pig slurries (Rivi+re et al., 1974; Acea & Carballas, 1983). A small percentage of aerobic bacteria were acidophilics or acid-tolerant and were aerobic spore-forming, and no cyanobacteria were found. The densities of actinomycetes, fungal
102
R. Nodar, M. J. Acea, T. Carballas
units and algae were three, four and six orders of lower magnitude, respectively, than bacterial density. Actinomycetes represented a percentage of the total microbial population lower than those given for most soils (Kfister, 1967; Acea & Carballas, 1986) and some manures (Allievi et al., 1987); also, there was a lower actinomycetes content than in other organic wastes (Diaz-Ravifia et al., 1989). The number of fungal propagules was lower than in soils (Dommergues & Mangenot, 1970) and manures. However, it may be pointed out that the ratio of propagules to mycelia was relatively high because fungal mycelium was almost undetectable. Therefore, poultry excreta showed negative conditions for fungal development, especially if compared with soils (West, 1988). Also unfavoured were autotrophic micro-organisms such as cyanobacteria and algae, less competitive than heterotrophic groups in materials with high organic-matter content (Dommergues & Mangenot, 1970). However, algae abundance was similar to some soils and cattle slurry (Acea & Carballas, 1983, 1986) and higher than in composted urban refuses (Diaz-Ravifia et al., 1989). The largest groups, by far, were those that carry out the breakdown of proteins and the ammonifying organisms; similar predominance was found in many soils and manures (Alexander, 1967; Acea & Carballas, 1983, 1986). By contrast, nitrifiers were scarce. These results are in accordance with the fact that almost all the inorganic nitrogen in poultry excreta is ammonium. This gap between the two steps of nitrogen mineralization may be explained for two reasons: on the one hand, it may be due to the biochemical heterogeneity and ubiquity of micro-organisms bringing about proteolysis and nitrogen ammonification, these two processes occurring in both aerobic and anaerobic environments; on the other hand, since oxygen is an obligate requirement for all species concerned with nitrification, where the O 2 supply is inadequate for both ammonium and nitrate oxidizers, there will be little ammonium oxidation. Denitrifiers were in relatively high densities, probably favoured by anaerobic conditions and a substrate rich in organic materials (Sprent, 1987; Groffman et aL, 1988). The same reasons could have made the density of anaerobic free-living nitrogen-fixers three orders of magnitude higher than the aerobic ones. Except for aerobic cellulolytics, all the studied physiological groups involved in the carbon cycle were present in relatively high densities in poultry excreta, in accordance with the high carbon content of this waste, although anaerobic cellulolytics clearly predominated over the other carbon cycle micro-organisms: results opposite to those found in soils, cattle slurry and composted urban residues (Alexander, 1967; Acea & Carballas, 1983, 1986; Diaz-Ravifia et al., 1989). The populations of amylolytics, pectolytics and aerobic cellulolytics were lower than in soils and other wastes. By
Microbial composition of poultry excreta
103
contrast, anaerobic cellulolytics were found in higher numbers than in soils and in composted urban residues (Acea & Carballas, 1986; Diaz-Ravifia et al., 1989). The anaerobic groups involved in the sulphur cycle clearly predominated over the aerobes. Sulphate reducers and anaerobic mineralizers of organic sulphur were in higher densities than in soils, and in similar number to other wastes (Acea & Caballas, 1986; Diaz-Ravifia et al., 1989). Nevertheless, the sulphate reducers were in lower densities than in cattle and pig slurries (Rivi6re et al., 1974; Acea & Carballas, 1983). The elementary sulphur oxidizers were present in poultry wastes in similar densities to those in soils and cattle slurry, and higher than in urban refuse compost (Acea & Carballas, 1983, 1986; Diaz-Ravifia, 1989). In conclusion, the distribution of the different taxonomic and physiological groups of micro-organisms showed that most of the organic matter of poultry faeces will tend to be incompletely oxidized by anaerobic organisms, and intermediate metabolic products will be produced in higher quantities than in well-aerated soils or wastes.
A C K N O W L E D G E M ENTS The authors thank Mrs Blanca Arnaiz for technical assistance and Dr E. Eiroa for drawing the figures for this paper.
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