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Applied Soil Ecology 34 (2006) 62–64

Tryptophan concentration of animal wastes and organic fertilizers

I.A. Arkhipchenko *, A.I. Shaposhnikov, L.V. Kravchenko
Research Institute of Agricultural Microbiology, Podbelsky Shossee 3, St-Petersburg-Pushkin 8, 189620, Russia
Received 15 June 2005; accepted 13 December 2005

The concentration of tryptophan in organic wastes and in several fertilizers produced from them via aerobic or anaerobic
transformation and drying was determined using a high-performance liquid chromatography method. The concentration of both free
and fixed tryptophan present in proteins and peptides was measured. Litter dung contained the highest level of tryptophan among the
organic wastes examined, followed by activated sludge, liquid dung, and wastewater sediment. Among the fertilizers, Bamil,
produced from a mixture of activated sludge and pig-farm wastewater sediment, contained the highest level of tryptophan
(388.5 mg g 1). The detected levels of tryptophan suggest that the application of organic fertilizers can increase the concentration of
tryptophan in soil to physiologically relevant values.
# 2006 Elsevier B.V. All rights reserved.

Keywords: Tryptophan; Activated sludge; Liquid waste sediment; Litter dung; Liquid dung

1. Introduction Organic fertilizers are one possible source of

tryptophan in soil. However, the effect of organic
Organic wastes contain compounds, which are fertilizers on IAA biosynthesis in soil has not been
capable of promoting plant growth (Baca et al., 1994; adequately studied, and only a few publications have
Day and Katterman, 1992). A considerable proportion of evaluated the tryptophan concentration in organic
plant growth enhancement can be attributed to processes substrates used as fertilizers (Hacking et al., 1977;
involving indole-3-acetic acid (IAA), and IAA concen- Vriens et al., 1989) In particular, analyses of tryptophan
tration has been associated with soil fertility (Franken- concentration in microbial fertilizers produced by
berger and Arshad, 1995). The main physiological fermenting animal and poultry wastes are lacking.
precursor of IAA in soil is a free L-tryptophan. Sources of To study potential use of organic fertilizers as a
free L-tryptophan include microorganisms and plant root source of exogenic tryptophan in soil, a determination
exudates (Kravchenko et al., 1994). Lebuhn et al. (1994) of the concentration of free tryptophan and tryptophan
detected low natural tryptophan levels in soils. This low in proteins and peptides, both in organic wastes and in
natural tryptophan concentration makes it necessary in the fertilizers produced from them, was conducted.
some cases to augment soils with additional tryptophan to
enhance the microbial biosynthesis of auxins (Arshad
et al., 1995). 2. Materials and methods

The analyzed materials included (Table 1): (a)

poultry plant litter dung; (b) pig-farm activated sludge;
* Corresponding author. Tel.: +7 812 476 30 17;
(c) sediment from pig-farm liquid waste; (d) liquid
fax: +7 812 476 16 10. plants dung from poultry; (e) microbial fertilizer
E-mail address: (I.A. Arkhipchenko). ‘‘Omug’’ produced from poultry litter dung with a

0929-1393/$ – see front matter # 2006 Elsevier B.V. All rights reserved.
I.A. Arkhipchenko et al. / Applied Soil Ecology 34 (2006) 62–64 63

Table 1 Table 2
Characteristics of organic wastes and fertilizers Tryptophan concentration in organic wastes and fertilizers
Fertilizers and Moisture Content of mineral Fertilizers and Tryptophan concentrationa (mg g 1)
initial wastes (%) elements (%) initial wastes
Free Conjugated Total
Litter dung 247.3  30.4 212.8  25.5 460.1  55.9
Litter dung 45 2.8 1.8 1.6
Activated sludge NDb 363.3  27.8 363.3  27.8
Activated sludge 96 8.8 5.0 1.2
Liquid waste ND 141.5  10.2 141.5  10.2
Liquid waste sediment 94 6.2 3.4 1.2
Liquid dung 75 5.7 2.7 3.2
Liquid dung 11.3  1.8 214.3  15.7 225.6  17.5
Omug 15 3.6 1.6 1.0
Omug ND 24.1  2.3 24.1  2.3
Ecud 11 1.6 1.8 0.4
Ecud 3.0  0.5 14.8  1.5 17.8  2.0
Bamil 10 5.0 1.8 0.8
Bamil 240.0  8.2 148.5  17.5 388.0  27.5
Data are shown as averages of five replicated experiments with
hot air (60 8C) blow through for 8 h; (f) microbial standard deviation.
pelleted fertilizer ‘‘Ecud’’ produced from the poultry ND: not detected.
dung following anaerobic fermentation at 40 8C for 7–8
days; (g) microbial pelleted fertilizer ‘‘Bamil’’ pro- wastes varied depending on the substrate type, with
duced from a 1:1 mixture of the activated sludge and values ranging from 1.2 (litter dung) to 0.05 (liquid
pig-farm waste sediment, dried at 80 8C. dung). The activated sludge and the wastewater sediment
The concentration of free tryptophan was determined had no detectable free tryptophan. The highest free
in water extracts for the substrates under study. tryptophan concentration was found in litter dung
Determination of fixed tryptophan was done after (247.3 mg g 1). These data much higher than results
isolation of the protein from the experimental samples of Hacking et al. (1977), who, using a microbiological
and its alkaline hydrolisis (Metodyi, 1962). The resulting method, determined the concentration of accessible
extracts and hydrolisates were evaporated to dryness, tryptophan in dried litter dung to vary between 0.3 and
dissolved in 0.5 ml methanol and passed through nylon 3.5 mg kg 1 depending on the storage duration before
membrane filters (pore size 0.45 mm). Quantitative drying. According to the results presented here, all initial
analyses of tryptophan were carried out using a JASCO wastes are characterized by a high concentration of the
LC-900 series HPLC system (JASCO International Co., fixed tryptophan, probably due to the high protein
Ltd., Japan) equipped with UV-975 UV detector, and a concentrations. Several authors have noted a high content
computer with BORWIN (JMBS Developments, France) of protein in an activated sludge (Lerch et al., 1992;
chromatography software. Tryptophan was separated Vriens et al., 1989), and large amount of protein in
with LiChrosorb RP-C18 (Sigma, USA), 5 mm column manure have also been observed (Iniguez-Covarrubias
(size 250 mm  4.6 mm) using an isocratic gradient of et al., 1986). Tryptophan, lysine, and threonine are often
water–acetonitrile–acetic acid (83:17:0.2; v/v/v). The growth-limiting amino acids for livestock, and diets
elution rate was 0.9 ml min 1 Column temperature was supplemented with high levels of these amino acids are
33 8C and elution pattern was detected at 220 nm. The indispensable in modern animal husbandry. High
presence of tryptophan in the effluent of the analyzed tryptophan levels might be expected in animal wastes,
samples was judged by comparison with retention times given that most domestic animals show low rates of
of pure tryptophan standards of known concentration. tryptophan assimilation (Cervantes et al., 1995; Schutte
Results are the mean of five experiments with et al., 1995).
standard deviation. The tryptophan concentration in fertilizers produced
from organic wastes varied considerably depending on
3. Results and discussion the type of fertilizer. The largest total concentration of
tryptophan, 388.5 mg g 1 (0.04% of the sample
The tryptophan concentration of the organic wastes weight), was observed for the Bamil fertilizer sample,
and the organic fertilizers produced from them are produced from a mixture of activated sludge and pig-
indicated in Table 2. All organic wastes had a high total farm wastewater sediment. The amount of this amino
concentration of tryptophan. Litter dung contained the acid in Bamil was more than 10 times higher than the
highest concentration of tryptophan followed by acti- fertilizers produced from poultry dung. This can
vated sludge, liquid dung, and sediment of the liquid probably be explained by the decrease in tryptophan
wastewaters. The ratio of free and fixed tryptophan in the concentration, which occurs during processing of the
64 I.A. Arkhipchenko et al. / Applied Soil Ecology 34 (2006) 62–64

poultry wastes into fertilizers (Table 2). For the Omug Baca, M.T., Femandezfigares, I., Denobili, M., 1994. Amino acid
and Ecud fertilizers, only 5.2% and 7.7% of the initial composition of composting cotton waste. Sci. Total Environ. 153
(1/2), 51–56.
tryptophan concentration were retained, respectively, Cervantes, M., Cromwell, G.L., Stably, T.S., 1995. Synthetic
after processing. amino acid supplementation in the low protein grain–sorghum–
According to the data of Lebuhn et al. (1994), as well soybean meal diet for pigs. Cuban J. Agric. Sci. 29 (2), 197–
as Lebuhn and Hartmann (1993), the concentration of 208.
Day, A.D., Katterman, F.R.H., 1992. Sewage sludge provides plant
tryptophan in different types of soil varies within the
growth factors in arid environments. J. Arid Environ. 23, 229–233.
range of 0.08–5.8 mg kg 1. Current recommendations Frankenberger Jr., W.T., Arshad, M., 1995. Auxins in phytohormones
for the application of organic fertilizers suggest use of in soils. Microb. Product. Funct. 17–136.
1–5 g fertilizers per kg of soil. According to our results, Frankenberger Jr., W.T., Chang, A.C., Arshad, M., 1990. Response of
this level of fertilizer use would augment the soil with Raphanus sativus to the auxin precursor, L-tryptophan applied to
0.02–1.9 mg exogenous tryptophan per kg soil. The soil. Plant Soil 129, 235–241.
Hacking, A., Dervish, M.T., Rosser, W.R., 1977. Available amino acid
physiological effect of exogenous tryptophan manifests concentration and microbiological condition of dried poultry
themselves within a range of 0.01–3 mg kg 1 of soil manure. Br. Poult Sci. 18, 443–448.
(Arshad et al., 1995; Frankenberger et al., 1990). Thus, Iniguez-Covarrubias, G., Franco-Gomez, M.D.J., Pena-Romero, M.,
the application of biofertilizers, such as those studied Ciurlizza-Guizar, A., 1986. Evaluation of the protein quality of
here, has the potential of raising the soil tryptophan solids recovered from hog manure slurry. Agric. Wastes 16, 113–
concentration to physiologically relevant levels. Kravchenko, L.V., Leonova, E.I., Tikhonovich, I.A., 1994. Effect of
root exudates of non-legume plants on the response of auxin
Acknowledgments production by associated diazotrophs. Microb. Release 2,
Lebuhn, M., Hartmann, A., 1993. Method for the determination of
We are grateful to Dr. G. Kowalchuk, Department of
indole-3-acetic acid and related compounds of L-tryptophan cat-
Plant–Microorganism Interaction, Netherlands Institute abolism in soils. J. Chromatogr. 629, 255–266.
of Ecology Centre for Terrestrial Ecology for his Lebuhn, M., Heilmann, B., Hartmann, A., 1994. Effects of drying/
comments and helpful criticism on the manuscript. This rewetting stress on microbial auxin production and L-tryptophan
study was carried out within the framework of the catabolism in soils. Biol. Fertil. Soils 18, 302–310.
Netherlands–Russian joint project No. 047.002.003 Lerch, R.N., Barbarick, K.A., Sommers, L.E., Westfall, D.G., 1992.
Sewage sludge proteins as labile carbon and nitrogen sources.
‘‘Development of biotechnological methods for manure SSSAJ 56, 1470–1476.
treatment focused on fertilizer production’’ financed by Metodyi, 1962. In: Pleshkov, B.P. (Ed.), Analiza belkov i aminokislot
the Netherlands Organization for Scientific Research v rasteniyakh, Metodicheskoye rukovodstvo, VIR, Leningrad,
(NWO). p. 40.
Schutte, J.B., Verstraten, A.J.M.A., Lenis, N.P., Dejong, J., 1995.
Requirement of young pigs for apparent ileal digestible trypto-
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