J Sci Food Agric 1996,72,493-500

A Comparative Study of the Effect on Barley Growth of Humic Substances Extracted from Municipal Wastes and from Traditional Organic Materials Miguel Ayuso, Teresa Hernandez,* Carlos Garcia and Jose A Pascual Department of Soil and Water Conservation and Organic Waste Management, CEBAS-CSIC, PO Box 4195, Murcia, Spain (Received 2 January 1996, revised version received 28 May 1996; accepted 28 June 1996)

Abstract: The effect on barley growth of humic substances extracted from municipal wastes with different degree of stability was studied. This effect was compared with that of the humic substances extracted from organic materials used in industry to obtain humic acids (peat and leonardite). The humic sub- stances extracted from municipal wastes had higher aliphatic character, lower oxygen group content and a more heterogeneous composition than the humic substances from peat and leonardite. All humic substances, regardless of their origin, nature and characteristics increased plant yields (from 38 to 62% of the increase) and macronutrient absorption with respect to the control when they were added at different amounts (5, 100 and 200 mg C kg-') to a calcareous soil. This increase was highest with the humic substances from municipal wastes, which in turn had the greatest positive effect on the absorption of nitrogen and phosphorus up to 74% increase of N and 72% increase P with sewage sludge HS at 200 mg C kg-' by barley plants.

Key words: barley plant, commercial humic acid, compost, humic substances, leonardite, macronutrient, peat, sewage sludge, yield.

INTRODUCTION

Humic substances constitute a fundamentally important fraction of organic matter, and many authors assign them a primary role in soil fertility because of the func- tions they carry out. They positively affect nitrogen and phosphorus dynamic in the soil (Figliolia et a1 1990; Biondi et al 1994), favour microbial metabolism (Alianello et al 1991), directly or indirectly stimulate a plant's absorption of nutrients (Guminski and Glabis- zewski 1983; Raina and Goswami 1988), stimulate plant photosynthesis and respiration (Flaig 1968; Chen and Aviad 1990), etc.

Generally, the greater or lesser activity of humic sub- stances has been related with the number of oxygenated groups, particularly carboxylic and phenolic groups (Piccolo et al 1992a). Some authors have suggested that

* To whom correspondence should be addressed.

the fractions of least molecular weight show the highest degree of bioactivity (Nardi et a1 1994), while others have suggested that it is the heaviest fractions which show such activity (Malcolm and Vaughan 1979).

The organic matter content of many soils of Mediter- ranean countries has diminished drastically during recent years, the following reasons being cited: unsuit- able agricultural practices such as the overapplication of mineral fertilisers, intensive cultivation, the non- rotation of crops (factors which provoke mineralisation of the soil's organic matter content) or the absence of organic amendment due to the scarcity of organic sources and the fall in animal manure production.

The relation between a soil's fertility and its organic matter content is clear and so it is not surprising that the restitution of the latter will benefit the former. For- tunately, many of the wastes produced by human activ- ity, such as sewage sludge and municipal solid wastes, can be used as organic amendment in agriculture and in

493 J Sci Food Agric 0022-5142/96/$09.00 0 1996 SCI. Printed in Great Britain

494 M Ayuso et a1

the recovery of degraded soils, either directly or after composting (Hernandez et al 1992). The use of such products is doubly beneficial since the environmental impact of storing them is lessened and a continuously produced product of little economic value can be put to profitable use.

There has been increased interest in using humic sub- stances as a way of incorporating organic matter in a soil because of the growing use of drip irrigation as a consequence of diminishing water supplies. Indeed, there is a wide variety of commercially available pro- ducts, which are alkaline extracts of the humic sub- stances from normally very humified organic materials such as leonardites, peat, etc. However, they are expen- sive and come from limited resources.

In this study, we made a comparative study of the characteristics of the humic substances extracted from municipal wastes of different degrees of maturity and those extracted from the organic materials usually used in commercial humic acid manufacture and we evaluate the effects of these substances on plant growth and nutrient absorption. For this, plant growth was studied in a calcareous soil to which different doses of these humic substances were added.

MATERIALS AND METHODS

The urban wastes used were an aerobic sewage sludge from a waste water depuration plant and a compost made from an aerobic sewage sludge mixed with the organic fraction of a municipal solid waste in a 1 : 1 total organic carbon ratio. Composting was carried out in an open air pile with occasional turning (Indore system). The composting phase lasted 105 days and the pile was turned seven times. The maximum temperature reached was 68°C and humidity was maintained at 45-

65% throughout. After composting, the material was left undisturbed for three months for the organic material to stabilise. The organic materials studied, which are widely used in industry to obtain humic acids, were a leonardite from Teruel (Spain) and a black peat from Granada (Spain). We also used a widely available peat based commercial humic acid (HA). The main characteristics of the materials used are shown in Table 1.

The humic substances (HS) of the organic materials were extracted by shaking the samples with 0.1 M P,O,Na, at pH 9.8 for 24 h. The extracts were centri- fuged at 13200 x g and the supernatant was dialysed until Na+ and K + cations were removed, and finally ly ophilised.

Elemental analysis

Elemental analysis of the humic substances was per- formed by gas chromatography on a C H N Hewlett- Packard 185 microanalyser. Oxygen was calculated by difference.

Functional analysis

Total acidity was determined by the barium hydroxide adsorption method and carboxyl groups by the Ca- acetate method (Schnitzer and Khan 1972). The amount of acidic-OH groups was calculated by subtracting the -COOH content from the total acidity content.

Gel filtration

A 500 mg sample of lyophilised humic substances were dissolved in 0.1 M NaOH and the pH adjusted to 8.3. After 3 days the suspension was centrifuged to remove

TABLE 1 Characteristic of the organic materials

Parameters Sewage Compost Peat Leonardite Commercial sludge H A

PH EC (dS m-') Ash (g kg- ') TOC" (g kg-') Extractable C (g kg- ') Humic C (g kg-') Fulvic C (g kg- ') Humic C/fulvic C Total N (g kg-') Total P (g kg-') Total K (g kg-') C P

6.52 3.41

279.50 364.40 75,70 16.60 59.10 0.28

50.50 8.10 2.80 7.20

7.52 3.01

668.00 122.50 19.10 6.00

13.10 0.45

13.20 9.10 5.50

13.28

5.17 1.23

59.60 487.40 40.80 29.80 11.00 2-64 9.50 1.20 3.00

51.30

3.20 2.75

361.00 347.40 190.50 175.40 15.10 11-60 5.60 0.20 3.00

62.03

12.98 56.50

410.40 151.20 ND*

133.00 18.20 7.30 5.60 0.60

123.00 27.00

TOC, total organic carbon. ND, not detected.

Effect on barley growth of humic substances extractedfrom municipal wastes 495

TABLE 2 Soil characteristics

PH Electrical conductivity (dS m- ') Water holding capacity (Yo) Organic matter (g kg-') Total organic carbon (g kg-') Extractable carbon (g kg-') Humic carbon (g kg-') Fulvic carbon (g kg- ') Water-soluble carbon (g kg- ') Total nitrogen (g kg- ') N-NO, (g kg- ') N-NHf (g kg-') Total phosphorus (g kg-') Available phosphorus (g kg- ') Total potassium (g kg- ') Available potassium (g kg- I)

7.94 0.19

17.00 6.71 3.90 0.60 0.40 0.20 0.1 1 0.26 0.06 0.02 0.30 0.02 4.76 3.42

insoluble materials, transferred to a 100 ml flask and made up to volume with distilled water. Sephadex gels were allowed to swell in distilled water for 24 h before they were placed into the columns. Two types of 'Sepha- dex' gels, G-100 and G-150, were used in this study. The column used was 2.5 x 45 cm. To permit the gel bed to stabilise, it was percolated with 300 ml of eluent. Its height was maintained at 40 cm at all times. Samples of 3 ml were carefully placed on top of the Sephadex gel and eluted with C0,-free distilled water following the procedure suggested by Schnitzer and Skinner (1966). The eluates were passed with continuous flow through a spectrophotometer and absorbance was registered at 465 nm.

The fractions corresponding to individual peaks were generally characterised by their K,, , determined by the ratio V , - V,/KV, (where V, is the void volume of gel evaluated with dextran blue 2000, is the total volume of gel, V , is the elution volume corresponding to the peak).

Plant growth experiment

For the plant growth experiment barley was planted in a calcareous soil (see Table 2), to which different

amounts of the products representing 50, 100 and 200 mg of C/Kg of soil were added. These growth experiments were carried out in quadruplicate, 10 barley (Hordeum vulgare) plants being planted in each 500 cm3 container. Growth conditions were as follows: 16 h of light (25°C and 75% RH) and 8 h of darkness (20°C and 80% RH).

Plant material was washed in distilled water, dried at 65°C and ground. The following analyses were made: N, using a colorimetric method based on Berthelot's reaction (Crooke and Simpson 1971); P and K in the nitric perchloric digestion extract, P by the Murphy and Riley (1962) method and K by flame photometry.

RESULTS AND DISCUSSION

The carbon content of the HS from municipal wastes (sewage sludge and compost) were within the levels indi- cated by other authors (Deiana et al 1990; Piccolo et al 1992b). The commercial HA and the HS from peat and leonardite showed a slightly higher carbon content than those of the municipal wastes (Table 3); the levels recorded were similar to those of the HS extracted from soil (50-65%) (Schnitzer 1978). The greater H content of the HS obtained from the sewage sludge and compost revealed their greater aliphatic character due to the dif- ferent intensities of the oxidation and humification pro- cesses undergone by the materials. The processes of aerobic digestion and composting (both of which last a comparatively short period of time) cannot be com- pared with the long natural processes of oxidation undergone by leonardites and peats. The highest N levels occurred in the municipal wastes (Table 3). This was to be expected since the municipal wastes showed the highest N values (Table I), the nitrogen coming from the bacterial cell proteins, polypeptides and other nitrogenated compounds present in residual waters and incorporated in sewage sludges (Ayuso 1995). Some of these nitrogenated compounds are not degraded during the short transformation processes suffered by these materials and remain associated to the humic molecule (Hernandez et al 1993).

TABLE 3 Results of the elemental analysis of the humic substances'

Humic substances YO C % N % H YO 0 HJCb O/Cb CJN

Sewage sludge HS 47.61d 7.51a 8.13a 36.75d 2.05 0.57 6.3

Peat HS 49.92b 2 .04~ 4 .74~ 43.30a 1.13 0.65 24.5 Leonardite HS 53.27a 1-01d 3.94d 41-78b 0.88 0.58 52.7 Commercial HA 52.85a 2 . 1 1 ~ 5 . 0 1 ~ 39.63~ 1.15 0.58 24.6

Compost HS 48.4% 6.02b 5.96b 39.57~ 1.48 0.61 8.0

' For each parameter data followed by the same letter are not significantly different at the 5% level according to the LSD test.

Atomic ratios.

496 M Ayuso et a1

The H/C ratio decreases as the aromatic nature and molecular condensation of the humic substances increases (Garcia et a1 1992). The low levels of this ratio in the HS from the leonardite, peat and commercial HA (Table 3) may be due to the fact that they have more aromatic and condensed compounds than the corre- sponding substances from municipal wastes. The high H/C ratio of the sewage sludge HS shows their highly aliphatic character and the possible extraction of non- humic compounds such as lipids, polysaccharides, etc. (Steelink 1985). The lower H/C ratio of the compost HS (Table 3) compared with those from sewage sludge, is due to the condensation and enrichment in aromatic structures that they undergo during composting (Diaz 1990; Garcia 1990). The C/N ratio varies with the quan- tity of nitrogen, this ratio pointing to the great differ- ences existing between the HS obtained from municipal wastes and those from more evolved materials (Table 3).

The total acidity values are higher in the HS from leonardite, peat and commercial HA, as is to be expected in materials which have undergone lengthy oxidation processes (Table 4). However, the difference between the total acidity of the HS from these materials and those from municipal wastes is not very great due to the higher proportion of fulvic acids, which are richer in oxygenated groups than humic acids (Roletto and Luda 1987), in the latter. Table 1 shows that the humic C/fulvic C ratios are considerably lower in sewage sludge and the compost ( c l ) than in the leonardite, peat and commercial HA.

The different degrees of oxidation of the various HS becomes clear when we look at the phenolic/carboxylic acidity ratio (Table 4), which decreases as the degree of oxidation increases (Garcia et al 1992). The values observed confirm that composting involves an increased degree of oxidation of the humic substances, while the HS of sewage sludge remain the least oxidised of all.

The oxygenated functional groups largely determine the behaviour of HS in the soil, since they influence the cationic exchange capacity of the organic matter, nutri- ent chelation, etc. According to this the most oxidised HS (in our case those of the leonardite, peat and com- mercial HA) would be the most effective when they are incorporated in the soil.

The more clearly defined peaks which are observed in the elution curves for the leonardite HS and commercial HA (especially those obtained with the G-150 gel) showed that these HS were the most homogeneous as regards molecular size (Fig 1). De Nobili et al (1989) pointed out that the fractions of greatest molecular size (excluded fraction) decrease in number and those of intermediate size (intermediate, included fraction) increase as the organic matter evolved. According to this, the HS extracted from the leonardite, peat and commercial HA are more humified than those of the sewage sludge. The molecular sizes of the HS from the compost were more similar to those of the more evolved materials than to those of the sludge, suggesting that the composting process breaks down the most poly- merised compounds with smaller more condensed com- pounds being produced.

The high percentage of the excluded fraction of the sewage sludge HS (Tables 5 and 6 ) suggests that their humic fraction is of large molecular size, probably due to the union of aliphatic chains to the humic nucleus. This excluded fraction may be due to the existence of compounds of a lipidic nature (Inbar et a1 1990) which have not been broken down because the sewage sludge has not undergone any process of oxidation. These same compounds are responsible for the excluded frac- tion observed in the HS from the compost (which also contains sewage sludge). The excluded fraction of the HS obtained from the peat and commercial HA is prob- ably due to the presence of partially degraded structures from plant remains such as lignins, carbohydrates, etc (Inbar et a1 1990).

Regardless of their origin, all the HS significantly enhanced plant growth compared with the control in every doses applied; however, except in the case of the HS from sludge and compost, increased doses did not result in any significant change in yield (Table 7). This suggest that HS introduce favourable elements for plant development (oxygenates functional groups, phytohor- monal effect, improvement in nutrient uptake, etc). On the other hand, the fact that increased doses do not nec- essarily bring about improved yields can be explained either by the fact that the HS also introduce some factor which limits plant growth or by a saturation effect.

TABLE 4 Functional groups of the humic substances"

Humic substances Acid total -COOH -OH OH/COOH

Sewage sludge HS 746c 312d 434a 1.39a Compost HS 681d 348d 333bc 0-95b Peat HS 823b 439c 384b 0.87b Leonardite HS 980a 696a 284c 0 .40~ Commercial HA 857b 577b 280c 0 . 4 8 ~

" For each parameter data followed by the same letter are not signifi- cantly different at the 5% level according to the LSD test.

Effect on barley growth of humic substances extracted from municipal wastes 497

G-1 00 G-150

SEWAGE SLUDGE AB SEWAGE SLUDGE

T n 0,W

0 50 loo 150 200 0 SO 100 150 200 250

COMPOST AbS

0.4

0 50 100 150 200 ml

0.1 iE!A 0 0 SO 100 150 200 ml

COllAIlYRClAL HA 1 .&P

0.4

0 50 100 150 200 ml

LEOWARDITE AbS

0 50 I00 150 200

AbS COMPOST

0 50 100 150 200 250

0 50 100 150 200 250 ml

COArrmERCIALHA 1..

0.2 iiu 0 0 50 100 150 200 250

LEOWARDITE Abs 3..

0 50 100 150 200 250 ml

Fig 1. Sephadex G-100 and G-150 filtration of a commercial humic acid and humic substances extracted from sewage sludge, compost, peat and leonardite using C0,-free distilled water as eluent.

The HS from the compost and sewage sludge not only produce the greatest increase in yield (by a signifi- cant margin when compared with the other HS studied), but they are the only substances whose effect on growth increases at higher doses (Table 7). This is probably because they are derived from materials with an unde- graded (sewage sludge) or only slightly degraded (compost) organic matter, which contain a higher con-

centration of low-molecular-weight compounds than the other materials (see the relative concentrations of fulvic and humic acids in Table 1). These compounds result in a higher degree of bioactivity in the short term (Nardi et a1 1994). There is a clear difference in the behaviour of the humic substances from sewage sludge and compost, increased doses of the former increasing plant yield while increased doses of the latter have the

498 M Ayuso et a1

TABLE 5 Elution constants (KaJ and fractions of the humic substances through Sephadex G-100

Humic ( K a J Distribution percentages substances

Fexcluded Fintermediate Fincluded Fercluded Fintermediate Fincluded

- - Sewage sludge 0 1.01 95.3 4.7 Compost 0 0.39 1 .oo 23.2 70.1 6.7 Peat 0 0.47 1.05 31.3 50.6 18.1 Leonardite 0 0.27 1.08 28.4 31.2 40.4 Commericial HA 0 0.38 1 .oo 31.1 34.7 34. I

opposite effect. This difference is probably due to the different capacity of the humic substances to affect plant metabolism, either because they favour to a greater or lesser extent nutrient absorption or because of their dif- fering phytohormonal action (Dell'Agnola and Nardi 1987; Muscolo et a1 1993). The fall in yield recorded with the compost HS may be caused by their acting as phytohormones in low doses and as phytotoxins in high doses, which does not occur with the sewage sludge HS.

In general, we think that a concentration of 50 mg of C kg-' should be considered as the optimal dose for producing a positive effect on plants, even in the case of the HS from sewage sludge, higher doses of which result in even higher yields (Table 7). However, it does not

seem worthwhile increasing the doses four fold (from 50 to 200 mg C kg-') to obtain an increase of 10% in yield.

The results obtained from analysing the nutrients in the plant material (Tables 8-10) show that application of all the humic substances favours nutrient uptake. There is little likelihood that the quantity of nutrients supplied by the HS would be responsible for the increased absorption of nutrients by plants since the quantity in question is negligible compared with that already existing in the soil (Table 2). For this reason any increased absorption must be the result of an indi- rect effect of the HS on the plants. The HS derived from originally stable material such as leonardite, peat and

TABLE 6 Elution constants (KaJ and fractions of the humic substances through Sephadex G-150

Humic ( K a v ) Distribution percentages substances

Fexcludcd Fintermediate Fincluded Fexcluded Fintermediafe Fincludcd

Sewage sludge 0 0.71 0.94 67.0 24.7 9.8 Compost 0 0.71 0.92 21.6 66.1 12.3 Peat 0 0.76 0.91 29.6 43.2 21.2 Leonardite 0.19 0.69 1.09 6.0 73.5 20.5 Commercial HA 0 - 0.90 19.0 81.0 -

TABLE 7 Barley yield in the soils treated with different doses of humic substances (% as regards control

yield)

Doses (mg C k g - ' ) Sewage Compost Peat Leonardite Commercial LSD sludge HS H S H S HS H A

0 100 100 100 100 100 - 50 145 163 153 141 143 10

100 150 162 143 I42 138 11 200 162 148 150 148 140 9

LSD" 10 14 11 10 11

LSD, least significant difference.

EfSect on barley growth of humic substances extracted f rom municipal wastes 499

TABLE 8 Nitrogen content in barley plants (YO dry wt) as affected by different doses of humic substances

Doses Sewage sludge Compost Peat H S Leonardite Commercial LSD (mg c k7-’1 H S H S H S H A

0 0.82 0-82 0-82 0.82 0.82 - 50 0.99 1.11 0.94 1.14 0.90 0.06

100 1.38 1.17 1.19 1.18 0.99 0.06 200 1.43 1.23 1.26 1.12 1.11 0.05

LSD 0.08 0.08 0-08 0.09 0.09

the commercial HA have a less favourable effect on nitrogen and phosphorus absorption as the doses increases, than the HS from sewage sludge and compost. This suggests that the latter group of materials contain humic substances partly formed of less stable compounds, which are therefore more active than those of the former group.

A plant’s uptake of nitrogen is greatly enhanced by the action of humic substances of small molecular weight (Dell’Agnola and Nardi 1987). This explains why the sewage sludge HS, which are richer in fulvic acids than the others, favour nitrogen absorption and leads to a greater growth when plants are treated with these substances. Phosphorus absorption is significantly stimulated by all the HS studied, particularly by those

obtained from the municipal wastes (which also have a greater effect as the doses increases). This is probably because the HS increase the availability of phosphorus by forming easily assimilable phosphohumates (Raina and Goswami 1988). The HS also favour potassium absorption, but there are no significant differences between the different doses.

The results demonstrate that humic substances extracted from municipal wastes (sewage sludge and compost) are more effective than those extracted from the commonly used materials. For this reason, it seems clear that the use of municipal wastes as new sources of organic matter in the production of liquid biofertilisers opens up new and commercially attractive perspectives for the biofertiliser industry. At the same time it will

TABLE 9 Phosphorus concentration in barley plants (mg kg- * dry wt)

Doses Sewage sludge Compost Peat H S Leonardite H S Commercial LSD (ms c kg - 7 H S H A

0 772 772 772 772 772 - 50 1118 939 1033 91 1 1005 88

100 1100 1097 1162 1214 1018 86 200 1331 1403 1205 1310 1060 125

LSD 138 75 105 140 95

TABLE 10 Potassium content in barley plants (% dry wt)

Doses Sewage sludge Compost Peat Leonardite S H Commercial LSD (mg c k7-7 SH S H SH H A

0 3.52 3.52 3.52 3.52 3.52 - 50 3.58 3.75 4.08 3-82 3.80 0.20

100 3.65 3.75 3.83 3.95 3.80 0.22 200 3.70 3.68 3.90 3.88 3.53 0.21

LSD 0.21 0.30 0.2 1 0.27 0.29

500 M Ayuso et a1

help avoid a potentially harmful environmental problem which would otherwise be created by their accumulation.

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