709

Acidophilic sulphate-reducing bacteria: candidates for bioremediation o f acid

mine drainage

A.M. Sen and B. Johnson

School of Biological Sciences, University of Wales, Bangor, Gwynedd, Wales, UK, LL57
2UW

Acid mine drainage (AMD) is currently one of the most widespread forms of pollution
worldwide. It is mainly anthropogenic in nature, resulting from mining activities. AMD
occurs as an end result of the oxidation of sulphide minerals, producing high concentrations of
sulphate. These oxidation processes are accelerated by activity of iron-oxidising
chemolithotrophic microorganisms. Acidophilic sulphate-reducing bacteria (aSRB) exist in
anaerobic sediments in acid mine drainage environments, and can reduce sulphate to sulphide.
This results in the precipitation of metal sulphides and the release of hydrogen sulphide.
Current biotechnological processes employ neutrophilic SRB to remove metals and sulphate
from acidic wastewaters; however, a pre-treatment step is necessary in order to produce the
circumneutral pH required by the bacteria, aSRB therefore have a potentially important role in
bioremediation of AMD, in view of their ability to withstand low pH. The culture and
potential use of aSRB for this purpose is examined.

Acidophilic sulphate-reducing bacteria were sampled from acid mine drainage environments
in Wales, UK, and the Caribbean island of Montserrat. These were used as inocula for bench-
scale bioreactors, where enrichments for aSRB and investigations into alkalinity generation
by the mixed cultures following acidification were carried out. This was demonstrated using
one mixed culture at a pH as low as 1.73. Sulphate reduction by two immobilised mixed
cultures in batch systems was determined in order to identify the optimum pH for
bioremediation using the aSRB. Samples taken from the bioreactors were used to inoculate
overlay plates at pH 3 and 3.6.

1. INTRODUCTION

Acid mine drainage (AMD) is widely recognised as a critical environmental problem facing
the mining industries. It has been recently estimated that in the U.S.A. alone, around 500
billion gallons are produced annually from acid-generating sites, affecting up to 17,000 km of
streams (21). The estimated cost for preventing or treating pollution from these sites is in the
order of billions of dollars (7). In the U.K., around 100 or more streams are affected by AMD,
710

and the problem has gained increased significance with the recent accelerated decline in the
mining industries (1, 26).
AMD is often highly acidic (pH < 3), or potentially acidic, and characteristically contains
elevated concentrations of iron and a variety of other metal species, as well as sulphate. It is
formed when sulphide minerals in mining spoil heaps, tailings or adits undergo chemical and
biological oxidation processes. Mining allows oxygen to be introduced into the deep
geological environment where such minerals are normally in a reduced state (26). The
oxidative dissolution of sulphide minerals (most notably pyrite, FeS2) is greatly accelerated
by acidophilic metal-mobilising bacteria such as Thiobacillus ferrooxidans and
"Leptospirillum ferrooxidans' (19). These reactions occur in several stages, though the overall
reaction may be summarised as:

FeS2 + 3.7502 + 3.5H20 h~ Fe(OH)3 + 2H2SO4 (1)

Streams impacted by AMD are characterised by their yellow-orange coloration, with

sediments coated by ferruginous deposits ('ochre') which are highly detrimental to benthic
fauna. In addition, the acidity of AMD contributes to the severe deterioration of water quality
both directly and through promoting elevated solutions of other heavy metals. This
combination of factors can result in considerable toxic effects on aquatic and riparian life
(11). AMD can also enter groundwater, which is often pumped to the surface for human use
(18).
Conventional ('active') treatment of AMD by adding an alkaline agent (usually lime) results
in an increase in pH and the formation of a voluminous sludge of metal hydroxides and
gypsum, which is difficult and expensive to dewater and dispose of (5). The costs of
maintaining an active system can be substantial, and since mines can discharge acidic
ferruginous discharge waters for many years after they cease active production, other cheaper
and long-term solutions to the problem have been sought. Bioengineering approaches for
treating AMD and similar effluents have been described, with the most well-known being the
"Thiopaq' process which currently treats about 5000 m 3 of acidic, zinc-rich effluent/day at the
Budelco zinc refinery in the Netherlands (5). However, the most significant alternative to
active treatment of AMD is the use of natural or constructed wetland ecosystems (8, 24).
These effect amelioration of AMD by the various alkaline-generating processes that occur
within them, particularly in the anaerobic sediments (12). Processes of dissimilatory iron
reduction and sulphate reduction have been postulated to be important reactions which effect
AMD remediation by lowering the acidity of inflowing AMD and concentrations of soluble
metals (19). The reduction of sulphate to sulphide by sulphate reducing bacteria (SRB) is
generally considered to be of particular importance. Dissimilatory sulphate reduction, under
anaerobic conditions, generates net alkalinity by converting a strong acid (sulphuric acid) to a
weak acid (hydrogen sulphide). Many heavy metals form highly insoluble sulphide minerals,
and biogenic sulphide production can result in a very effective removal of these metals:

2CH20 + SO42""-1"H + - - - ~ 2H20 + 2CO2 + HS (2)

M 2+ + HS'------~ MS + H § (3)

The dissimilatory reduction of ferric iron and sulphate reverses, in essence, the reactions of
pyrite oxidation, which are responsible for the production of AMD. The metal sulphide
 711

precipitates formed by this process are much denser than the bulky ferric iron precipitates
formed by active treatment of AMD, and are stable so long as contact with oxygen is
precluded.
Recent studies have shown that dissimilatory reduction of ferric iron can be carried out by a
wide variety of acidophilic bacteria, which colonise AMD and impacted watercourses (19).
However, truly acidophilic SRB have not been isolated and characterised, despite the process
of sulphate reduction having been demonstrated in acidic, metal-rich environments (9, 25).
Indeed, sensitivity to even moderate acidity (pH <6) is a feature of known SRB. Johnson et al.
(16) used glycerol to enrich for acidophilic SRB (aSRB) using anaerobic "acid streamer'
growths as inoculum; a culture was obtained which was able to reduce sulphate in a medium
poised initially at pH 2.9 (though no lower). A spore-forming, Desulfotomaculum-like
bacterium was observed in the culture, which was not characterised further. More recently,
Hard et al. (10) demonstrated bioremediation of acid mine drainage using SRB, with
methanol as an electron donor. The use of non-acidic substrates for enriching for aSRB rather
than compounds such as lactic acid (which is widely used in enrichment cultures for
neutrophilic SRB) has particular advantage; organic acids are known to be toxic, in general to
acidophilic bacteria (13). The use of aSRB in a bioengineered system for treating AMD
would have the major advantage that these bacteria (unlike those used in present systems)
would not be inhibited or killed by direct contact with the acidic effluent, thereby reducing
operating costs (e.g. lime addition).
This paper describes new methodologies that have been developed for isolating and
culturing aSRB, and demonstration of sulphate reduction and acid consumption by mixed
populations of aSRB in acidic, metal-rich liquors under laboratory conditions.

2. METHODS

2.1. Enrichment cultures of acidophilic SRB

Enrichment cultures of aSRB were set up using a basal salts/yeast extract medium
containing (in g/1 distilled water): (NH4)2SO4, 0.45; KC1, 0.05; MgSO4.7H20, 0.5; KHEPO4,
0.05; Ca(NO3)E'4H20, 0.014, and yeast extract, 0.2. Glycerol (at 10 mM, f'mal concentration)
was provided as the electron donor. The medium was adjusted to about pH 4 with sulphuric
acid and dispensed (-~ 25 ml) into universal tubes and heat sterilised (120~ for 20 minutes.).
After cooling, ferrous sulphate from a 1 M pH 2.0 filter-sterilised stock solution was added to
tubes to give a final concentration of 10 mM ferrous iron, the inoculum sediment or streamer
sample added, and the tubes completely filled with basal salts/yeast extract medium. The final
pH of inoculated tubes varied with the nature of the inoculum added, but was generally
between pH 3.0 and 3.5. Cultures were incubated at 30~ for up to three weeks, with periodic
inspection for SRB activity (blackening of cultures as a result of the production of FeS and an
increase in pH). Positive tubes were subcultured into larger (250 ml) bottles containing the
same medium.
The materials which formed the inocula for these cultures were: (a) acid streamer material
taken from about 30 cm depth within the main stream in the derelict Cae Coch pyrite mine
(14,15); blackened sediment from about 20 cm depth below the Afon Goch stream draining
the Parys copper mine; (c) samples of sediment from the White River and various geothermal
sites on the Caribbean island of Montserrat (Johnson et al., unpublished data).
712

2.2. Bioreactor cultures of acidophilic SRB

Three cultures of acidophilic SRB obtained by the above enrichment technique were
subsequently grown as batch cultures in laboratory-scale bioreactors. No attempt was made to
obtain pure cultures of aSRB at this stage in the research programme. Two 2 L fermenters
were used: (i) LH Fermentation Series 502D; (ii) Electrolab 300 Series. These were run in
two modes. In the first instance, the cultures were adjusted to a pre-determined pH value, and
the ability of cultures to generate alkalinity under these conditions was recorded. Temperature
was maintained at 25~ and cultures were unstirred except immediately prior to sampling (to
ensure homogeneity). If a positive result was obtained (i.e. a significant rise in culture pH),
the medium was drained (-~25% of total volume), replaced with fresh medium and the culture
poised at a lower pH value using 1M sulphuric acid. This experiment was continued to the
point at which alkali generation by each of the mixed aSRB cultures was inhibited by low pH.
In a second series of experiments involving aSRB in bioreactor cultures, the enrichment
cultures obtained from the Parys mine sites and from Montserrat were grown in batch mode at
constant pH, and rates of sulphate reduction, acid consumption (0.1 M hydrochloric acid) and
substrate disappearance were monitored. The medium used in these experiments was the same
as used in the enrichment cultures but with 0.02 g/1 yeast extract and 10 mM ethanol as
electron donor instead of glycerol. Cultures were sparged with nitrogen to remove hydrogen
sulphide. Temperature was maintained at 30~ and very slow stirring (20 rpm) was provided.
Three cultures of acidophilic SRB obtained by the above enrichment technique were
subsequently grown as batch cultures in laboratory-scale bioreactors. No attempt was made to
obtain pure cultures of aSRB at this stage in the research programme.

2.3. Growth of acidophilic SRB on solid media

A variety of solid media were screened for their ability to support the growth of aSRB. Most
of these were based on the "overlay' technique, which has been shown to promote colony
growth of heterotrophic as well as autotrophic acidophilic microorganisms (17). A medium
based on the ferrous iron overlay formulation (17) proved particularly successful. This
medium contained 0.02% yeast extract, 5 mM ferrous sulphate and 5 mM potassium sulphate,
gelled with Sigma type 1 agarose (0.5%, final concentration) and with a final pH (of the
combined medium) of pH 3.5. Glycerol (at 10 mM) was included in some solid media
formulations. Samples from enrichment cultures were streaked onto solid media and
incubated at anaerobic jars ('Anaerogen' system, Oxoid, U.K.) for 1-3 weeks, at 30~

2.4. Analytical techniques

Solution pH was measured in situ in bioreactors, using Ingold of Mettler-Toledo InPro
combination pH electrodes, and checked ex situ on an occasional basis using a pHase rapid
renew glass electrode (BDH Ltd., U.K.). The surface pH of gelled solid media was measured
using a combined pH/reference cell with a flat-surfaced membrane (Russell pH Ltd., U. K.).
Redox potential was measured in situ using a Metier-Toledo combination redox electrode,
and ex situ using a combined platinum/silver-silver chloride redox cell (Russell). Sulphate
was determined using the turbidometric method described by Rand et al. (23). Glycerol was
measured using a colorimetric method using formazan (22), and ethanol using an enzyme-
coupled assay in a standard analytical kit (Boehringer Mannheim Gmbh).
 713

3. RESULTS

3.1. Establishment of enrichment cultures of aSRB

Positive enrichments were obtained from all three source materials. These were identified
by the blackening of cultures (due to the formation of FeS), increased pH and the obvious
small of hydrogen sulphide when the culture bottles were opened. In the case of the
Montserrat inocula, positive results were obtained with about 25% of those used. The cultures
were pooled to provide the "Montserrat' aSRB culture. This culture and those obtained from
the Parys and Cae Coch mines, were repeatedly subcultured in glycerol/yeast extract/ferrous
sulphate medium, though no attempt at obtaining pure cultures was made at this stage.

3.2. Demonstration of the acid neutralisation potential of mixed aSRB cultures

Bioreactors, containing glycerol/yeast extract/ferrous sulphate medium were inoculated with
about 500 ml of active aSRB cultures. Once the cultures had established (i.e. blackening, due
to formation of FeS), the potential of the mixed cultures to generate alkalinity following
exposure to episodes of acidification was investigated, as described above. The result
obtained with the mixed aSRB culture from the Parys mine is shown in Figure 1. Whereas all

pH (units) Redox Potential (mV)

4.80 . - 350

,.. :', ,, : -,,,

4.00 / "'\ ~t / '"',,\ ~,,, / / ' 250

/ i ) ,: ,, r
3.20 150

2.40 50

1.60 -50
15 25 35 45 55 65

Time (days)

Figure 1. Alkalinity generation by aSRB following acidification. Arrows indicate

replacement of medium and acidification of culture. Changes in pH ( ) and redox
potential ( ................) in the bioreactor culture are shown.

three aSRB cultures were able to effect a pH rise in cultures that were acidified to a lower
limit of pH 3.0, only the Parys culture showed a positive response when acidified to values of
714

pH < 3.0. Net alkalinity was produced with cultures of the Parys aSRB in media adjusted to
pH values as low as 1.73, but was notably less effective at pH values of <2.0.

3.3. Sulphate reduction and glycerol utilisation by a mixed aSRB culture

Following preliminary experiments using bioreactor cultures, others were set up to examine
more closely the relationship between the activities of aSRB and acid neutralisation. In the
first of these, the relationship between sulphate reduction, glycerol utilisation and alkali
production by the culture obtained from the Cae Coch mine was examined; again, there was
no pH control on the culture. Data, shown in Figure 2, confirmed that reduction of sulphate
and catabolism of glycerol occurred in this culture and therefore, by inference, in the others.
The pH of the culture increased during the phase of active sulphate reduction, and continued
to increase slowly after the time that sulphate was apparently depleted.

Sulphate/Glycerol (mM) pH (units)

16 7.50
14
- o/..ty.
12 i 6.50

10
5.50
8
6
4.50
4~
2 3.50
0 P

-2 ~ ~ . . . . ~ 2.50
0 2 4 6 8 10 12 14 16 18

Time (days)

Figure 2. Sulphate reduction and glycerol utilisation by mixed aSRB (Cae Coch culture).
Disappearance of sulphate ( 9 ) and glycerol (O), and changes in pH ( O ) are shown.

3.4. Sulphate reduction by immobilised aSRB grown in pH-controlled batch cultures

Batch culture bioreactor systems were set up to conduct experiments with immobilised
aSRB at controlled pH. Acid-washed porous glass beads (2-4 mm diameter, Dennert Poraver
Gmbh, Germany) were used to immobilise aSRB in bath cultures. Bioreactors were
inoculated with colonised beads,-200 ml of sterile larger (5-6 mm diameter) beads added,
together with culture medium (pH 3.5) and the cultures allowed to establish ahead of
experimental runs. The culture medium was also modified in these experiments (section 2.3).
Ethanol (10 mM) had been shown to be an effective electron donor for all three aSRB cultures
 715

and was used in place of glycerol, and the concentration of yeast extract used was lowered by
an order of magnitude. Data from one of these experimental runs, in which pH was controlled
at pH 4.0 are shown in Figure 3. Sulphate reduction was found to be concomitant with
hydrogen consumption and the disappearance of ethanol in this culture. The aSRB culture
from the Parys mine site has displayed active reduction in bioreactor cultures set at pH 2.5,
3.0, 3.5 and 4.0, while the Montserrat culture appears to be less acidophilic at pH 2.5.

Sulphate/Ethanol (raM) H+(mM)

12 25
c

10 - 20
20

15

)
100

0
0 2 4 6 8 10 12 14

Time (days)

Figure 3. Sulphate reduction, acid consumption and ethanol utilisation in a bench-scale

bioreactor system with immobilised aSRB (Parys culture). Disappearance of sulphate ( o ) and
ethanol ( § and consumption of acid (H+; V ) are shown.

3.5. Growth of acidophUic SRB on solid media

Successful growth of aSRB from all three cultures has been achieved using overlaid solid
media. In contrast, colony growth on equivalent non-overlay media was found to be very poor
and non-reproducible. A medium formulation containing yeast extract and ferrous iron has
been particularly successful in promoting rapid (within one week) growth of aSRB. Larger
colonies were o~en obtained by the addition of 10 mM glycerol to plate media. Acidophilic
SRB grew as small, white to off-white, round, entire colonies on the acidic (pH 3.6) solid
media; with time these became increasingly blackened due to the deposition of FeS as the pH
increased with the developing colonies (Fig. 4a). With more prolonged incubation (and
especially in glycerol-containing plates) the entire plate surface became blackened, and the
aSRB colonies themselves frequently developed a metallic sheen (Fig. 4b).
716

Figure 4. (a) colonies (- 2 weeks old) of acidophilic SRB from Cae Coch mine, grown on
ferrous sulphate/yeast extract overlay medium; (b) older (-~ 4 weeks) colonies of acidophilic
SRB from Montserrat, grown on ferrous sulphate/yeast extract overlay medium supplemented
with l0 mM glycerol, showing extensive deposition of ferrous sulphide within colonies and
on the plate surface; (c) scanning electron micrograph of acidophilic SRB from Parys mine,
grown on ethanol/ferrous sulphate medium.

4. DISCUSSION

Acid mine drainage (AMD) is a worldwide pollution problem which requires cost-effective
treatment solutions. The two mostly widely used remediation strategies (liming and the use of
wetlands) both have major drawbacks in terms of recurring costs (liming) and effectiveness
(wetlands). Bioremediation of AMD using SRB provides an alternative, and in many ways
more effective, solution to the problem, as has been demonstrated in bench-scale, pilot-plant
and full-scale operations (3, 4, 5, 6, 10, 21, 25). However, current technologies necessarily
use neutrophilic SRB, which means that, at least in some situations, pre-treatment of acidic
effluents with alkali is required upstream of the SRB reactor in order that the biomass is not
damaged or rendered inactive. The main attraction of using acidophilic SRB in such a process
is that they would be adapted to, and tolerant of, the acidity and (presumably) the elevated
concentrations of iron and other metals in AMD, so that pre-treatment of AMD could be
avoided. Whether, in this theoretical scenario, aSRB alone would be capable of effecting
adequate improvement in AMD quality to its release into the environment is not known. It is
 717

conceivable that they could be used in conjunction with neutrophilic SRB, in which the initial
stages of AMD amelioration is brought about by aSRB, beyond which neutrophilic species
assume a more important role.
The results from the experiments described in this report show clearly that SRB isolated
from acidic environments are capable of actively reducing sulphate in def'med acidic culture
media. The three isolates demonstrated different degrees of acid tolerance, with that of the
Parys mine culture being appreciably greater than that of the Montserrat and Cae Coch
cultures. Interestingly, the SRB in the latter two cultures were morphologically very similar
(both were spore-forming Desulfotomaculum-like bacteria, similar to that described by
Johnson et al. (16), whereas the Parys isolate grew as much larger (5 ~tm + in length) motile
rods which also formed terminal endospores (Fig. 4c). Future work will include screening for
more acidophilic strains of aSRB from environmental samples.
On occasions, particularly during the early stages of this work, problems had been
experienced in maintaining and transferring cultures of aSRB. One way in which the handling
of these bacteria could be greatly improved was to immobilise them onto a solid matrix. Other
researchers, working with neutrophilic SRB, have experienced similar results (2). The support
material selected, "Poraver' beads, had earlier been used successfully as a matrix for SRB in
fixed bed bioreactors (20). They have the advantage of being highly porous (about 80% pore
space) and chemically inert, following acid-washing to remove the small amounts of
carbonate present.
The minimum redox potential values recorded in these experiments were considerably
higher than those normally associated with SRB cultures. However, given that the pKa of
hydrogen sulphide is 6.88, the major product of sulphate reduction in the aSRB cultures
would be gaseous H2S rather than soluble HS (which is more prevalent with neutrophilic
SRB). Sparging cultures with nitrogen gas provides a ready method of removing much of the
dissolved hydrogen sulphide gas from culture solutions.
One reason for the success in obtaining cultures of aSRB described in this report probably
stems from the use of a non-acidic organic substrate (glycerol) in enrichment cultures.
Standard media for enriching for SRB often contain large concentrations (- 10 mM) of
organic acids, such as lactic acid which, for reasons described earlier, is highly toxic to many
acidophilic micro-organisms (though it is possible that organic acids are metabolised by aSRB
when supplied in small concentrations). Results from the current study suggest that both
glycerol and ethanol are incompletely oxidised by the three aSRB cultures (data not shown).
The batch culture experiments so far have all involved aSRB in cultures that are assumed to
be mixed. However, it is now possible to obtain pure cultures of these bacteria by single
colony isolation on overlay solid media. Future work will seek to characterise these bacteria
both in terms of their physiologies (e.g. substrate utilisation and metabolic products) and
phylogenetically (from sequence analysis of their 16S rRNA genes).

ACKNOWLEDGEMENTS

This work was funded by an Industrial CASE studentship from BBSRC, supported by BNFL.
The authors thank BNFL scientists for their support and input into this research programme,
and Dr. K. B. Hallberg (ofUWB) for providing invaluable discussions and assistance.
718

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