CHARACTERISTICS OF ORGANIC CARBON AND NITROGEN FRACTIONS IN WORTS

Dorota Kalembasa
Soil Science and Plant Nutrition Department, Academy of Podlasie, Siedlce, Poland

ABSTRACT

In worts (rye and potato) the total contents of nitrogen and carbon and their fractions were determined for fertilization purposes in agriculture. The rye wort contained more carbon and nitrogen, 442.0 and 32.0 (g.kg^-1), than the potato wort; 340.3 and 28.9. 0.1 mol.dm^-3 of NaOH solution was used for the extraction of humic acids from worts. The carbon extracted by alkaline extract reached 51.0% of its total content in the rye wort and 75.2% in the potato wort. The extracts were acidified to the value of pH 1.5 and got separated into precipitated solid and liquid. The content of carbon in precipitated (solid) phase accounted for 3% of the total carbon in the rye wort and for 19.5% in the potato wort. The precipitated solids were analyzed with the spectrophotometric methods and the results have shown very similar properties as the humic acids extracted from soils. The application of acid hydrolysis allowed for a separation of nitrogen compounds as the following fractions expressed as a percentage of total nitrogen in rye and potato worts were respectively: 1) in non-hydrolysable compounds 88.7 and 87.9; 2) in hydrolysable compounds 11.3 and 12.1. Taking the nitrogen determined in hydrolysable compounds as 100%, the following fractions were separated: a) easily hydrolysable compounds 30.5 and 29.5%; b) not easily hydrolysable compounds 54.0 and 5.5%; c) nitrogen in mineral compounds 15.5 and 65.0%.

Key words: rye and potato worts, fraction of carbon and nitrogen, alkaline and acidic extractions.

INTRODUCTION

Big changes during the last years observed in the fertilization management have been mainly due to a decreasing production of organic fertilizers. The changes have had a negative effect not only on the yield of the plants fertilized but also on the content of organic matter in soils. A deficiency of plant nutrients can be supplemented by the application of mineral fertilizers yet other organic materials should be searched for to substitute for organic fertilizers to maintain an adequate amount of organic matter in soil, which is very important for the equilibrium of organic carbon in soil and even, in some cases, for increasing the level of organic matter in soil, especially in light sandy soils. All that is also important for environmental protection. Over the last few years more attention has been paid to the possibilities of utilization of organic waste in agriculture as the source of plant nutrients and organic matter, for reclamation of polluted land and soil amendment.

A considerable amount of waste is produced by the agrifoods industry; the waste varies a lot as it comes from different sources and therefore contains different amounts of nutrients and organic carbon. Most of them can be utilized directly as fertilizers or after biological processing, including composting or vermicomposting.

For the last few years in Poland the production of ethanol, which could be added to gasoline, has been very much discussed about. In this situation a lot of distilleries will increase the production of wort as a waste in the biotechnological process. Right now in Poland ethanol is produced in distilleries and a total amount of worts produced accounts for 2.5 m m³ and is likely to go up. In Poland ethanol is mainly produced from winter rye and potato and therefore as a waste product from distilleries - it will be a rye and potato wort [8]. The results published recently have pointed out that worts contain big quantities of plant nutrients and low quantities of heavy metals [3,11,12,13]. The application of worts significantly increased the yields of plants, content of carbon and nitrogen in soils as well as improved the chemical composition of the plants harvested [18]. Following German reports [6,7], the mineralization of worts in soils enhances the uptake of nutrients by plants. Similarly worts probably contain humic substances which facilitate the plant growth [5]. In agricultural literature there is not sufficient information about the content of carbon, nitrogen or their fractions in worts.

The aim of the present study was to determine the total content of carbon and nitrogen and their fractions in rye and potato worts for fertilization purposes in agriculture.

MATERIAL AND METHODS

The wort samples were collected over 1999-2002 at the Krzesk and Mordy distilleries in the vicinity of the city of Siedlce, eastern Poland. The worts were taken as fresh samples once they were produced (12 samples from each distillery). Winter rye grain and potato tubers were purchased from local farmers and used as a raw material for the production of ethanol. The chemical composition results for the worts investigated have been published in other papers [11,12,13]. The worts were produced from completely different plant parts in which also the content of carbon and nitrogen differed a lot. The wort samples were dried in the vacuum using the rotary evaporator, ground and screened through the 0.25 mm diameter-mesh sieve.

1. The samples dried were exposed to the following analyses:

Fractional composition of carbon according to IHSS method [1], briefly: the samples of 1 g of wort were shaken with 100 cm³ of 0.1 mol.dm^-3 NaOH over 4 hours and separated by centrifuging into two fractions: liquid and solid, which was repeated twice. The liquid was collected into 250 m³ volumetric flasks and filled up with 0.1 M NaOH up to the mark. This was the first alkaline fraction of the worts which was taken for further analysis. The residue in the test-tube for centrifuge was three times rinsed with deionized water and 50 cm³ of 0.25 mol.dm^-3 of sulphuric acid (VI) was added and acid hydrolysis was carried out over 24 hours. Once the sulphuric acid (VI) had been removed, the residue was three times rinsed with deionized water and the alkaline extraction was repeated exactly as described for the first extraction. This is how the second alkaline fraction was separated. In both fractions the total content of carbon in organic compounds was determined [14]. Simultaneously from the dried worts samples 1 gram samples were extracted with 100 cm³ of 0.1 mol.dm^-3 of NaOH following the Schnitzer method [20], modified by Persons [19]. In extracts the content of total carbon in organic compounds was determined with the oxidation method [14]. The extracts were acidified to the pH 1.5 with sulphuric acid (VI) and left for 24 hours, humic acids (HA) were precipitated, separated from fulvic acids (FA) by centrifugation. In the solution of fulvic acids the content of organic carbon was determined and the content of carbon in humic acids was calculated as the difference between the content of carbon in the extracts and in the solution with fulvic acids.

The humic acids after precipitation and centrifugation were treated at room temperature for 36 hours with aqueous solution of the mixture of hydrochloric and hydrofluoric acids (990 cm³ H₂O + 5 cm³ HCl + 5 cm³ HF). The ash content in the humic acids was below 1.0%. The purified humic acids were dried in vacuum and used for further investigation which involved the following analyses:

1. contents of C, H and N with the method of elemental analysis, using the C/H/N analyzer by Perkin Elmer. The content of oxygen was calculated by the subtraction of the sum of the above elements from the 100% value. The content of the elements determined was presented as mass and atomic percentage. Based on the content of atomic percentage, the degree of internal oxidation of humic acids was calculated using the following equation:

where:
O, N, H and C - the content of elements determined expressed in atomic percentage;

2. VIS spectra of humic acids solutions in the range from 400 to 600 nm;

3. value of Δlog K = log K₄₀₀ - log K₆₀₀, where K is the absorbance of humic acids solution at the wavelength of 400 and 600 nm;

4. ratio of the value of absorbance measured at 465 and 665 nm wavelength expressed as E₄/E₆ coefficient.

2. In the fresh samples the fractional composition of nitrogen compounds in worts was carried out separating the following fractions [10]:

1. nitrogen in mineral compounds: 10 grams of fresh sample material was extracted over 1 hour with 100 cm³ of 2 mol.dm^-3 of KCl. In the extracts the ions of NH₄⁺ were distilled off with MgO and NO₃^- ions after their reduction with Devard´s alloy;

2. nitrogen in easily hydrolysable compounds was separated by the extraction of the samples with 100 cm³ 0.25 mol.dm^-3 of H₂SO₄ over 24 hours at room temperature. The extracts were mineralized with the Kjeldahl method following Olsen´s method [10];

3. nitrogen in not easily hydrolysable compounds was separated by the extraction of wort samples with 100 cm³ of 2.5 mol.dm^-3 of H₂SO₄ over 24 hours at room temperature. The extracts were mineralized and the amount of nitrogen determined with the Kjeldahl method and by the subtraction of the amount of nitrogen in fraction c - nitrogen in fraction b, the amount of nitrogen was calculated in not easily hydrolysable compounds fraction [10];

4. nitrogen in non-acid-hydrolysable compounds was calculated as a difference between the total content of nitrogen and the nitrogen in not easily hydrolysable compounds.

The significance between the mean values of the parameters investigated was calculated using the analysis of variance (test F Fisher-Snedecor). Where significant, the value of LSD_0.05 was calculated with the Tukey test.

RESULTS AND DISCUSION

The content of carbon (Table 1) in organic compounds (C org) in the rye wort as the mean of all the samples investigated was 442.0 g.kg^-1 of D.M. and was significantly higher than that in the potato wort 340.3 g.kg^-1 of D.M. Out of all the investigated samples of worts, a higher content of organic carbon was extracted in the first fraction than in the second one (Table 1). Taking the total amount of organic carbon as 100%, the following amounts of carbon were extracted: from the rye wort - 45.5% in the first fraction and 5.50% in the second one, which makes up a total of 51.0%, while from the potato wort - 67.3% and 7.90%, which makes up a total of 75.2%, respectively. It means that the amount of organic carbon compounds extracted from the rye wort was significantly lower than that from the potato wort. The organic carbon extracted in the first fraction is loosely bonded with the mineral fraction of worts, similarly like with other organic compounds which are not extracted with the 0.1 mol.dm^-3 of NaOH. In extracts from both worts the content of carbon in fulvic acids (FA) was a few-fold higher (significant difference) than in humic acids (HA) and the values expressed as percentage were: for the rye wort - 97.0 and 3.0, while for the potato wort - 80.5 and 19.5% of the total carbon extracted. The ratio of carbon in HA and FA was very low and differed significantly; it accounted for 0.03 and 0.24 for rye and potato wort, respectively.

The content of the total nitrogen N_t (Table 2) in both investigated worts was similar and in the rye wort amounted to 32.0 and 28.9 g.kg^-1 of D.M. in the potato wort, respectively. In both worts the ratio of C:N was very narrow and in the rye wort it was 13.8 and in the potato wort - 11.8, respectively. The value of the C:N ratio indicates that after the application (introduction) of worts into soils a quick mineralization takes place and therefore a lot of available forms for plant nutrients appear in the soil solution. The fractioning of nitrogen compounds has shown that only from 11.3 to 12.1% of the total nitrogen was observed in hydrolysable form compounds and nitrogen was present mainly in nitrogen compounds which were non-hydrolysable. All that is easily accountable for as following fermentation non-hydrolysable compounds are the only ones which remain in the wort, whereas others undergo different transformations. Such a high amount of nitrogen in non-hydrolysable compounds shows that these forms are rather stable proteins and can undergo mineralization in soil. In both worts the amounts of nitrogen in easily hydrolysable compounds were similar and amounted to 30.5% and 29.5% of the total amount of nitrogen in hydrolysable nitrogen compounds.

Very big (significant) differences were observed between the two worts analyzed in the amount of nitrogen in not easily hydrolysable compounds and in mineral forms. The nitrogen in not easily hydrolysable compounds was in the rye wort nearly 10-fold higher than in the potato wort, which must have been due to different proteins in rye grain from those in potato tubers. It was quite opposite for the mineral forms of nitrogen. This fraction of nitrogen in the rye wort reached 15.5% of the total hydrolysable compounds nitrogen, whereas in the potato wort it was up to 65%, which must have been due to the fact that in cereal grain the content of mineral nitrogen forms is always very low and usually lower than in potato tubers.

The elemental analysis of humic acid extracted from worts (Table 3) has shown the differences in their chemical structure. The content of elements determined expressed in atomic percentage (a) ranged for the rye and potato worts analyzed as follows: C - 33.4 and 28.3; H - 46.7 and 47.7; N - 3.0 and 2.5; O - 16.9 and 21.5, respectively. The content of the elements determined in the humic acids extracted from worts was very similar to the content of those elements in humic acids extracted from soil humus [16,17,20] and from vermicomposts [9,15].

The humic acids extracted from the wort of rye contained more C (+ 5.16%) and N (+ 0.43%) but less H (-0.97%) and O (- 4.65%) than from the potato wort, which must have been due to more compounds of carbon and nitrogen contained in rye grain being very resistant to degradation during fermentation.

Based on the value of H:C ratio, it is possible to define the degree of condensation of humic acid nuclei, as the value is inversely proportional to the aromatic bonds of carbon. According to van Krevelen´s theory [1950], the value of H:C ratio in the range of 0.7-1.5 confirms the existence of aromatic rings coupled with aliphatic chains which contain up to 10 atoms of carbon.

The value of the internal oxidation degree (ω) in the humic acids investigated was negative (-0.117 for the rye wort) and positive (+ 0.099 for the potato wort). The positive value of the internal oxidation coefficient (ω) has shown that humification is connected with carbon oxidation in the molecule of humic acid and indicates a superiority of the aromatic over aliphatic structures in humic acid molecules [17]. Obviously in this case positive and negative values are very low and therefore the discussion is rather difficult.

The VIS spectra of the sodium humate solutions investigated from both worts did not differ considerably (Table 4). The highest absorbance values 0.070 were stated at the wavelength of 400 nm for the humic acids extracted from the rye wort and slightly lower - 0.063 for the potato wort. The optical density of humic acids solution depends upon their chemical structure [2]. An increase in the molecular weight and the degree of condensation of humic acid nucleus increase the optical density of the compounds investigated [2,17]. Schnitzer and Kahn [20] reported on the absorbance of humic acids solution in the visible light increasing with an increase in the following parameters (i) the ratio of carbon in aromatic nucleus to carbon in aliphatic chain (ii) the total carbon content in humic acids (iii) the molecular weight of humic acids. The values of Δ log K for both humic acids analyzed did not differ considerably and amounted to + 0.6586 and + 0.7395 for rye and potato wort, respectively. The value of Δ log K indicates the degree of humification of humic acids [17]. When the values decrease and are close to 0.1-0.2, the humification degree of humic acids increases. The numbers for the humic substances investigated have pointed out to a very low degree of humification. The E₄/E₆ coefficient values representing the ratio of absorbance measured for humic acids solutions at the wavelength of 465 and 665 nm differed slightly and were higher for humic substances extracted from the potato wort (7.50) than from the rye wort (6.33). Lower values of E₄/E₆ coefficient indicate a higher molecular weight of humic acids and a higher degree of condensation of their structure [2,16]. The value below 5 indicates a relatively high degree of condensation, whereas a higher value - a low degree of condensation of humic acids as well as a predominance of aliphatic chains over aromatic rings in the structure of humic acids.

CONCLUSIONS

1. The total content of carbon was higher in the rye than in the potato wort, however the content of carbon extractable with 0.1 mol.dm^-3 of NaOH was higher in the potato than in the rye wort.

2. In extracts from worts out of the total extractable carbon more carbon was determined in fulvic than in humic acids.

3. The total content of nitrogen was higher in the rye that in the potato wort.

4. The content of nitrogen in hydrolysable compounds of the total nitrogen was very low and accounted for 11.3 and 12.1% in the rye and potato wort, respectively.

REFERENCES

1. Aiken G.R., 1985. Isolation and concentration techniques for aquatic humic substances [In:] Humic Substances in Soil, Sediment and Water. Eds. G.R. Aiken, D.M. Mc Knight, R.L. Wershaw, P. Mc Carthy, Wiley, New York, 363-385.

2. Chen Y., Senesi N., Schnitzer M., 1977. Information provided on humic substances by E₄/E₆ ratios. Soil Sci. Soc. Am. J. 41, 352-358.

3. Dechnik I., Filipek T., 1998. Preliminary evaluation of distillery rye wort potential for fertilization. Wyd. Politechniki Szczec., Pr. Nauk. 547, 260-263 [in Polish].

4. Gonet S.S., 2003a. Humus, humic substances, organic carbon - definitions, comments and determination methods [W:] Humic substances in soils and fertilizers. Polskie Towarzystwo Substancji Humusowych Wrocław, 21-29 [in Polish].

5. Gonet S.S., 2003b. Wpływ substancji humusowych na rośliny [W:] Substancje humusowe w glebach i nawozach [Effect of humic substances on plants [In:] Humic substances in soils and fertilizers], Polskie Towarzystwo Substancji Humusowych, Wrocław, 95-104 [in Polish].

6. Gutser R., 1998. Stickstoffumsatz, Lagerverhalten und optimale Verwertung von Schlempe auf landwirtschaftlichen Flächen [Nitrogen transformations, storage and optimum worts use on agricultural areas]. Die Branntweinwirtschaft 1 [in German].

7. Gutser R., Amberger A., 1989. Verwertung von Schlempe als organischer Dünger in der Landwirtschaft [Use of worts as an organic fertiliser in agriculture]. Die Branntweinwirtschaft 129 (11), 178-180 [in German].

8. Jarosz L., 1998. Spirit production and utilization trends in Poland. Przem. Ferm. Owoc. Warz. 8, 29 [in Polish].

9. Kalembasa D., Kalembasa S., Godlewska A., Makowiecki K., 1993. The content of nitrogen and carbon in different fractions of biohumus. Polish J. Soil Sci. 26 (2), 87-95.

10. Kalembasa S., 1995. Application of isotopes ¹⁵N and ¹³N in soil and chemical-and-agricultural studies. WNT Warszawa [in Polish].

11. Kalembasa S., Chromiński P., 2002a. The content of nutrient elements in rye and potato wort. Rocz. Glebozn. 53 (3/4), 113-119.

12. Kalembasa S., Chromiński P., 2002b. The content of B, Cu, Mn, Mo, Zn, Co and Li in rye and potato wort. Chemia i Inżynieria Ekologiczna 9 (12), 1605-1611.

13. Kalembasa S., Chromiński P., 2003. The content of Pb, Cr, Cd, Ni, Fe and Al. in rye and potato wort. Arch. Environmental Protection 29 (1) 141-146.

14. Kalembasa S., Kalembasa D., 1992. A quick method for the determination of ratio in mineral soils. Polish J. Soil Sci. 25 (1), 41-46.

15. Kalembasa S., Kalembasa D., Makowiecki K., Godlewska A., 1994. Elemental composition and spectrophotometric characteristics of humic acids extracted from vermicomposts. Polish J. Soil Sci. 27 (2), 93-101.

16. Kononova M., 1968. Soil organic matter. Pergamon Press Oxford.

17. Kumada K., 1987. Chemistry of Soil Organic Matter. Developments in Soil Science 7, Elsevier Amsterdam.

18. Łabętowicz J., Stępień W., Gutowska A., 1999. Comparison of fertilization value of three distillery worts: potato, rye and vinasse in microplot experiments. Fol. Univ. Agric. Stetin., Agricultura 77, 213-218 [in Polish].

19. Persons J.W., 1989. Isolation of humic substances from soils and sediments [In:] Humic substances and their role in the environment. Eds. F.H. Frimmel, F. Christman, Wiley and Sons, Life Science Research Report 41.

20. Schnitzer M., 1978. Humic substances; Chemistry and reactions [In:] Soil organic matter, Elsevier Amsterdam.

21. Schnitzer M., Kahn S.H., 1972. Humic Substances in the Environment. Marcel Dekker, New York.

22. Van Krevelen D.W., 1950. Graphical statistical method for investigation of the structure of coal. Fuel 29, 269-284.