The loch eil project: Cellulose-degrading bacteria in the sediments of Loch Eil and the Lynn of Lorne

J. esp. mar. Biol. Ecol.. 1982. Vol. 56. Elsevier Biomedical PresT

THE LOCH EIL PROJECT: THE SEDIMENTS

261

pp. 2677218

CELLULOSE-DEGRADING

BACTERIA IN

OF LOCH EIL AND THE LYNN OF LORNE

I. VANCE’

and S. 0. STANLEY

Scottish Marine Biological Association, Dunstaffnage Marine Resrarch Laboratory, Ohan, Arg,v/l, Scotlund and

C. M. BROWN’ Department of Biological Sciences. Universit_vqf DunderJ, Dunder, DDI 4HN, Scotland

Abstract: Cellulose-degrading

bacteria were enumerated in the sediments of Loch Eil and the Lynn of Lorne, Scotland, using the most probable number technique. Highest population densities of aerobic and anaerobic cellulolytic bacteria were found in the deep basin of Loch Eil. In the sampling site closest to the effluent outfall from a pulp and paper mill the fluctuations in bacterial population density correlated with the discharge of suspended solids. Within the sediments, the population density of cellulolytic bacteria decreased with depth but cellulolytic activity appeared to be concentrated in the upper 10 mm. Cellulolytic bacteria isolated from the sediments were shown to utilize pulp fibre (a constituent of the mill effluent) as the sole carbon source and to produce extracellular j-1,4-glucanase. The interstitial water from sediments in Loch Eil also contained low levels of extracellular p-1,4-glucanase activity but the ecological significance of this is uncertain.

INTRODUCTION

Microbial decomposition of cellulose has been estimated to return 85 x 10’ tonnes of carbon dioxide to the atmosphere each year (Cowling, 1963) and hence is an important step in the global carbon cycle. In the marine environment, some 34 species of cellulolytic bacteria have been identified (Jones et al., 1976) and of these the genera Cytophaga and Sporocytophaga are thought to predominate in aerobic degradation, whilst the genus Clostridium is thought to be most active in anaerobic cellulolysis (Rheinheimer, 1974). Most samples of marine sediment contain cellulolytic bacteria (ZoBell, 1946) and there the population density tends to be greater than in the overlying water (Laurent, 1969). ZoBell (1938) gives lo3 cellulolytic bacteria in each g of wet sediment as a typical result. The population density of heterotrophic bacteria, however, has been shown to be influenced by the discharge of industrial effluents and it has been suggested that high population densities might be an indication of pollution (Rokosh et al., 1977). ’ Present address: BP Research Centre. Chertsey Road, Sunbury-on-Thames, England. ’ Present address: Department of Brewing and Biological Sciences. Edinburgh, EHI I HX, Scotland. 0022-0981/82/0000-0000/$02.75

0 1982 Elsevier

Biomedical

Press

Middlesex, Heriot-Watt

TW16 7LN, University.

I.VANCEETAL.

268

Effluent from a pulp and paper mill is discharged into Loch Eil and electron microscope studies on material from model sediments have shown that the major component of the effluent was degraded by cellulolytic bacteria (Vance et al., 1979). The present work is an investigation of the cellulolytic bacteria within the sediments of Loch Eil and Lynn of Lorne, with particular reference to the effects of the influx of organic material from the pulp and paper mill at Annat Point. A description of the sampling stations together with relevant background data are given in Pearson (1981). MATERIALSANDMETHODS

Sample collection A Craib corer (Craib, 1965) was used to take undisturbed sediment samples from the four sampling stations which were permanently marked with buoys. The samples of the upper 100-200 mm of sediment were taken in transparent acrylic core tubes (240 mm long, 57 mm diameter). On removal from the corer the tubes were stoppered with rubber bungs and stored in crushed ice prior to analysis (usually within 48 h). Enumeration of anaerobic bacteria All manipulations were carried out in a flexible PVC glove bag, described fully by Leftley & Vance (1979) containing an atmosphere of oxygen-free nitrogen, from which the final traces of oxygen had been removed in a gas scrubber containing B.A.S.F. calalyst R3 - 11 (B.A.S.F., U.K. Ltd., London). The overlying water was removed and the upper 50 mm of sediment was extruded with a plunger into a graduated tube of the same internal diameter as the core tube. The sample was homogenized with a sterile spatula and a IO-ml subsample removed using a truncated, disposable 20-ml syringe. The subsample was added to 90 ml of sterile phosphate buffer, pH 7.4, which contained sodium chloride (0.4 M) and was reduced with sodium dithionite (0.03% w/v). Resazurin (0.001% w/v) was included as a redox indicator. Further serial ten-fold dilutions were made, each dilution being well shaken by hand. From each dilution 5 inoculations of 1 ml were made into separate test tubes (16 x 150 mm) containing sterile, modified Omeliansky’s medium (Kadota, 1956). This was further modified by preparing the medium with a synthetic sea water (“Instant Ocean”, Gallenkamp Ltd.) containing sodium dithionite (0.03% w/v) and resazurin (O.OOll;/,w/v). Strips (100 mm x 10 mm) of Whatman No. 1 chromatography paper served as the cellulose substrate. Prior to inoculation, the reduced medium was stored in McIntosh and Fildes jars (Gallenkamp Ltd.) under hydrogen. After inoculation, the tubes were replaced in the anaerobic jars and incubated at I5 “C for 21 days in the dark. From the number of tubes of each dilution which

CELLULOSE-DEGRADING

showed

ccllulolytic

original

sample was computed

Enumwation

Aerobic

qf‘lwut

activity.

rrsistunt

and anaerobic

BACTERIA

the most probable

269

IN SEDIMEKI‘S

number

(MPN)

using the tables of Alexander

of bacteria

in the

(1965).

spews

dilution

bottles

used in the MPN

technique

wcrc heated

to 80 ‘C for 20 min. The contents of the bottles were then inoculated into tubes ol media as described for the MPN technique. The tubes were incubated for 21 days at I5 “C in the dark. after which time they were scored for cellulolytic

activity.

Subsamples from the sediment cores were removed from the glove bag and serial dilutions were made under a normal atmosphere. The same diluent and medium described for the anaerobic enumeration were used. with the omission of reducing agent and rcdox indicator. The MPN technique was also used for the enumeration of the aerobic bacteria. after an incubation period of 21 days at I5 “C in the dark. Both the aerobic and anaerobic population densities were reported in terms of the dry weight of the sediment studied.

A known volume of the IO-’ dilutions was passed through a weighed Whatman GF-C filter. The filter and the retained sediment were oven dried at 100°C for 24 h and then rc-weighed. C’wticul

distribution

Transverse lytic bacteria

The dry weight

of the sediment

by difference.

0J’huctcri~i

sections of sediment cores were taken at various depths and cellulowere cnumeratcd using the MPN technique. To minimize contami-

nation of the different levels due to the shearing extrusion of the sediment. subsamplcs were taken transverse section (Wood. 1965). Cdldol,~~tic

was obtained

effect of coring and subsequent from the central portion of cdch

uctivit!’

To determine the vertical distribution of cellulolytic activity, strips of Whatman No. I chromatography paper were wound around glass tubes (100 mm long, I5 mm diameter). secured with autoclave tape and inserted into sediment cores. The cores were incubated at 10°C in the dark for 28 days. On removal of the test strips from the sediment arcas of cellulolysis were noted with rcfcrence to the depth below the sediment -water interface.

270

I. VANCE ETAL.

Labelled substrates

The method of Poincelot & Day (1972) was modified for use with wood pulp as the cellulose substrate. Bleached Stora pulp, obtained from Scottish Pulp and Paper Mills Ltd., was washed three times with distilled water on a Buchner funnel fitted with Whatman No. 1 filter paper. The excess water was removed and 12 g of moist pulp was added to 500 ml of distilled water at 80 “C. With constant stirring, 1.5 g of Remazol Brilliant Blue (Raymond A. Lamb, 6 Sunbeam Road, London, WI4 8UE) was added to the pulp suspension. Sodium sulphate solution (30”/, w/v in distilled water) was added in 5 aliquots of 20 ml, at intervals of 2 min. Trisodium orthophosphate (2.64 g in 15 ml distilled water) was then added and the suspension stirred at 80 “C for 20 min. The stained pulp was washed on a Buchner funnel with distilled water until the filtrate was colourless. Excess stain was removed by autoclaving the pulp in two changes of distilled water (1 litre) at 103.5 kPa (15 lb . in-‘) for 15 min. Excess water was removed on a Buchner funnel and the moist pulp was removed and stored at 4 “C. A sample of known weight was oven dried at 100 “C for 24 h and the dry/wet wt ratio determined. Cellulose solubilization rates

The rates of cellulose solubilization by bacteria from enrichment cultures in the most probable number tubes were determined using salts medium described by Berg et al. (1972), which was modified by the addition of NaCl to a concentration of 0.4 M. Cultures were grown in 250 ml Erlenmeyer flasks containing 100 ml of salts medium and bleached wood pulp (0.1 “/:, w/v) as the sole carbon source. The pulp was sterilized by autoclaving (103.5 kPa for 15 min) in the salts medium. Incubation was at 20 “C in the dark in an orbital incubator (Gallenkamp Ltd.) at 100 r .min’. At intervals, cellulose in cultures was determined after the gravimetric method of Fahraeus (1947), except that the cellulose was collected on weighed Whatman GF-C filters. When labelled substrate was used, the extent of the release of the label was quantified by centrifuging a 5-ml sample of the medium at 2700 g for 10 min and measuring the absorbance of the supernatant at 596 nm. Extracellular p-1 ,I-glucanase activity in bacterial cultures

Ten-fold dilutions of sediment samples were prepared in sterile 80% sea salts (T. Gerrard & Co., Worthing Road, East Preston, West Sussex) and 5 ml of these dilutions were used to inoculate aerobic enrichment cultures which consisted of 100 ml of the basal medium used in the MPN tubes in 250-ml Erlenmeyer flasks. Whatman No. 1 filter paper or Avicel SF cellulose powder (Koch-Light Laboratories, Colnbrook, Bucks) was added at 1% as the carbon source. The cultures were incubated at 20 “C for 20 days in the dark on an orbital shaker operating at

CELLULOSE-DEGRADING

BACTERIA

271

IN SEDIMENTS

100 r .min-‘. Culture samples were centrifuged for 30 min at 10000 g and 4°C and the supernatant was assayed for /?-1,Cglucanase activity. /I-I ,4-glucanase assay

Viscometric assay. The decrease in viscosity of a carboxymethylcellulose (CMC) solution when incubated with samples was determined by the method of Vance et al. (1980). DNS assay. The production of reducing sugars from a CMC solution when incubated with samples was determined using a dinitrosalycilic acid (DNS) reagent by the method of Vance et al. (1980). One unit of activity was designated as the amount of enzyme which produced a net increase of 0.1 absorbance units in a l-cm cell at 550 nm.

RESULTS

In the aerobic MPN tubes the degradation of the chromatography paper was restricted to the medium-air interface, whilst in the anaerobic tubes the whole area of submerged paper showed discrete zones of cellulolytic attack. Pigmentation was particularly obvious in the aerobic tubes, ranging from light yellow through ochre to light brown. Occasionally, creamy-pink and purple pigmentation was observed which corresponded to the observations of Kadota (1956) which he attributed to Cytophaga rosea and Vibrio purpureus, respectively. Sediment samples which were heated to 80 “C lost all cellulolytic activity indicating that the bacteria were present as viable vegetative cells rather than heat resistant spores. TABLEI

Vertical distribution of aerobic cellulolytic bacteria in sediments: figures represent log MPN bacteria g-’ of dry sediment; ND, none detected; the 95% confidence limits are log MPN + 0.496; results are significantly different when (log MPNa/0.3471) -(log MPNb/O.3471) >2.0. Depth in sediment

Station

E70

Station

E24

(cm) t&l I-2 5-6 9910

4.78 2.38 1.80 ND

3.75 3.15 1.55 ND

The vertical distribution of cellulolytic bacteria in sediment samples is shown in Table I. The decrease in population density with depth in the sediment was obvious in all of the samples examined, for both aerobic and anaerobic bacteria. Although

Mean

April May June 3 uly August September October __--.----

February March

I975 1975 1976

--

and anaerobic

November December January

Aerobic

--

3.45

3.67 3.23 3.34 3.68 3.98 4.36 3.21

3.04 4.26

3.47 2.36 2.77

Aerobic ------

_ .--

cellulolytic

1.82

3.4

l____l_-

3.58 2.94 2.95 3.89 4.0 3.61 3.07

3.42 4.12

2.99 2.71 3.52

Aerobic

Station

--1.54

ND 1.79 2.64 2.04 1.86 2.88 ND

2.21 ND

2.99 2.04 ND

Anaerobic

E70

2.87

_____l-ll

2.92 3.11 2.23 3.22 4.14 2.91 2.80

2.67 3.58

1.72 2.74 2.44

Aerobic

---I_.__

1.68 1.69

1.36

-__

1.87

- ---

2.14 1.76 2.78 2.26 2.36 1.53 1.81

1.41 ND ND 1.83 2.60 1.19 2.70 2.72 2.13

0.85 1.69 1.87

I

bacteria

0.37

ND ND ND ND ND ND 1.11

ND

1.65 1.67 ND

Anaerobic

- --_

----

LY

log MPN

Station Aerobic

ND 1.74 ND

___-__-.

---

represent

_- __

figures

Anaerobic

_I_-

E24

of Lorne:

Station

50 mm of sediment from Loch Eil and Lynn of dry sediment; ND, none detected.

____-

1.32 2.14 2.89 2.22 1.22 2.51 ND

2.90 1.34

1.30

2.26 1.77

Anaerobic ___-.

-.-

E2

Station

-.

in upper

bacteria

TABLE II

-g-t

k

z

z

5

?

CELLULOSE-DEGRADING

BACTERIA IN SEDIMENTS

213

viable cellulolytic bacteria were found 50 mm below the sediment surface, celiulolytic activity, as indicated by macroscopic examination of the test strips, was restricted to the upper 10 mm of sediment. The temporal and spatial distributions of cellulolytic bacteria are shown in Table II. The mean population densities of both aerobic and anaerobic bacteria found in Loch Eil sediment samples were greater than those from a control station in the Lynn of Lorne. Within Loch Eil greater population densities were found in sediment samples from the deep basin (Stations E2 and E70) than from the head of the loch (Station E24). In most of the samples the aerobic bacteria were more numerous than the anaerobes although the relative activities of the two populations were not determined. A positive correlation (Y= 0.52), which is significant at the 1)‘c confidence level, was found between the population density of aerobic cellulolytic bacteria from Station E2 and the mean effluent discharge during the previous month. Wood pulp of the type discharged by the mill at Annat Point was utilized as the sole carbon source by mixed cultures of aerobic bacteria from positive MPN tubes. Pulp which had been labelled with remazol brilliant blue, however, was shown to be more resistant to bacterial cellulolysis (Fig. 1). Despite this resistance to

t

I

0

I 5

I 10

I

i

15

20

I 25

DAYS Fig. 1. Degradation of wood pulp by cellulolytic bacteria: II. pulp labelled with Remazol Brilliant Blue; 0. unlabelled pulp.

cellulolysis such labelled substrates are extremely useful as a means of monitoring relative degradation rates without sacrificing cultures. The degradation rates of dried and non-dried labelled pulp fibre were compared and Fig. 2 shows that pulp which had been dried after labelling was degraded at a slower rate. Extracellular p-1,4-glucanase activity was found in enrichment cultures of cellulolytic bacteria from sediment taken from Stations E24 and E70. Greatest activity was found in cultures supplied with Avicel rather than filter paper as the carbon source (Table III). Parallel investigations using the medium described for

274

I. VANCE

ETAL

the solubilization studies showed a similar result. although all of the activities were lower. Using the viscometric assay, which is more sensitive for endo-acting enzymes, no activity was found in unconcentrated samples of interstitial water from Station E24 or E70 nor in samples from E70 which were concentrated 75fold. By concentrating samples from E24 170-fold the activity was high enough to be assayed (Fig. 3). Heating at 100°C for 15 min was found to destroy all activity. The

The1 E III

Extracellular

B-1 ,Cglucanase

Source of inoculum

activity Carbon

in enrichment

cultures:

source

ND, not detected

Activity

(DNS units

E70 E70

Avicel Filter paper

2.1 0.81

E24 E24

Avicel Filter paper

2.4 ND

ml-‘)

0.9-

0.8-

0.7 -

0.6-

0” t=

0.5 -

a=

ma 0”$ 0.L2; a 0.30.2

0 .l

0 I 11

IIll

0

l

10

20

I

J

III

30

LO

50

DAYS Fig. 2. Release of Remazol

Brilliant Blue from labelled wood q, dried wood pulp; 0, non-dried

pulp in cultures wood pulp.

of cellulolytic

bacteria:

CELLULOSE-DEGRADING

215

BACTERIA IN SEDIMENTS

DNS assay was also used on samples from Station E24 which had been concentrated 125fold and the activity was equivalent to 1.98 x 10-I DNS units. ml-’ of unconcentrated interstitial water.

07 0

L

/

I

I

,

5

10

15

20

25

REACTION

TIME

(MINUTES1

Fig. 3. Reduction in viscosity of CMC solution by 170-fold concentration Station E24 in Loch Eil.

of interstitial water from

DISCUSSION

The population densities of cellulolytic bacteria observed in the present work were of the same order as those found by Goman (1973) and ZoBell (1938) when allowance is made for the water content of the sediment. Theoretical considerations, however, suggest that the MPN technique is likely to under-estimate true population densities (Cochran, 1950; Alexander, 1965) and in practice DiGeronimo et al. (1978) have shown that plate counts of bacteria from natural environments give higher values than the MPN method. In the present work the choice of dilution factor and number of replicates gives a low order of precision with a standard error of log MPN = 0.25. Consequently, a large difference in MPN must exist in order to be certain that populations are significantly different, as detailed in Table I. Despite these limitations, the MPN technique is still a valuable tool for the microbial ecologist interested in gross population differences. Analysis of the mean values of population density of aerobic cellulolytic bacteria, shows for example,

I.VANCE ETAL.

276

no significant

difference

between

those from the deep basin

E70 and E2) but there is a significant station

(LYl)

between

those

from the control

in the Lynn of Lorne and the other three stations.

The high population

density

is of interest,

and in particular,

MPN

from

values

difference

of Loch Eil (Stations

Station

of cellulolytic

bacteria

the correlation

E2. Wyatt

(1979)

in the deep basin of Loch Eil

between

the effluent

has also

demonstrated

discharge

and

a positive

correlation between the effluent discharge and the population density of ciliated protozoa from Station E2 during the same sampling period. Pearson (1975) considered that the greater part of the effluent from the mill at Annat Point is retained within the Loch E&Loch Linnhe system and has shown that the organic carbon content of the sediment at Station E2 has increased since the start of effluent discharge (Pearson, 1971). There is some evidence, therefore, that the mill effluent enhances the carbon content of the sediment close to the outfall and this may explain the apparent influence upon the bacterial population, particularly as we have shown that the major component of the effluent can serve as the sole carbon source for bacterial cultures (Fig. period the mean cellulose

1). It is notable, however. that during the sampling concentration in the upper 50 mm of sediment at

Station E2 was 607” of that at E70 (Vance, 1977). In laboratory sediment models Stanley et al. (1978) have shown that chemical analysis may give no indication of the influx of cellulose to a system because under some conditions most of the cellulose is rapidly degraded. This may be the situation at Station E2 where measurements of pH, Eh and sulphide concentration indicate high levels of microbial activity (Vance, 1977). The vertical distribution of cellulolytic bacteria in the sediments of Loch Eil is similar to that described by Laurent (1969) for freshwater sediments. In the case of Loch Eil the oxygen diffusion rate and the pore space volume have been shown to decrease with sediment depth (Vance, 1977) and both factors may be expected to restrict bacterial growth in the lower horizons. The concentration of labile organics organic obscured

is also likely to influence carbon

content

decreased

by, for example,

the bacterial

distribution.

with depth (Vance,

the disruptive

In some samples

1977) but this trend

effects of coring

upon

sediment

the

is often stratifi-

cation (McIntyre, 1971). Cellulolytic activity was only observed in the upper 10 mm of sediment despite the presence of viable cellulolytic bacteria down to 50 mm. Jones (1979) has also observed that microbial hydrolytic activity in the sediment of Lake Windermere was greatest at the sediment-water interface. The cause of this distribution is presumably a higher concentration of suitable substrates on the sediment surface resulting in a bacterial population with a high activity. The cellulose solubilization studies illustrate that the impact a cellulosic material has on a bacterial population depends upon the state of the substrate. Substituted celluloses such as the labelled pulp fibre will be catabolized more slowly and high degrees of substitution will aggravate the situation, particularly if the substituent

CELLULOSE-DEGRADINGBACTERIAINSEDIMENTS

277

groups are of high molecular dimensions (Cowling, 1975). Dried celluloses will also be slowly degraded as a result of the irreversible closer re-orientation of the cellulose microfibrils (Jayne & Hunger, 1961). This implies that non-dried cellulosics from pulp mills will be degraded faster in the environment than the dried wastes from paper mills. The type of cellulose supplied to cultures of bacteria isolated from the sediments of Loch Eil was also shown to influence the extracellular b-1,4-glucanase activity. Similar results have been shown for the soil bacterium Sporocytophaga myxococcoides (Vance et al., 1980). It is not clear, however, if this effect is caused by enhanced enzyme synthesis or by differential adsorption of the enzyme to the residual substrates. The extracellular /I-1,4-glucanase found in the interstitial water of sediments showed 1 x 10e2 of the activity in the enrichment cultures which, in turn, was 2.5 x 10e2 of the maximum activity found in fermenter grown cultures of S. myxococcoides (Vance et al., 1980). The significance of these apparently low levels of activity in the natural environment is difficult to assess, particularly since the conditions of the enzyme assays were not optimized. Preliminary results show, for example, that greater activity is found at higher pH values. Further work on the kinetics of these enzymes would be of considerable interest since extracellular enzymes are believed to play a significant part in the oxidation of organics in the marine environment (Waksman & Hotchkiss, 1938).

ACKNOWLEDGEMENTS

I. V. is grateful for a N.E.R.C. research studentship and financial support from the Royal Society. REFERENCES ALEXANDER, M., 1965. Most-probable-number method for microbial populations. In, Methods @‘soil analysis, Part 2,Chemical and microbiological properties. edited by C. A. Black, American Society of Agronomy, Madison, pp. 1467-1472. BERG, B., B. v. HOFSTEN & G. PETTERSSON, 1972. Growth and cellulase formation by Cellvibriofilvus. .I. appl. Bact., Vol. 35, pp. 201-214. COC.HRAN, W.G., 1950. Estimation of bacterial densities by means of the most probable number. Biometrics, Vol. 6, pp. 105-l 16. COWLING, E.B., 1963. Structural features of cellulose that influence its susceptibility to enzymatic hydrolysis. In, Advances in enzymatic hydroly.sis of cellulose and related materials, edited by E.T. Reese, Pergamon Press, New York, pp. l-32. COWLING, E. B.. 1975. Physical and chemical constraints in the hydrolysis of cellulose and lignocellulose materials. Biotechnology and Bioengirteering Symposium No. 5. Wiley Interscience, New York, pp. 163-181. CRAIB, J.S., 1965. A sampler for taking short undisturbed marine cores. J. Cons. perm. inf. E.uplor. Mer, Vol. 30, pp. 34-39. DIGERONIMO, M. J., M. NIKAICIO & M. ALEXANDER, 1978. Most probable-number technique for the enumeration of aromatic degraders in natural environments. Microb. Ecol., Vol. 4, pp. 263-266.

278 FAHRAEUS, G., 1947. Studies pp. I-128.

I. VANCE in the cellulose

ET AL.

decomposition

by Cytophaga. S.vmh.

her.

upsal., Vol. 9,

GOMAN, G.A., 1973. Aerobic cellulose decomposition in the bottom of Lake Baikal. Mikrobiologiya, Vol. 4, pp. 148-153. JAYNE, G. & G. HUNGER, 1961. Rearrangement of microfibrils in dried cellulose. In, Fundamentals of‘ pupermaking,/ibres. edited by F. Bolam. Technical Section of the British Paper and Board Makers’ Association Inc.. London, pp. 263-271. JONES. E. B. G., R. D. TURNER, S. E. J. FURTADO & H. KOHNE, 1976. Marine biodeteriogenic organisms. 1. Lignicolous fungi and bacteria and the wood boring Mollusca and Crustacea. 1121. Biodeterior. Bull., Vol. 12, pp. 120-134. JOFUES,J. G.. 1979. Microbial activity in lake sediments with particular reference to electrode potential gradients. J. gen. Mierobiol., Vol. 115,pp. 19-26. KADOTA, H., 1956. A study on the marine aerobic cellulose-decomposing bacteria. Mem. Coil. Agric. Kyoto Univ., No. 74, Fish. Ser. No. 6, 128 pp. LAURENT, M., 1969. Experimental investigation of cellulolysis in mud. In, Proc. 4rh in/. COO/:on ,lucrrer pollution research. Prague, pp. 9 17-925. L~FTLEY. J. W. & I. VANCE. 1979. An inflatable anaerobic glove bag. In, Cold tolerant microbes in .spoilage and rhr environment, edited by A. D. Russell & R. Fuller, Academic Press, London, pp. 51-57. MCINTYRE. A. D., 1971. Meiofauna and microfauna sampling. In, Merhods lor the srudy q/’ marine hen/has, I.B.P. Handbook No. 16, edited by N. A. Holme & A. D. McIntyre, Blackwell. Oxford, pp. 131-139. PEARSON. T. H., 1971. The benthic ecology of Loch Linnhe and Loch Eil, a sea loch system on the west coast of Scotland. III. The effect on the benthic fauna of the introduction of pulp mill efnuent. J. exp. mar. Biol. Ecol., Vol. 6, pp. 21 l-233. PEARSON, T. H., 1975. The benthic ecology of Loch Linnhe and Loch Eil, a sea loch system on the west coast of Scotland. IV. Changes in the benthic fauna attributable to organic enrichment. J. exp. mar. Biol. Ecol, Vol. 20. pp. l-41. PEARSON, T. H., 1981. The Loch Eil Project: introduction and rationale. J. exp. mar. Biol. Ecol., Vol. 55, pp. 93-102. PoINcEt.or, R. P. & P. R. DAY, 1972. Simple dye release assay for determining cellulolytic activity of fungi. Appl. Microbial., Vol. 23, pp. 875-879. RHEINHEIMER, G., 1974. Aquatic microbiology, Wiley, London, 184 pp. ROKOSH, D.A., S. S. RAo & A.A. JURKOVIC, 1977. Extent of effluent influence on lake water determined by bacterial population distributions, J. Fish. Res. Bd Can., Vol. 34, pp. 844849. STANLEY. S. O., T. H. PEARSON & C. M. BROWN. 1978. Marine microbial ecosystems and the degradation of organic pollutants. In, The oil industry und microbial ecosystems, edited by K. W. A. Chater & H. I. Someville, Heyden, London, pp. 60-79. VANCE, I., 1977. Bacterial degradation of cellulose in marine sediments. Ph.D. thesis, Dundee University, Scotland, 201 pp. VANCE, I., S.O. STANLEY & C. M. BROWN, 1979. A microscopical investigation of the bacterial degradation of wood pulp in a simulated marine environment. J. gen. Microbial., Vol. 114, pp. 69-74. VANCE, I., C. M. TOPHAM, S. L. BLAYDEN & J. TAMPION, 1980. Extracellular cellulase production by Sporocyrophaga myxococcoides NCIB 8639. J. gen. Microbial., Vol. 117, pp. 235-241. WAKSMAN, S.A. & M. HOT~HKISS, 1938. On the oxidation of organic matter in marine sediments by bacteria. J. mar. Res., Vol. 1, pp. 101-117. WOOD, E. J. F., 1965. Marine microbial ecology. Chapman and Hall, London, 243 pp. WYATT, C. E., 1979. The ecology of ciliated protozoa from organically enriched marine sediments. Ph.D. thesis, Dundee University, Scotland, 314 pp. ZOBELL, C. E., 1938. Studies on the bacterial flora of marine bottom sediments. J. sedim. Petrol., Vol. 8, pp. l&18. ZOBELL, C. E., 1946. Marine microbiology. Chronica Botanica, Waltham, Massachusetts, 240 pp.