Central Ontario JUDITH F

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1985, p. 315-322 Vol. 50, No. 2 0099-2240/85/080315-08$02.00/0 Copyright C) 1985, American Society for Microbiology Microbial Decomposition of Cellulose in Acidifying Lakes of South- Central Ontario JUDITH F. M. HOENIGER Department of Microbiology, Faculty of Medicine, University of Toronto, Toronto, Ontario, MSS IA8, Canada Received 11 February 1985/Accepted 16 May 1985

The rate of ceHlulose breakdown and density of bacterial populations were measured in the epilimnetic sediments and water columns of lakes in central Ontario that differ in pH, alkalinity, and nutrient status and are particularly sensitive to acidic inputs from atmospheric decomposition. There was no significant difference in decomposition rate in either oxic or anoxic sediment when mean epilimnetic pHs were in the range 5.5 to 6.9. The importance of these findings for the breakdown of autochthonous detritus in Canadian Shield lakes is discussed. Furthermore, the results of these experiments, in which dyed strips of cellophane (regenerated cellulose) were used as substrate, were compared with results of earlier decomposition studies carried out with coarse litter (leaves, twigs). Acridine orange direct counts of bacteria in the top 1 cm of sediment ranged from 5.5 x 108 to 1.0 x 109 per g and in planktonic water samples from 1.1 x 106 to 1.8 x 106 per ml. Bacterial densities were significantly higher in both the shallow sediment (P < 0.01) and the water column (P < 0.05) of dystrophic lakes than at these sites in oligotrophic lakes.

The acidification of soft-water lakes in North America and water were measured, and total numbers of bacteria were Scandinavia is one of the most serious environmental prob- counted in both the water column and the sediment. lems today. Although a direct cause and effect relationship has yet to be documented, it is likely that acidic precipitation MATERIALS AND METHODS affects both structure and function ofaquatic biota at various trophic levels: phytoplankton and attached algae, Site description. The five lakes selected for study are zooplankton and benthic invertebrates, amphibians, and fish located in the Muskoka-Haliburton region of central Ontario (6, 9, 15, 17, 25, 29, 40, 43, 44). (Fig. 1), near the southern fringe of the Precambrian Shield. Few studies have dealt with microbial processes in acidi- They are underlain by granitic bedrock with variable surficial fied a in view the deposits of till. Three lakes differing in nutrient status, pH, lakes, surprising fact of essential roles that and alkalinity were monitored during each of two successive microbes play in decomposition and nutrient recycling. Both summers: in 1983 Red Chalk, Plastic, and Chub Lakes were in the Adirondacks and in Scandinavia, breakdown of leaves monitored; in 1984, Harp, Plastic and Dickie Lakes were and twigs (monitored by weight loss in litter bags) occurred monitored (Table 1). Red Chalk and Harp Lakes are more slowly at reduced pH over the pH range ca. 5.0 to 7.0 oligotrophic and circumneutral and hence serve as controls; (11, 18) and 4.0 to 6.0 (26, 48), respectively. However, when Plastic Lake is oligotrophic and moderately acidic, while decomposition was measured in terms of end products (CO2, Chub and Dickie Lakes are dystrophic. CH4) in an artificially acidified lake at the Experimental Oxic and anoxic sites were studied in the first and second Lakes Area in northwestern Ontario, there was no signifi- years, respectively, the change of lakes being necessitated cant difference in rate as the epilimnetic pH dropped from by the fact that suitable anoxic sediments could not be found 6.7 to 5.1 over a 6-year ppriod (24). Further studies at the near the original oxic sites in Red Chalk and Chub Lakes. Experimental Lakes Area have suggested that reduction of The sediment of the control lakes was very sandy close to sulfate and nitrate in anoxic sediments is unaffected by the shore, but consisted of a mixture of sand and pebbles at acidification and that these reduction processes can increase greater depths. At Plastic Lake the sediment was made up of buffering capacity, at least over the short term (23, 41). fine silt with a surficial admixture of decaying detritus; in the The purpose of the present study was to directly examine dystrophic lakes plant litter, sand particles, and small stones the effect of reduced pH on microbial decomposition in were present. Canadian Shield lakes, which are particularly sensitive to All samplings were carried out in the littoral zone close to acidic inputs because of low alkalinity values. The lakes are an inflowing stream which is acidic, especially in the spring. situated in central Ontario, where deposition of strong acid Physicochemical measurements. Water samples were taken 70 to 100 per The at 1- to 2-week intervals by means of a glass van Dorn averages meq/m2 year (27). decomposition apparatus (2 liters), transferred to brown Nalgene bottles rate of cellulose (in the form of dyed cellophane strips) was (300 ml) equipped with conical plastic cap liners, and placed monitored because this polymer is the principal cell wall in a cooler for transport to and analysis at the Dorset constituent of most macrophytes and some attached algae Research Center, Ontario Ministry of the Environment. The and because these plants are conspicuous components of the pH was measured in the field with a gel combination elec- littoral zone in Shield lakes where, it is assumed, they trode and a portable Fisher Accumet 640 meter in 1983 and contribute large quantities of particulate organic matter to by a computerized automatic procedure at the Dorset Re- the detritus food chain (10, 49, 50). Thus a decrease in search Center in 1984. In 1983, inflection point alkalinity was cellulose breakdown could profoundly affect carbon cycling determined manually by potentiometric titration with 0.02 N within other trophic levels of the ecosystem of a lake. H2SO4 (33) and the same equipment. The following year, In addition, the pH, alkalinity, and temperature of the both pH and alkalinity were measured with a combination 315 316 HOENIGER APPL. ENVIRON. MICROBIOL.

overlying water (Fig. 2); the Eh reading was taken after 1 min of equilibration at each depth. The position of the oxic- anoxic interface in the sediment was confirmed by burying a 6-in iron spike for 3 to 4 weeks. Dissolved organic carbon was measured colorimetrically with phenolphthalein after the sample had been stripped of inorganic carbon and then oxidized by a UV digestor in an acid-persulfate medium (34). Total bacterial numbers. Water samples were taken in sterile, acid-washed, brown Nalgene bottles, and surface sediment was withdrawn with an alcohol-sterilized 10-ml plastic syringe. A 10-ml sample of water was fixed immedi- ately with glutaraldehyde (final concentration, 0.25%; Eastman Kodak Co.), and then the water and sediment samples were returned to the laboratory on ice. The top 1 cm of sediment was diluted 103-fold with cell-free water, vortexed, and blended in a mini-Osterizer jar; a 10-ml sample of the suspension was fixed with glutaraldehyde and refrig- FIG. 1. Map of the region in central Ontario showing the loca- erated. Aliquots (1 ml) were filtered through an unstained tions of the lakes. Nuclepore membrane (pore diameter, 0.2 ,um), stained with freshly prepared acridine orange (made up in 50 giM phos- phate buffer [pH 7.0] to a final concentration of 0.01%; pH probe, a Radiometer pHM64 pH meter, and a Radiom- Sigma Chemical Co.) for 5 min, and further processed by the eter ABU13 autoburette connected via a Differential Titrator method of Geesey et al. (14). The membrane was observed ABU13 control relay to a Hewlett-Packard calculator model (with focussing on the pores) under a Reichert Neopan 10. The program was designed by F. D. Tomassini and microscope (magnification, x 1,250) equipped for M. M. Rawlings (unpublished data). epifluorescence and illuminated with an HBO 50 W mercury The temperature of the water and sediment was measured lamp (filter combination: excitation, BP 455-490; beam split- with a standard glass alcohol thermometer or a metal probe ter, DS 520; barrier filters, LP 418, BP 515-560). Between designed for darkroom work. Electrode potential (Eh) gradi- 200 and 400 orange-fluorescing bacteria were counted in 20 ents were measured on undisturbed cores of sediment plus fields when a 10 by 10 reticule was used. Bacterial densities overlying water by using small Perspex tubes (length, 20 cm; in the water column and sediment of the three lakes sampled internal diameter, 2.5 cm; external diameter, 3.6 cm; Fig. 2) each summer were analyzed by the F test and Student t test which were inserted carefully into the sediment, withdrawn, (42). and capped in situ at either end with butyl rubber stoppers. Cellulose breakdown. Precut strips (2 by 5 cm) of transpar- Holes (diameter, 4 mm) had previously been drilled in the ent cellulose film (cellophane, grade 325 P, not moisture tubing at 0.5-cm intervals in a 450 spiral and then sealed with proof; obtainable from T. and R. Graham Ltd., Paisley, Polythene adhesive tape (no. 371; 3M Canada, Inc.) before Scotland) were boiled to remove plasticizers, stained with coring. The cores were placed in a cooler and returned to the Remazol brilliant blue R (Sigma) by the method of Moore et Dorset Research Center, where the Eh was measured with a al. (31) and Swift (45), and then placed individually in litter bright platinum electrode (21) and a fast-flow calomel refer- bags made from 10-cm lengths of pantyhose (gray or tan; ence electrode (Beckman Instruments, Inc.) connected to a mesh size 100 ,um) which were tied at both ends with Fisher Accumet 210 meter after standardization with ZoBell monofilament fishing line. Quadruplicate strips were at- solution (available from Ingold Electrodes, Inc.) (52). The tached to bricks and buried with a trowel in the sediment or platinum microprobe was inserted through a hole into the suspended in the overlying water. For breakdown in the oxic core, while the reference electrode was immersed in the zone, the strips were immersed by hand in 0.4 to 0.6 m of

TABLE 1. Physicochemical measurements at study sites in Dorset lakes during mid-June to late August 1983 and 1984

Year Lake site Initial Final Meana (Alkq/linteyr Temp (MC)C DOC (mg/liter)d

1983 Red Chalk, surface 6.7 7.0 6.9 44.2 24.8 5.5 Plastic, surface 5.4 5.7 5.5 2.7 24.6 18.7 Chub, surface 6.0 6.1 6.1 10.9 24.6 22.3 1984 Harp, surface 6.6 6.9 6.7 68.8 23.2 18.6 Harp, water/sediment 6.6 6.9 6.8 69.0 NDe Plastic, surface 5.4 5.7 5.6 1.2 22.8 22.4 Plastic, water/sediment 5.2 5.9 5.7 3.4 ND Dickie, surface 5.4 6.0 5.8 10.0 24.9 34.2 Dickie, water/sediment 5.9 6.2 6.1 6.6 ND a Mean surface pH based on 12 measurements in 1983, 8 measurements in 1984; water-sediment values based on 6 measurements. b Means of four values. c Mean of 14 measurements in 1983 and 8 in 1984. d Mean of weekly values for the inflowing stream from Ontario Ministry of the Environment data. DOC, Dissolved organic carbon. eND, Not determined. VOL. 50, 1985 CELLULOSE DECOMPOSITION IN ACIDIFYING LAKES 317 water at a distance of 2 to 3 m from the shore; they were Eh (mV) buried in anoxic sediment by a skindiver in 1.5 to 2.5 m of water approximately 4 to 8 m from shore. A different dye lot of Remazol brilliant blue R-stained cellulose strips was used in each ofthe three lakes. In all, eight samplings were carried E out at ca. 1-week intervals after placement of the strips in mid-to late June. The litter bags were refrigerated and 0 returned to the laboratory, where residual Remazol brilliant blue R was extracted with 0.35% KOH in a portable Sediment-water interface autoclave, and A595 was read with a Bausch & Lomb w Spectronic 20 spectrophotometer (45). Quadruplicate unex- z posed strips from the appropriate dye lot were extracted each time as controls. The percent weight of cellulose lost 0 (W) was calculated from the equation W = [(Acontrol - UI. Aexposed)IAcontroldx100 and the rate of decomposition was determined for each site by using multiple linear regression z techniques. The data were further analyzed by the chi- square test for homogeniety of variance using Bartlett's test and for significance of slopes and elevations using the F test FIG. 3. Eh gradients in cores of littoral sediment from Harp Lake (42). removed in either 0.5 m (--- -) or 2 m ( ) of water. In addition to the four extractable cellulose strips, one was immersed for mounting and subsequent examination with phase contrast optics to determine the type(s) of microor- primary source of acidity in Plastic Lake on an annual basis, ganism responsible for cellulolysis. with sulfate being the major proton-contributing anion during peak streamflow (March to May) and organic anions RESULTS AND DISCUSSION (humates and fulvates) predominating during summer months. Since the inflowing stream at Dickie Lake drains a Physical and chemical parameters. Physicochemical data sphagnum-conifer swamp, there is a particularly heavy load- obtained on water samples from the lakes during 1983 and ing of organic acids during the summer; this is reflected in 1984 are presented in Table 1. The pH regimes of the inshore both the pH and the dissolved organic carbon (Table 1). surface waters in the two control lakes, Red Chalk and Harp There was no significant difference between the pH of the Lakes, were analogous, whereas Dickie Lake was slightly overlying water and that at the sediment-water interface more acid than Chub Lake. Plastic Lake had identical except in Dickie Lake, where the increase may have been surficial pHs in the two years. due to activities of sediment bacteria (which were present in Lazerte and Dillon (27) showed that sulfuric acid is the high numbers; Table 2) or benthic algae, or to some other process altogether. Alkalinity measurements show that Plas- tic Lake and Chub and Dickie Lakes are extremely sensitive to acidification (17). This is particularly serious for Plastic Lake, an oligotrophic lake. Red Chalk and Harp Lakes are moderately sensitive to acidic inputs. Temperatures in the littoral water were high in 1983, a very hot and dry summer. In 1984 the water temperature was somewhat lower in both Harp and Plastic Lakes, but high at the Dickie Lake site which is shallow (0.1 to 0.7 m) up to 10 m from shore. The range of dissolved organic carbon concentrations for the five lakes, i.e., ca. 6 to 34 mg/liter (Table 1) is typical of natural waters (50). Although the dissolved organic carbon in Plastic Lake was similar during successive years, the concentrations in paired control lakes (Red Chalk and Harp Lakes) and brownwater lakes (Chub and Dickie Lakes) differed to some extent. Eh gradients. Typical Eh gradients monitored on small cores of littoral sediment removed at two different depths (ca. 0.5 and 2.0 m) in Harp Lake are shown in Fig. 3. Eh decreased rapidly below the sediment-water interface in both cases, while the Eh discontinuity layer (32) or oxidized microzone (50) occurred quite far down in the core taken in shallow water but close to the interface in that from the deeper epilimnion. Since no measurable oxygen is present when the Eh falls below +100 mV (16), Eh profiles provided a guideline for burying cellulose strips in oxic and anoxic sediments and for studying their subsequent decomposition. Thus, a core re- FIG. 2. Diagram of the assembly used for measuring Eh in moved in 0.5 m of water at Red Chalk Lake, the 1983 control sediment cores. site, showed oxidizing conditions throughout, whereas one 318 HOENIGER APPL. ENVIRON. MICROBIOL.

Eh ( mV) on cores from Adirondack lakes, i.e., 2.9 x 108 to 9.9 x 108 -100 0 +100 +200 +300 +400 per g (3). 4 l | l l l *r: Direct counts cannot differentiate between metabolically active and inactive populations of bacteria. However, cer- E tain active populations could be affected by decreasing pH, 2 / as suggested (37) for respiring and heterotrophic bacteria at w pHs below ca. 5.5. Heterotrophs play essential roles in the 0 O/ cycling of carbon within a lake, in particular the mineraliza- Sediment - water Ao tion of organic matter such as cellulose. Therefore, any n i t rfac . z 0 f~~~~~~~~~~~~~ pH-related reduction in this process could have profound effects within the ecosystem. w I.- Decomposition. When litter bags containing dyed cellulose z U. strips were buried in the sediment or immersed in the /0i overlying water, the breakdown rate could be followed t0 4 ~~~~~~*1@ under either oxic (see Fig. 5) or anoxic (see Fig. 6) condi- (0 tions. In these graphs, sediment data have been plotted over 4- I0~~~~ the usual range of 0 to 100% and water column data have 6~~~~~~~~~~~~~~~~~~~~~~~ been expanded to maximize any differences. As noted above (Table 1), control clear-water lakes (Red Chalk and Harp FIG. 4. Eh gradients in littoral sediment cores taken at Plastic Lakes) and humic lakes (Chub and Dickie Lakes) had quite Lake in 0.5 m (----) or 2 m ( ) of water. similar physicochemical properties. In 1983, cellulose was decomposed to a greater extent in from deeper water (ca. 2.5 m) had a discontinuity layer at 6 the nearshore sediment (Fig. 5a) than in the water column cm below the interface, all readings above being in the + 150- (Fig. Sb), as would be expected from the bacterial densities to +300-mV range. Therefore, a new control site was at the two sites (Table 2). Preliminary analysis of the selected at Harp Lake for the 1984 experiment on cellulose sediment data by the chi-square test demonstrated that breakdown under anoxic conditions. residual variances between the lakes were homogeneous (X2 Figure 4 shows the Eh gradients in cores from the two sites = 4.0 with df 2). Furthermore, F testing showed that there at Plastic Lake which were monitored in consecutive years. was no significant difference between slopes (F = 3.4 with df The gradient decreased quite gradually in both cases; the 2, 15) but that there was a significant difference between nearshore sediment was oxic throughout, whereas that from elevations (F = 18.0 with df 2, 17; P < 0.01). With regard to deeper water was anoxic. With reference to the dystrophic the elevations, it is clear that decomposition began earlier in study lakes, the shallow sediment in Chub Lake had an Eh Red Chalk Lake than in the other two lakes (Fig. Sa). gradient similar to that in Plastic Lake (Fig. 4), but because Finally, when the water column data (Fig. Sb) were ana- the lake bottom fell off rapidly an anoxic site could not be lyzed, residual variances were again found to be homoge- reached by skin diving. When the Eh gradient was measured neous (X2 = 3.2 with df 2), and there was no significant in deeper sediment from Dickie Lake, the alternative lake, it difference between either slopes (F = 0.9 with df 2, 18) or likewise resembled that in Plastic Lake. elevations (F = 0.8 with df 2, 20). Iron spikes in sediment with the head protruding showed Several factors may have resulted in low breakdown rates rusting above and no change below the level of the discon- in the water column. Because of the hot, dry summer, the tinuity layer, thus confirming results obtained with the Eh water level fell so far in the nearby Trent-Severn Waterways microprobe. System used by pleasure boats that additional water had to Bacterial density. Acridine orange direct counts ofbacteria be drawn off from the surrounding watersheds, including the from the two sets of study lakes are presented in Table 2. lakes under investigation. Thus, on several occasions the Counts ranged from 8.8 x 105 to 1.8 x 106 per ml of littoral litter bags containing the cellophane strips were found water and from 5.5 x 108 to 1.0 x 109 per g (dry weight) of sediment. When the data were analyzed statistically, surface water (0.5 m) counts were significantly higher (P < 0.05) in the humic lakes (Chub and Dickie Lakes). Likewise, bacte- TABLE 2. Total bacterial counts by epifluorescence microscopy of Dorset lakes (mean standard deviation) rial populations in shallow sediment were higher (P < 0.01) in Dickie Lake. Cell counta in: These data are similar to pelagial water counts carried out Lake site, depth No. of Date samples (per Sediment on acidified lakes in the Sudbury, Ontario, region (37), the Water (n) X 106) (per g [dry Adirondack Mountains, N.Y. (11), Norway (47, 48), and wtl x 108) Sweden but are 20 to 100 times higher than counts (2, 20) August Red Chalk, 0.5 1 1.14 NDb (i.e., 1.3 x 104 to 7.1 x 104 per ml) reported by Boylen et al. 1983 Plastic, 0.5 3 1.33 (0.17) 10.43 (0.24) (3) for nine remote Adirondack lakes with pHs of 4.3 to 7.0. Chub, 0.5 4 1.76 (0.10) ND Furthermore, the finding that water counts were greater in the dystrophic lakes (Chub and Dickie Lakes) than in the September Harp, 0.5 2 1.10 (0.04) 7.85 (0.02) oligotrophic lakes (Red Chalk, Harp, and Plastic Lakes) is in 1984 Harp, 2.0 2 1.07 (0.01) 5.49 (0.58) agreement with observations on a large number of Scandi- Plastic, 0.5 2 1.08 (0.04) 8.12 (0.12) navian lakes, most of which are acidified (2, 47, 48). Plastic, 2.0 2 1.08 (0.02) 5.91 (0.11) The total number of bacteria in the top 1 cm of sediment in Dickie, 0.5 2 1.26 (0.03) 8.89 (0.13) the Dorset lakes cannot be compared with the results of Dickie, 2.0 2 0.88 (0.47) 7.05 (0.36) some other studies in which grab samples were taken (11, a Cell count given as mean standard deviation of n determinations. 36). However, bacterial densities are similar to those made b ND, Not determined. VOL. 50, 1985 CELLULOSE DECOMPOSITION IN ACIDIFYING LAKES 319 floating at the surface, where the bacteria would be exposed to intense sunlight and heat. Since fecal coliforms and fecal a streptococci in marine environments are probably killed by solar radiation (13, 22), the same factor could be responsible 1984- ANOXIC SEDIMENT -75-0 for the inactivation of cellulose decomposers on the strips. z Furthermore, the littoral water temperature was higher than 0 24°C (Table 1); it was sometimes as high as 28°C. It was a therefore above the optimal range for Cytophaga-like bacte- 4 ria (39), which have since been isolated (manuscript in : 50-0 preparation). When the experiment was carried out the following year at mu-I 25-0 a site farther from the shore in anoxic epilimnetic sediment 0 (Fig. 6a) and in the overlying water (Fig. 6b), the cellophane strips were broken down at the same rate in the three (o) different lakes. Statistical analysis of sediment data showed no residual variance (X2 = 4.0 with df 2) as well as no significant difference between slopes (F = 1.4 with df 2, 18) TIME (DAYS) or elevations (F = 1.1 with df 2, 20). The water column data (Fig. 6b) gave similar results regarding residual variance (X2 = 3.8 with df2), slopes (F = 2.7 with df2, 18), and elevations (F = 0.5 with df 2, 20). - 40-0 No clear pH-related depression of cellulose breakdown in z anoxic sediment was evident. This finding was perhaps due 0 to the presence of buffering end products (particularly -9 30-0 HCO) generated by sediment anaerobes (such as sulfate m w o4 020-0 -J U 0*

z TIME (DAYS) 8 FIG. 6. Rate of cellulose breakdown in littoral zone farther from -m4S shore of 1984 study lakes (plotted as mean ± 95% confidence .I intervals). Regression equations are as follows. (a) Anoxic sedi- w ment: Harp Lake, y = 1.26x - 4.88; Plastic Lake, y = 1.51x - 14.89; mu Dickie Lake, y = 1.70x - 14.47. (b) Water column: Harp Lake, y = on 0 0.51x - 1.45; Plastic Lake, y = 0.85x - 7.76; Dickie Lake, y = 0.82x - 10.64.

reducers which can tolerate low epilimnetic pH [19]). The 30 40 50 60 above explanation may be supported by the fact that the pH TIME (DAYS) at the mud surface in Plastic Lake rose from 5.2 to 5.9 during the sampling period. As a result, sediment heterotrophs (b) 1983 - WATER-COLUMN would be exposed to a pH that is less inhibitory to their metabolic activities.

0 Figure 6 shows a lag in the onset of decomposition which a u 49 doubtless reflects the time required for bacterial coloniza- m tion. From phase contrast observations of strips (manuscript ob in preparation) it is likewise concluded that the principal

0 cellulose degraders were Cytophaga-like bacteria. Similar J4j organisms have been enriched from several other aquatic mu sites (5, 38, 39). Similar observations of the effect of pH on decomposition in anaerobic sediment in an experimentally acidified lake were made by Kelly et al. (24). They measured the rate of 10 breakdown of organic matter in terms of methane and TIME (DAYS) inorganic carbon released from sediments and found no FIG. 5. Rate of cellulose breakdown in nearshore littoral zone of decline over a 6-year period when the epilimnetic pH fell 1983 study lakes; data points are plotted as mean + 95% confidence from 6.7 to 5.1. Since they found that the pH of sediment intervals. Regression equations are as follows. (a) Oxic sediment: porewater was much (6.7 to than that of the water Red Chalk Lake, y = 2.21x - 7.14; Plastic Lake, y = 1.46x - 16.07; higher 6.9) Chub Lake, y = 1.38x - 16.43. (b) Water column: Red Chalk Lake, column (5.0 to 5.5), it is clear that sediment anaerobes, y = 0.17x - 0.03; Plastic Lake, y = 0.11x + 3.02; Chub Lake, y = including cellulose decomposers, are not exposed to low pH 0.04x + 3.23. and can therefore function normally. Williams and Crawford 320 HOENIGER APPL. ENVIRON. MICROBIOL.

(51) also observed that methanogenesis in peatlands is pH was broken down at two sites with average pH 3.7 and 5.7, dependent. In their experiments, the optimal rate occurred respectively, than in the reference stream (pH 6.3), and the when peat samples with initial pHs of 3.8 and 4.3 were rate varied with leaf species. Although the same density of adjusted to pH 6.0. bacteria (ca. 1010 per g of dry detritus) was found on leaves In my experiments, cellulose was used in the form of at each site, there were significant differences in insect cellophane or "regenerated cellulose" (35), which has es- abundance. sentially the same chemical structure as native cellulose Clear trends in the structure of insect communities can be present as microfibrils in plant cell walls. It is therefore not discerned from experiments described in the above papers a model substance like polyguaiacol, used for studying (4, 12, 28): mayflies are absent, and the number of stone flies biodegradation of lignin (7). The colorimetric assay of cellu- is greatly reduced in acidic streams (below ca. pH 5.2) where lose breakdown with dyed cellophane strips has definite caddis fly and chironomid larvae predominate; several advantages over earlier methods involving the use of coarse mayfly and stone fly genera as well as dragonfly nymphs litter (leaves, twigs) in which about one-third of the cellulose occur under moderately acidic (ca. pH 5.7) conditions, is shielded from microbial attack by relatively recalcitrant together with large numbers of caddis flies and chironomids; lignin. richer and more balanced communities are found in streams In litter bag experiments, dry weight change or metabolic closer to neutral pH. Breakdown of leaf litter in stream activity of colonizing microorganisms, or both, were fol- ecosystems involves an initial phase of a biotic leaching lowed over a pH range of ca. 4.0 to 7.0 under natural and followed by two interacting biological processes: microbial laboratory conditions. Traaen (46, 48) found that birch conditioning and macroinvertebrate feeding (1, 8, 50). Dur- leaves and aspen twigs decomposed more slowly both in ing conditioning, bacteria and fungi colonize the surface, flowthrough tanks at pH 4.0 and 4.5 to 5.2 than at pH 6.0 and increase in biomass, and partially degrade the leaf tissue, in acidic Norwegian lakes (pH 4.5 to 5.5) when compared releasing substances which are then consumed by insect with less acidic ones (pH 5.6 to 6.5). Furthermore, leaves larvae. At the same time, shredder groups comminute the were more readily broken down than twigs. Laake (26) litter and thus vastly increase the surface available for added allochthonous detritus to cores of littoral sediment in microbial colonization. flow through tanks identical with those of Traaen (see Although the trophic interactions of detritus breakdown in above); both oxygen uptake and glucose turnover were lakes have been investigated far less thoroughly than those reduced at pH 4.0 to 4.5, whereas lignocellulose components in streams, a variety of potential detritivores may be found remained intact and filamentous fungi accumulated on the on the surface of the littoral sediment; these include insect core surface. In a 2-year field experiment, Francis et al. (11) larvae, amphipods, isopods, and crayfish (50). Previous (40) placed leaves of five species (beech, sugar maple, red maple, and ongoing (P. M. Stokes, personal communication) re- red spruce, and leather leaf) in the water column of three search indicates that as the epilimnetic pH falls below 5.5, Adirondack Mountain lakes with pHs of ca. 5.0, 6.0, and 7.0. changes occur in the benthic macroinvertebrate community; They found that the decomposition rate varied with both pH these changes, in turn, could result in reduced processing of and leaf species and that the density of colonizing bacteria litter, as has already been observed in acidic Norwegian (46, also varied with leaf type. The effects of low pH on break- 48) and Adirondack (11) lakes. down of autochthonous detritus (leaves of a sedge, Carex Leaves are progressively skeletonized as they decompose, sp.) were investigated by McKinley and Vestal (30) in until only the vascular elements (containing lignin in close microcosms with mean pHs of 3.4 to 7.4. Significant de- association with cellulose and hemicellulose) remain. The creases with declining pH occurred both in microbial activity rate of breakdown of a particular leaf species (e.g., dogwood (incorporation of radiolabeled acetate) and in dry weight, > birch > oak) appears to be correlated with its lignin nitrogen content, and C/N ratio of litter, while changes were content (4). This highly cross-linked, aromatic polymer is also observed by scanning electron microscopy in the com- quite resistant to biodegradation and thus blocks access to munity structure of the microbiota. cellulases of microbial origin. For this reason, a much Particularly relevant are field experiments in which the simpler substrate than litter, namely cellulose (as dyed strips rate of litter breakdown and changes in macroinvertebrate of cellophane), was selected for measuring microbial decom- populations were examined in acid and nonacid streams in position in the present field study. As noted above, the strips Sweden (12) and North America (4, 28). Friberg et al. (12) have an additional advantage: they can be viewed by phase reported that packs of leaf litter were decomposed more contrast microscopy, and the extent of colonization by slowly and that the biomass of insect larvae per pack was microorganisms can be assessed. Finally, it now seems much lower in a Swedish stream, with the pH varying likely that too much emphasis has previously been placed on between 4.3 and 5.9, than in a reference stream with pH the significance of reductions in coarse litter breakdown for varying between 6.5 and 7.3. Mackay and Kersey (28) plankton productivity in lakes impacted by acidic precipita- observed a ca. 50% loss in the dry weight of autumn-shed tion (9). sugar maple leaves over the winter in alkaline or broad-pH- The choice of cellulose was also predicated by the impor- range streams flowing into several Dorset lakes (including tance of macrophytes and attached algae as primary produc- Harp and Dickie Lakes, monitored by myself during the ers in Shield lakes (9, 18, 43, 44). It has been observed that course of the present study), compared with a 20 to 30% loss macrophyte biomass increases greatly with acidification, in acidic (pH 4.3 to 4.5) or moderately acidic (pH 5.3 to 6.2) while species diversity decreases (I. Wile, G. E. Miller, streams. Furthermore, a survey of insect larvae carried out G. G. Hitchin, and N. D. Yan, Can. Field Nat., in press). during the following spring and summer revealed clear Likewise, extensive mats of benthic algae (notably filamen- pH-related differences in taxa and abundance. Carpenter et tous greens like Mougeotia) are encountered in acidifying al. (4) examined the impact of acid mine drainage in a lakes, where they may replace pelagial forms as principal Virginia reservoir system on the decomposition of various producers of organic carbon (44). When these two groups of leaf species (dogwood, birch, oak and rush) as well as on littoral plants die, they doubtless contribute large amounts of leaf-colonizing bacteria and macroinvertebrates. Less litter autochthonous detritus to a lake. It has been found that in VOL. 50, 1985 CELLULOSE DECOMPOSITION IN ACIDIFYING LAKES 321 eutrophic lakes ca. 90% of the particulate organic matter eutrophic lake. Oikos 23:167-177. originates from macrophytes (49, 50) and is converted by 17. Harvey, H. H., R. C. Pierce, P. J. Dillon, J. R. Kramer, and heterotrophic microorganisms to inorganic carbon and min- D. M. Whelpdale. 1981. Acidification in the Canadian aquatic erals. That such particulate organic matter as environment: scientific criteria for an assessment of the effects (provided of acidic deposition on aquatic ecosystems. National Research cellulose in this study) is degradable under pH conditions Council Canada report no. 18475. National Research Council, currently encountered in most Shield lakes is of fundamental Ottawa. importance to carbon cycling and thus affects all trophic 18. Hendrey, G. R. 1982. Effects of acidification on aquatic primary levels within the ecosystem. producers and decomposers, p. 125-134. In R. E. Johnson (ed.), Acid rain/fisheries. Proceedings of the International Symposium ACKNOWLEDGMENTS on Acidic Precipitation and Fish: impacts in Northeastern North I thank Dino Fallavolita, Stefano Monardo, and Robin Pellow for America. American Fisheries Society, Bethesda, Md. summer assistance and Peter Dillon for hospitality at the Dorset 19. Herlihy, A. T., and A. L. Mills. 1985. Sulfate reduction in Research Center, Ontario Ministry of the Environment. I am freshwater sediments receiving acid mine drainage. Appl. particularly indebted to Edward de Grosbois and Steve Smith for Environ. Microbiol. 49:179-186. statistical help, to Ronald Hall for helpful suggestions on the 20. Johansson, J.-A. 1983. Seasonal devclopment of bacterio- manuscript, and to both Ann Zimmerman and Norman Yan for many plankton in two forest lakes in central Sweden. Hydrobiologia stimulating discussions. 101:71-88. This work was supported by contracts from the Associate Com- 21. Jones, J. G. 1979. Microbial activity in lake sediments with mittee on Scientific Criteria for Environmental Quality, National particular reference to electrode potential gradients. J. Gen. Research Council of Canada. Microbiol. 115:19-26. 22. Kapuscinski, R. 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