Effects on the molecular weight distribution of coal-derived humic acids studied by ultrafiltration

FUEL PROCESSING TECHNOLOGY ELSEVIER

Fuel Processing Technology 52 (1997) 225-237

Effects on the molecular weight distribution of coal-derived humic acids studied by ultrafiltration K. Henning, H.-J. Steffes, R.M. Fakoussa

*

Institute of Microbiology and Biotechnology, l/nicer&y of Bonn, Meckenheimer Allee 168, D-53115 Bonn, Germany

Abstract Low-rank coal derived humic acids were fractionated by ultrafiltration to investigate the effects of various experimental conditions on their apparent ‘molecular weights’. With decreasing pH the apparent molecular weight of the humic acids increased dramatically. Aggregation seems to be the reason for this effect. However, detergents, organic solvents or EDTA had a negligible effect on the fragmentation pattern. Oxidation of the coal with H,O, before the extraction of the humic acids and also the alkaline strength during the extraction influenced the molecular weight of the extracted humic acids to a remarkable degree. Fractionation obtained by ultrafiltration was compared with data of size exclusion chromatography as another technique to determine the molecular weight distribution. 0 1997 Elsevier Science B.V. Keywords: Coal-derived humic acids; Molecular

weight distribution;

Ultrafiltration;

SEC

1. Introduction Humic substances, ‘the dark coloured substances extracted by alkaline solutions from geological deposits’ [l] are widely distributed in nature. They are a heterogeneous mixture of a large number of molecules with different molecular weights and different structures [2,3]. The molecular weight distribution, the elemental composition and the quality and quantity of functional groups like hydroxyl-, carboxyl- or carbonyl groups of this heterogeneous mixture have been investigated. Mainly humic substances from soil or water sources were used in these experiments [4-81. However, only little information is available about humic substances from coal origin. Wildenhain summarized the knowledge in this field for the first time [9]. Polman

* Corresponding author. Fax: +49-228-737576 037%3820/97/$17.00 PII SO378-3820(97)0003

0 1997 Elsevier Science B.V. All rights reserved l-3

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and Quigley described, that by size exclusion chromatography (SEC) the molecular sizes of soluble coal molecules can be reproducibly determined [lo]. Stefanova et al. reported on the concentration of O-containing functional groups in humic acids from different lignites and compared the molecular weight distribution by SEC, too [l]. Piccolo et al. published the elementary composition of molecular weight fractions of coal-derived humic acids obtained by ultrafiltration [ 111. Ralph and Catcheside described also the use of SEC for the determination of the molecular mass of solubilised low-rank coal, but got very high values [12]. All groups used different stationary and mobile phases. We investigated the effects of various experimental conditions on the apparent molecular weight distribution of alkaline extracted humic substances from a Rhenish low-rank coal. This provides a better understanding of how microorganisms solubilize and depolymerize coal at room temperature and normal pressure [13- 181. The structure of the coal itself (e.g. about 70% of this Rhenish lignite are extracted by alkaline solutions, see Section 2) and certain properties of the coal are important to clarify the mechanism of solubilization by these microorganisms. In this paper, the effects of detergents, solvents, pH and other parameters on the molecular weight distribution of coal-derived humic acids are presented. We chose ultrafiltration as a useful technique for the fractionation to investigate the relative distribution of the molecular weights under different conditions and compared the results with those obtained by SEC.

2. Materials and methods For the most experiments a lignite (Rheinbraun AG, Cologne, Germany), called Bergheim, lithotype A, was used. This coal has the following elemental composition: C 100 H**,, Nl,, so.1 %56 and an ash content of 1.3%. In one experiment the lithotypes B-F from the same origin were used. Extraction: 150 mg of the powdered (> 0.2 mm) coal was extracted with 20 ml of 0.5 N NaOH for 48 h at room temperature under nitrogen on a rotary shaker. The dark-coloured supematant was separated from insoluble residue by centrifugation and stored up to 24 h under N, at 4°C. Fractionation: Generally the supematant (humic and fulvic acid) was diluted around 1:10 (adjusting to an optical density (OD) of 7 at 280 nm) and the pH was adjusted to 7.5 with HCl. The dilution to OD = 7 was necessary to get reproducible results. Then different agents (see Results and discussion) were added to the solution. The ultrafiltration process was carried out in a stirred ultrafiltration cell (Amicon Model 8010) under N,. Using Amicon membranes (XM300, YM 100, XM50, PM30, YMlO, PMlO, YM3) different molecular weight (MW) fractions were obtained. Depending on the specific experiment 3, 4 or 5 cutoffs (pore sizes) were used. Ultraviolet absorbance of filtrate and concentrate at 280 nm and 450 nm, respectively, were measured with a Cary 1 spectrophotometer (Varian, Australia) to determine the concentration of the humic acids in every fraction. The absorbance and the dry weight of the fraction, obtained from the desalted freeze-dried sample were proportional to each other and allowed us to use the absorbance as a measure of the concentrations of the humic acids in the fractions.

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Processing Technology 52 f IY97) 225-237

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Size exclusion chromatography (SEC): The HPLC system (LiChroGraph series, Merck, Darmstadt, Germany) consisted of a gradient pump, an autosampler, a diode array-detector and HSM-software. The separation was carried out in a column (PSS-Gel, Hema Bio 40, 10 pm, 8 X 300 mm, PSS-GmbH, Mainz, Germany) with a mixture of 20% acetonitrile and 80% water containing 2 g/l KH,PO, and 5 g/l NaNO, (about pH 9) as mobile phase for aqueous samples (Method 1) and with a Plgel 5 pm column (Hewlett Packard, Waldbronn, Germany) for organic solvents and THF as mobile phase (Method 2). The flow rate was set at 1 ml/min. 20 ~1 aliquots of the fractions, diluted to the same optical density at 450 nm (about l-3 AU), were automatically injected and the absorbance at 450 nm detected in the DAD was used for evaluation. Standards of polystyrene sulfonic acids, sodium salt (PSS-GmbH, Mainz, Germany) were compared with the humic acid samples by their retention time.

3. Results and discussion Preliminary studies with membranes from different companies gave the best reproducible results with membranes from Amicon (data not shown). Amicon membranes with a hydrophilic (YM) and hydrophobic surface (XM or PM) gave the same separation pattern. This fact was very important, because not all cutoffs (pore sizes) from only one type of membrane are commercially available. Additionally these results showed that the charge of neither the surface of the membrane nor the humic acids itself seems to influence the fractionation. 3.1. Influence

of the concentration

for alkaline extraction

There is no general method of alkaline extraction for humic acids! Different reagents and concentrations were used. Fig. 1 shows the fractionation pattern depending on the concentration of aqueous sodium hydroxide. Apart from the absolute amount of extractable substances the molecular weight distribution also differs considerably. When the concentration of NaOH was lowered from 1 mol/l to 0.01 mol/l, the fractions had higher molecular weights. However, when it was further lowered to 0.0001 mol/l and 1 X 10m6 mol/l, the molecular weights decreased. At higher concentrations, an additional breakdown of bonds seems to be responsible for this effect, while very low concentrations loose their strength to dissolve humic acids. Khairy and Ziechmann observed that the fragmentation decreased with the increase of the concentration of dissolved humic acids [8]. The results confirm the importance of using equal concentrations of the alkaline reagents and conditions (temperature, atmosphere [S]) for the extraction and for comparison of any investigation which is made by extracting humic acids. 3.2. Influence

of pH

The influence of different pH conditions on the association behaviour of the humic acids was examined. The pH of the diluted humic acid solutions was adjusted to pH

K. Henning et al. / Fuel Processing Technology 52 (19971225-237

228

100% f MW (kDa)

??
._

~300-100 w >300

7

c NaOH [mol/l] Fig. 1. Effect of the alkali concentration used for the extraction of humic acids from coal” on the molecular weight distribution of the humic acids obtained by ultrafiltration. (* Rhenish brown coal, source: district Bergheim, called lithotype A).

10.0, 9.0, 8.0, 7.0, 5.5, 4.5 and 3.5. A further decrease in pH was not possible because of the precipitation of the humic acids. The molecular weight distribution changed dramatically to higher pretended weights with decreasing pH (see Fig. 2). The carboxylate anion changes to the protonated form and H-bridges are formed to a high degree. The increase in the size of molecules can be caused by aggregation only. In general, the aggregation of molecules has to be considered for all experiments with humic acids because of the high amount of O-containing functional groups and metal ions (ash content), which are involved in several kinds of hydrogen bonding and charge transfer complexes. Similar results were reported about humic substances from fresh waters, admittedly with much lower differences in the molecular weight distribution [4,19]. Visser [20] observed an increase in the viscosity for solutions of humic acids from aquatic and terrestrial origin at lower pHs, which he also explained as a result of aggregation. Although humic acids from fresh water or soil should not be compared with those from lignites (see below). A pH above 7 did not influence the fractionation in any way. This confirms that the change from carboxylate anion to the protonated form is responsible for the increase of the size only. Another kind of confirmation for the assumption of aggregation is the fact that repeated precipitation and dissolution of the humic acids leads to drastic differences in the fractionation at pH 7. 3.3. Effect of detergents, organic solvents and chelators Different concentrations of sodiumdodecylsulphate (SDS, 0.03% and 0.3%) and Tween 80 (0.25%, l%, 2.5% and 5%) were added to the diluted pH adjusted solutions of

K. Henning et al. /Fuel

Processing Technology 52 (1997) 225-237

MW II

229

[kDa]

>30 50-30 100-50

w 300-l

00

?? z-300

O%V pH=3,5

/

/ 4.5

/ 5.5

/ 7

50%

25%

0%

Fig. 2. Apparent molecular weight distribution (coal: lithotype A, Bergheim, Cologne).

of humic acids at different pH values obtained by ultrafiltration

humic acids. The decrease in pH value of the sample after addition of the detergents was readjusted to pH 7.5 with sodium hydroxide. A control without detergents was also investigated. Using the membranes YM 100 and YM 30 three fractions were obtained. With increasing concentration of added SDS the apparent molecular weight shifted mainly from the fraction MW > 100 kDA to the fraction 100 - 30 kDa. While 32% of the sample (refered to the optical density at 280 nm) had a molecular weight of > 100 kDa without SDS, the same fraction contained 22% with 0.03% SDS and 11% with 0.3% SDS only. We did not detect any changes in the smallest fraction (< 30 kDa). In general it was difficult to reproduce these results exactly. Probably especially the detergent is responsible for this effect, because we had this problem in these experiments only. However, the addition of Tween 80 successively increased the percentage of the fraction with the molecular weight > 100 kDa from 20% without detergent to 39% with 1% of Tween 80. The detergent is charge transfer bonded to the humic acids and it is decreasing the association between the molecules. But on the other hand every molecule of the detergent itself has a molecular weight of 1309 Da and so the molecule itself also increased the molecular weight of the detergent-humic acid associate to a high degree.

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It is suggested that Tween 80 may prevent an association between humic acids, but it could not be recommended for this purpose because of its own molecular size. Some other detergents were not useful for ultrafiltration because of the rapid clogging of the pores of the membrane (e.g. N-cetyl-N, N, N-trimethylammonia bromide). Methanol (MeOH), tetrahydrofuran (THF) and dimethyl sulphoxide (DMSO) were chosen to study the influence of organic solvents. Concentrations of 1, 5 and 10% (and MeOH additionally 25%) were used in the present study. Methanol (25% cont.) had no effect on the molecular weight distribution of the humic acids. THF and DMSO decreased the molecular weights only slightly. For instance, the amount of humic acids changed in the fraction MW > 100 kDa from 45.9% without solvent to 38.5% with 10% DMSO. The influence of these solvents is negligible especially if compared to other effects. The effects of chelators on the microbial solubilization of lignite are well described [21,22]. EDTA (ethylene diamine tetraflouroacetic acid, cont. up to 1%) was added as chelator. The pH was readjusted with NaOH after addition. Only very small effects were observed as a shift up to 5% from the fraction MW 100 - 30 kDa to MW < 30 kDa. A possible explanation may be that we used a lignite with a low ash content and a very diluted humic acid solution (see Section 2). 3.4. Preoxidation

with hydrogen peroxide

(H, 0, )

Preoxidation of the lignite with H,O, is a common method in the field of solubilization by microorganisms to sterilize the coal and to make an attack on the coal more easily. The changes that occur in the molecular weights caused by preoxidation were investigated. Powdered coal, (2g), was shaken with 10 ml hydrogen peroxide in concentrations of 0, 1, 3, 6 and 10% for 17 h at room temperature. Later the solid coal was separated by filtration and washed with 10 volumes of water. Dried samples were extracted as described in Materials and methods and the alkaline extracts ultrafiltrated with membranes with the following cutoffs: 10, 30, 100 kDa. Fig. 3 shows the results of the mean of three replicates. A general trend to smaller molecules with higher concentrations of the hydrogen peroxide could be observed. The shifting is not limited to two adjacent fractions, but it can be recognized in all fractions. Hence, it can be concluded that the peroxide is attacking molecules of all sizes. This would facilitate the attack of the microorganisms. Further investigations of those extracts will show if additional effects (O-containing functional groups etc.> are responsible for the easier degradation of such pretreated coal by the microorganisms. 3.5. Preextraction

with an organic solvent

Coal contains aliphatic compounds and waxes besides the humic substances, which can be extracted with organic solvents before the alkaline extraction. In order to know how this hydrophobic ingredients of low molecular weight influence the molecular weight (association) of the humic substances, the coal was extracted one to three times with ethyl acetate before the alkaline extraction (0.5 N NaOH). The following four

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100% /

I

75%

MW (kDa) cl<10

50%

30-10

,

100-30

?? >lOO 25%

,

7 0% L

/

/ 0%

1%

/ 3%

/ 6%

/ 10%

cont. hydrogen peroxide Fig. 3. Effect of preoxidation of lignite with hydrogen peroxide in different concentrations on the molecular weight distribution of the alkaline extracted humic acids measured by SEC (see Materials and methods, coal: lithotype A, Bergheim, Cologne).

fractions were obtained by ultrafiltration: MW > 100, 100 - 30, 30 - 10 and < 10 kDa. The smallest fraction did not show significant differences after the extraction (see Table 1). The removal of the hydrophobic ingredients caused a greater shift from the fraction 100 - 30 kDa to the fraction of > 100 kDa, compared to the total amount of the humic acids; without extraction 21% were found in the largest fraction (> 100 kDa). After the first extraction 28% and after the third extraction 30% were found in this fraction. It could be that the hydrophobic aliphatic compounds and waxes seem to prevent the association between the humic substances. 3.6. Different

lithotypes

All results described were done with one litho type of coal. Lignite from one source is subdivided into various lithotypes based on the colour and stratification. Lithotypes A to F from Rhenish lignite (Bergheim, Cologne) were used in the following experiments. Table

1

Percentage molecular ultrafiltration Times of extraction

0 1 3

weight distribution

of humic acids after preextraction

with ethyl acetate obtained

MW fraction (kDa) >lOO

100-30

30-

21.35 28.15 30.15

64.9 60.0 57.6

10.25 8.5 8.15

10

< 10 3.5 3.35 4.8

by

K. Henning et al./ Fuel Processing Technology 52 (1997) 225-237

232

25%

7 0%

A

B

C

D

E

F

88.5

51.2

Lithotype OD [280 nm]

136.9

46.9

51.2

38.6

Fig. 4. Molecular weight distribution of humic acids from different lithotypes (A-F) (Bergheim, Cologne) and amount of alkaline extract compared by OD[280 nm].

of Rhenish brown coal

Differences between the lithotypes could be detected by the amount, which can be extracted with 0.5 N NaOH (see Fig. 4). It is also shown that the molecular weight distribution (cutoffs 100, 30, 10 kDa) differs to a remarkable degree. The size of the molecular weights extracted correlated with the amount of the humic acids which could be extracted (alkaline extract). Comparing data with other characteristics of the lithotypes like aromaticity, content of water, elementary composition did not lead to more correlations. 3.7. Comparison

with size exclusion chromatography

The results presented above are reproducible with less than 10% total deviation. But it is not useful to compare the values of different methods absolutely, only relative comparisons are possible. As shown very impressively by Linehan and coworkers, it is difficult to get absolute values of the actual molecular weights of humic acids (Table 2, [23]). They compared 8 different methods, where the molecular weights differed from thousands to million Daltons for the same sample! Too many factors like association or adsorption influence the molecular weight of the hydrophilic highly charged humic acids. We chose the size exclusion chromatography (for conditions see Section 2) for comparison to evaluate the results obtained by ultrafiltration. Samples of the above described ultrafiltration experiments were examined. In Fig. 5 the changing of the molecular weights as a result of preoxidation with hydrogen

233

K. Henning et al. / Fuel Processing Technology 52 (1997) 225-237 Table 2 Apparent

average

Trametes

versicolor,

molecular

weights

and molecular

ranges

for leonardite

which has been solubilized

by

taken from Ref. [21] M, (Da)

Method Vapor phase osmometry Ultrafiltration: pH 3.0 or 6.4 pH 10.4 Laser light scattering Dynamic Static Mass spectrometry Gel permeation chromatography: tetrahydrofuran eluent Aqueous eluents Unbuffered pH 7.0 pH Il.5 pH 11.5 using IHSS Suwannee fulvic acid as molecular weight standard “Number average molecular

Molecular

340”

?

> 5oooo

1

50000

weight range

‘1

< 50000 ? <2ooo Cl000 1800

Narrow

800000 11000 6100 570

600000 to 1000000 200 to 50000 200 to 40000 0 to 2500

200

to 20000

weight

peroxide is shown. In all cases the relative shifting of the molecular weight distribution agreed with the results from the ultrafiltration. Molecular weight fractions, obtained by ultrafiltration, were analyzed by SEC to standardize the data. Table 3a compares the molecular weights, obtained by calibration with polystyrene sulfonic acids with the results from ultrafiltration. The structure from those polystyrene sulfonic acids is in our opinion more similar to the humic acids due to their hydrophilic and acid character than in former times often used dextrans [24] or proteins [ 1I.

Fig. 5. Size exclusion chromatography (Method 2) of humic acids after preoxidation hydrogen peroxide (coal: lithotype A, Bergheim, Cologne).

of the brown coal with

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Processing Technology 52 (1997) 225-237

Table 3 a: Comparison of the retention times in size exclusion chromatography (SEC, Method 1) of molecular weight fractions of humic acids obtained by ultrafiltration with those of polystyrene sulfonic acids. b: Fractionation of polystyrene sulfonic acids (PSS) obtained by ultrafiltration with three membranes (used membranes had pore sizes for 100000, 30000, 10000 Daltons, measured by size exclusion chromatography). Left bar: amount of the PSS standard solution of a certain molecular weight, which passed the ultrafiltration membrane, right bar: amount of the PSS standard solution of a certain molecular weight, which was retained by the membranes, Figures in the bars: MW of PSS standards

It can be seen that the molecular weights, determined by standardization with polystyrene sulfonic acids, considerably differ from the cutoffs of the ultrafiltration. A result which is distinct from that of Ref. [12].

Or6

.!?0.3

a J Hc 0,2

0.1 030

117-I, 0

-T”~‘~“l~“~,~~~~r--y-T-

““, 2

1

;

4

5

:““,““,“T”‘,”

6

Retention

2

1

I”“““‘1 8

9

10

11

12

Tiatn (r&i)

04

i:' 'a 0,3 J I? 6,2 O,l o,o ’ “‘I

0

1

2

f”’ i 3

,““q 4

5

6

1

8

9

10

11

12

Retention Tima Wn)

Fig. 6. Size exclusion chromatography lithotype A, Bergheim, Cologne).

(Method

1) of humic acids from (a) coal and (b) soil origin (coal:

K.

Henninget al. /Fuel Processing Technology 52 (1997) 225-237

235

One possible explanation would be that the pore sizes in the ultrafiltration are influenced by association or adsorption of the humic acids to the membranes and therefore are not actual. Another possibility may be due to the interaction between the column material and the humic acids which could be different from that between the sulfonic acids and the column material in the column of the SEC. To answer this question, the polystyrene sulfonic acids were investigated with different ultrafiltration membranes. The results show (see Table 3b) that the polystyrene sulfonic acids do not pass in every case the membranes with a specific cutoff as expected. For instance the sample with MW = 18 500 Da still passed the membrane with a cutoff of 100 000 but not the membrane with a cutoff of 30000. Provided that the commercial standardized samples have the right molecular weight, there must be an interaction between the calibration substances and the membranes. Comparing Tables 3a and 3b, it is evident that these differences do not have the dimension of those observed by SEC. 3.8. Comparison

of humic acids from coal and soil origin

Most of the characterization reported in the literature is done with humic acids from aquatic or soil origin. Humic acids were prepared by alkaline extraction of coal and soil under the same conditions. We could prove by SEC (see Fig. 6) that the molecular weights from the soil humic acids are remarkably smaller than from coal derived humic acids. First peak represents the large molecules, which do not enter the pores of this type of column material. Comparing the height of both peaks, 22.4% of the soil humic acids, but 65.7% of the coal humic acids are located in the first fraction. These results show that more work is necessary to investigate coal derived humic substances for their own characterization compared with the humic acids from soil or aquatic environments.

4. Conclusions Further experiments with additional methods are necessary to get results for the absolute molecular weights of humic acids. One possibility would be the derivatization of carboxylic and hydroxylic groups after the demineralization of the coal to prevent other kinds of possible interactions (H-bonding, chelator effects) between the molecules. But nevertheless both ultrafiltration and size exclusion chromatography are useful methods to assess the relative molecular weight distribution. Also the effects of various parameters on the molecular weights, also caused by microbial depolymerization of coal, can be determined [25,26]. Our results make it easier to evaluate those effects on the molecular weight during incubation of microbial cultures with coal.

Acknowledgements Financial support from the Ministry of Science and Culture of Mecklenburg-Vorpommern and the Ministry of Economics, Small Business and Technology of NorthrhineWestphalia is gratefully acknowledged. We are thankful to Prof. H.G. Triiper, the Max

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Processing Technology 52 f 1997) 225-237

Planck-Institut ftir Kohlenforschung and the Rheinbraun AG for their support. We also thank W. Fritsche and M. Hofrichter of the University of Jena, Germany, for the method of size exclusion chromatography.

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[23] J. Linehan, A.C. Scott, J.A. Campbell, R.M. Bean, Molecular weight determinations of biosolubilized coal, in: Proc. 2nd Int. Symp. on the biological processes of coal, San Diego, USA, Sept. 25-3 1, 1991, p. 23. [24] T. Vartiainen, A. Liimatainen, P. Kauranen, The use of TSK size exclusion columns in determination of the quality and quantity of humus in raw waters and drinking waters, Sci. Total Environ. 62 (1987) 75. [25] G. Willmann, R.M. Fakoussa, Biological bleaching of water soluble coal macromolecules by a basidiomycetes strain, Appl. Microbial. Biotech. 47 (2) (1997) 95. [26] J.P. Ralph, D.E.A. Catcheside, Depolymerisation of macromolecules from Morwell brown coal by mesophilic and thermotolerant aerobic microorganisms, Fuel Proc. Techn. 40 (1994) 193.