The effect of acidity is most marked in nutrient-poor waters and this isreflected in its impact on the biota at all levels. The effect may be direct,for example on the physiology of fish and invertebrates, may involvemobilization of the toxic element aluminium, may reduce primaryproduction by altering the community structure of the algae, or may beindirect through its impact on the processing of organic carbon. We donot intend to provide a major review of the acid rain literature, as thisinformation is readily available elsewhere (Norton et al. 1989) as arereports of detailed case studies (Kerekes 1989). In this brief report weshall concentrate on the effect of low pH on the initial stages ofdecomposition and the conditioning of incoming particulate carbon ordetritus by microbes, particularly certain genera of filamentous bacteria

Bernard N. Simon

J. Gwynfryn Jones

FBA Journal System (Freshwater Biological Association)

200 B. M. SIMON AND J. G. JONES 
SOME OBSERVATIONS ON THE ABSENCE OF BACTERIA FROM 
ACID WATERS IN NORTHWEST ENGLAND 
BERNARD M. SIMON AND J. GWYNFRYN JONES 
(Mr B. M. Simon and Professor J. G. Jones, 
Institute of Freshwater Ecology, The Windermere Laboratory, 
Far Sawrey, Ambleside, Cumbria LA22 OLP, England) 
Introduction 
The effect of acidity is most marked in nutrient-poor waters and this is 
reflected in its impact on the biota at all levels. The effect may be direct, 
for example on the physiology of fish and invertebrates, may involve 
mobilization of the toxic element aluminium, may reduce primary 
production by altering the community structure of the algae, or may be 
indirect through its impact on the processing of organic carbon. We do 
not intend to provide a major review of the acid rain literature, as this 
information is readily available elsewhere (Norton et al. 1989) as are 
reports of detailed case studies (Kerekes 1989). In this brief report we 
shall concentrate on the effect of low pH on the initial stages of 
decomposition and the conditioning of incoming particulate carbon or 
detritus by microbes, particularly certain genera of filamentous bacteria. 
In nutrient-rich waters, primary production and the sedimentation of 
the remains of the primary producers result in the development of an 
organic-rich benthic layer. This, in turn, permits the development of 
anaerobic zones; indeed, the depth to which aerobic decomposition 
occurs rarely exceeds a few millimetres. In the presence of suitable 
electron acceptors, particularly nitrate and sulphate, populations of 
denitrifying and sulphate-reducing bacteria develop, and the end-
products of their metabolism result in a net generation of alkalinity 
(Hanselman 1986; Kelly et al. 1982; Schindler et al. 1986; Davison & 
Woof 1990). These processes have been put to good effect in the 
recovery and management of extremely acid waters (Davison 1990; 
Davison et al. 1989). 
The effects of acidification are therefore most likely to be observed in 
well-oxygenated, nutrient-poor waters, in which there is less scope for 
the full range of bacterial metabolism to be expressed. This is not to 
imply that the inhibition of anaerobic metabolism is the sole controlling 
factor; there is an even greater wealth of evidence for the direct impact 
of low pH and aluminium toxicity on the biota of such waters. However, 
with a few exceptions (Gahnstrom & Fleischer 1985; Hoeniger 1985), in 
which the methods used were either environmentally irrelevant or of 
ABSENCE OF BACTERIA FROM ACID WATERS 201 
insufficient sensitivity, most studies have shown that decomposition is 
slower in such susceptible, nutrient-poor acid waters (Hoeniger 1986; 
Hildrew et al. 1984; Burton et al. 1985; Mackay & Kersey 1985; Rao et 
al. 1984; Rimes & Goulder 1986; Traaen 1980). In some of these 
investigations, low pH apparently had no effect on the numbers of 
bacteria present, but.activity per unit cell number and/or the mean cell 
volume were both reduced under acid conditions. 
Although bacterial decomposition processes have provided the focus 
for attention in many of the above reports, little attention has been given 
to the composition of the bacterial community and the role of its 
component parts, particularly in nutrient-poor waters which are provided 
with sources of organic carbon and reducing power in the form of poor 
quality detritus. 
The study sites 
Previous long-term research conducted at Windermere, particularly that 
of Carrick & Sutcliffe (1983) and Sutcliffe & Carrick (1983, 1986) 
provided a wealth of background information which permitted the 
selection of sites of widely ranging pH and alkalinity in Cumbria, north-
west England. The subjective assessment of many researchers had been 
that detritus decomposed more slowly in the acid waters and that this 
was associated with changes in the invertebrate fauna. The purpose of 
our brief investigation was to determine whether any differences in the 
bacterial flora might account for these observations. We decided to 
examine the aerobic bacterial populations of detritus in fast-flowing, 
well-oxygenated streams. These would be the most susceptible to acid 
conditions as their metabolic end-products would not generate any acid-
neutralizing capacity. 
The sites were chosen to include the major underlying geological 
strata found in the region (Fig. 1). A preliminary study was undertaken in 
the upper basin of the River Duddon (on bedrocks of the Borrowdale 
Volcanic Series). Two streams, designated 6 and f in Fig. 1, were 
approximately 1 km apart and drained the same side of the valley (Figs 
2, 3). These are small streams ranging from 30 cm to 1 m in width, with 
fast-flowing well-aerated water. The water in stream b had a mean 
annual pH of 6.7 whereas that in stream /was 4.9. The vegetation on the 
banks of both streams and on the surrounding land was largely 
composed of Nardus stricta L. (moor mat grass) but mixed swards of 
other grasses were also present. The main input of detritus into the 
streams occurred during the winter in the form of dead leaves and stems 
of N. stricta, particularly after snowfall and ensuing freeze-thaw cycles. 
This material and the water in the streams were sampled. 
202 B. M. SIMON AND J. C.JONES 
ABSENCE OF BACTERIA FROM ACID WATERS 203 
204 B. M. SIMON AND J. G.JONES 
The bacterial population and its activity 
Although it is recognized that dilution plating of natural samples onto 
artificial media wil l not provide quantitative information about the 
bacterial population, the technique may provide an early indication of 
gross differences in the composition of the microflora. In this instance the 
samples were diluted and spread on casein-protein-starch agar (Jones 
1970). A cursory examination of the agar plates showed immediately 
that there was a marked difference in the bacterial populations which 
had developed. The bacterial colonies from the acid stream /were small 
and mostly white or colourless. The bacteria from the circumneutral 
stream b, on the other hand, often produced spreading, pigmented 
colonies, most of which were yellow. The colonies are shown in the 
colour plate on the front cover of this issue of the Forum (with a legend 
on the inside front cover at the bottom of the page). This provides only a 
qualitative assessment and therefore both water and detritus were 
examined in more detail. Direct counts were performed by 
epifluorescence microscopy (Jones & Simon 1975) and microbial activity 
was measured as oxygen uptake, carbon dioxide evolution and 
tetrazolium dye reduction. The results (Table 1) show that, in spite of 
ABSENCE OF BACTERIA FROM ACID WATERS 205 
the marked difference in pH and alkalinity between the two streams, 
there was little difference in the total bacterial population of the water 
and only a slightly smaller bacterial count under acid conditions. 
However, all measurements of activity indicated that decomposition was 
slower at the lower pH. 
As mentioned above, one of the most marked differences was in the 
absence of yellow spreading colonies on plates inoculated with detritus 
from the more acidic waters. These colonies are distinctive and are often 
found when freshwater samples are examined. However, the organisms 
responsible belong to a taxonomic group which is currently the subject 
of some debate. We have tentatively assigned them to the genus 
Cytophaga and give our reasons for doing so later in this report. 
The above results were obtained from streams on bedrocks of the 
Borrowdale Volcanic Series. However, almost identical findings were 
obtained from an acid stream, pH 4.7 (Fig. 4), and a circumneutral 
stream, pH 6.6, which drain Skiddaw Slates further north in Cumbria 
(sites s and t in Fig. 1). Direct counts of bacteria on the detritus were 
2.2 x 10'° and 2.0 x 1010 per g dry wt respectively, and carbon 
206 B. M.SIMON AND J. C.JONES 
mineralization was approximately halved in the acid stream. Cytophaga 
spp. were undetectable in the detritus of the acid stream yet colony 
counts were the equivalent of 7.8 x 107 g~' in the other, circumneutral 
stream. Clearly, it was necessary to compare a wider range of sites. After 
consultation with Dr D. W. Sutcliffe, 25 streams were examined. To 
reduce the variability caused by sampling "detritus" of uncertain age, 
dead stems of N. stricta were introduced into the streams and examined 
after 1 month. The results (Fig. 5) show clearly that the Cytophaga spp. 
were absent from the more acid waters. On the occasions when a few 
Cytophaga isolates were obtained this usually coincided with streams 
being in spate, or collapse of the banks onto the introduced grass stems. 
We have yet to determine the source of these bacteria. Similar results 
were obtained when the same procedure was applied to 10 lakes and 
tarns of varying pH. 
ABSENCE OF BACTERIA FROM ACID WATERS 207 
Further evidence for the poor pH tolerance of the bacteria was 
obtained with the steady loss of Cytophaga spp. when detritus from a 
neutral stream was transferred to an acid site. The bacteria also 
recolonized detritus moved from acid to neutral water. Addition of 
limestone chips to an acid stream resulted in the rapid appearance of 
Cytophaga spp. In a study of two lakes in Canada, Hoeniger (1986) 
reported that bacteria which resembled Cytophaga were restricted in 
their activity at pH values < 5. 
The taxonomic status of the bacteria 
Although we have tentatively assigned the bacteria which produced the 
yellow spreading colonies to the genus Cytophaga we accept that further 
study wil l be required before positive identification can be confirmed. In 
this instance we considered that it was more important to determine how 
similar the organisms were than to assign them to a particular genus. 
To this end 450 isolates were obtained. Of these, 291 had been 
specifically selected because of their characteristic spreading growth, 
although we must record that other, less numerous, colonial 
morphotypes show a similar, but less marked, site distribution. We chose 
34 of these isolates from a wide range of sites and characterized them on 
the basis of 57 tests. These were conventional phenotypic tests which 
defined growth characteristics (colour, spreading growth, motility, 
gliding, flexirubin pigment, Gram stain, growth at 30°C and 37°C), 
physiology and substrate utilization (oxidation and fermentation of 
glucose, pectin, aesculin, gelatin and cellulose hydrolysis, N 0 3 
reduction to N 0 2 and N2, indole production, assimilation of glucose, 
arabinose, mannose, mannitol, n-acetyl-glucosamine, maltose, 
gluconate, caprate, adipate, malate, citrate and phenyl-acetate), 
possession of certain enzymes (arginine dihydrolase, urease, oxidase, 
catalase, acid and alkaline phosphatase, esterase, lipase, leucine-, 
valine- and cystine-arylamidase, trypsin, chymotrypsin, naphthol-as-B1-
phosphorylase, α and p galactosidase, (3 glucuronidase, α and p 
glucosidase, n-acetyl-β-glucosamidase,, α mannosidase/ α frucosidase), 
and sensitivity to selected antibiotics (ampicillin, oxytetracycline, 
kanomycin, chloramphenicol, streptomycin, erythromycin and 2, 4-
diamino-6, 7-di-iso-propylpteridine phosphate (0/129)). 
All the isolates responded in an identical manner to 38 of the tests and 
all those tested were incapable of growth below pH 5.5 on laboratory 
media. These results indicate a high degree of relatedness and raise the 
possibility that bacteria which are very similar in function are sensitive to 
acid conditions in geographically widely separated sites. The bacteria 
208 B. M. SIMON AND J. G.JONES 
were tentatively assigned to the genus Cytophaga on the basis of the test 
results but particularly because of their yellow pigment, gliding motility 
and growth on glucose mineral salts medium which contained 
ammonium as the sole source of nitrogen (Hayes et al. 1979). Individual 
cells were usually filamentous, being 3 - 5 0 (μm long (Fig. 6) and active 
gliding was observed at the edges of colonies. The isolates obtained 
were clearly very similar, but in the case of such a taxonomically 
"difficult" group as the genus Cytophaga, further characterization is 
required. Small differences in colonial morphology (surface texture, 
depth of colour) often corresponded to differences in response to the 
remaining 19 tests upon which there was not complete agreement. We 
are currently applying the methods of numerical taxonomy to a wider 
range of isolates while, at the same time, comparing the chromosomal 
DNA restriction fragments of some isolates from geographically widely 
separated sites. We thus hope to provide a genetic and phenotypic 
ABSENCE OF BACTERIA FROM ACID WATERS 209 
analysis of a group of bacteria (tentatively assigned to the genus 
Cytophaga) which consistently exhibit sensitivity to acidic conditions. 
The role of the bacteria 
Given the taxonomic uncertainty of the isolates and the degree of 
heterogeneity observed we can only speculate about their role in 
freshwater systems. The order Cytophagales contains gliding filamentous 
bacteria, usually growing or isolated at neutral pH. Many members of 
the group are characterized by their ability to degrade natural polymers 
and we noted that, of our isolates, all were capable of degrading pectin 
and 68% also degraded cellulose, and therefore could play an important 
role in plant polymer decomposition in the streams. This is not to imply 
that bacteria were the only microbes which might contribute to the 
differences in decomposition observed, although some fungi are 
reportedly absent from grass stems in streams of this region with 
pH < 5.5 (Chamier 1987). The role of microbes in invertebrate nutrition 
under such circumstances is worthy of further study, but we must now 
also consider their ability to accumulate elements such as aluminium 
under acidic conditions (Chamier et al. 1989). 
While certain aquatic animals are clearly more sensitive to pH than 
many microorganisms, perhaps the lower level of detritus "conditioning" 
due to the absence of these bacteria and other microorganisms in some 
acid waters also contributes to a reduction in invertebrates of 
intermediate sensitivity. Detritus "conditioning" generally refers to partial 
decomposition, with an attendant increase in nitrogen content associated 
with microbial growth. It was of interest to note that the C:N ratio of 
detritus in acid streams averaged 46 whereas that in circumneutral 
streams was 34, and this presumably contributed to their degree of 
"palatability". 
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Some observations on the absence of bacteria from acid waters in northwest England

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Some observations on the absence of bacteria from acid waters in northwest England

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