Active bacterial taxa linked to the degradation of [ 13 C]cellulose

A total of 989 bacterial 16S rRNA sequences were analyzed and assigned to 94 family-level OTUs from ‘light’ and ‘heavy’ fractions of [¹³C]cellulose treatments at 15°C and 5°C (Table 19). Family-level coverages ranged between 85-94% in the different clone libraries indicating sufficient sampling. Rarefaction curves were lower in clone libraries from ‘heavy’ fractions at the end of the incubation with [¹³C]cellulose at 15°C and 5°C compared to clone libraries of

‘light’ and ‘heavy’ fractions at the start of the incubation or in ‘light’ fractions at the end of the incubation (Figure 11). This indicates that the RNA of only a subset of bacterial families was labeled with ¹³C-carbon derived from [¹³C]cellulose during the incubation.

High amounts of propionate, acetate and CO2 were produced during cellulose degradation (Figure 9). Certain Bacteroidetes species can hydrolyze cellulose and produce the observed fermentation products [23, 173, 353], and this phylum was involved in cellulose degradation in anoxic incubated agricultural soil [372] and in the human gut [48]. In this regard, OTU4a, which was enriched in ‘heavy’ fractions at 15°C, was closely related (96% maximum identity to FN434002; Figure 12) to sequences of unclassified Prolixibacteraceae that were labled in [¹³C]cellulose but not in [¹³C]glucose treatments of agricultural soil [372]. OTU4a had 95%

maximum identity to the rice soil bacterium PB90-2, which was highly abundant in its habitat and could hydrolyze xylan and pectin but not cellulose [51, 162]. OTU4b was labeled at 5°C and 15°C and was closely related to an uncultured bacterium from a paper pulp degrading consortia (EF562547; Figure 12). Recently, Mangrovibacterium diazotrophicum, a cellulolytic facultative aerobe of the Prolixibacteraceae was isolated (91% and 90% identity to OTU4a and 4b, respectively) [173], validating that this family indeed harbors cellulolytic fermenters. The hydrolytic nature of cultured relatives, the presence of uncultured relatives in various cellulose-degrading environments, and the incorporation of [¹³C]cellulose derived ¹³C-carbon in peat soil microcosms indicate that members of a novel genus within the Prolixibacteraceae are likely to contribute to cellulose degradation and propionate production in contrasting ecosystems.

Table 19 Number of sequences, OTUs, and coverages of bacterial 16S rRNA clone libraries

Clone library^a 15°C 5°C

total t0 L t0 H t40 L t40 H t80 L t80 H

No. of Sequences 150 165 171 170 174 168 989

No. of OTUs^b 45 54 43 25 45 19 94

Coverage [%] 85 85 91 94 90 94 98

at0 and t40, 0 d and 40 d of incubation, respectively, after 17 d of preincubation at 15°C; t80, 80 d of incubation after 22 d of preincubation. L and H; ‘light’ and ‘heavy’ fractions, respectively (Figure 6).

bOTUs were calculated based on 87.5% similarity cutoff (‘family level’ [492]). Modified from ref [387].

The highly novel OTU1 was abundant in ‘heavy’ fractions of [¹³C]cellulose treatments at 5°C and 15°C and could only be distantly affiliated to the Fibrobacteres (79% maximum identity to Fibrobacter succinogenes) (Figure 12). Taxa assigned to the Fibrobacteres were abundant cellulose degraders in the digestive tract of ruminants, anaerobic digesters, and municipal waste landfill sites [281, 360, 455], but this phylum was previously not recognized in peatlands.

F. succinogenes is the type species and represents one of only two cultured species of the Fibrobacteres. This microbe is a well studied hydrolytic fermenter that produces succinate and acetate exclusively from the breakdown products of cellulose [302, 426]. Based on the affiliation (although distinct) to a cellulose degrader, the accumulation of high amounts of propionate and acetate, and the high abundance of OTU1, on may suggest that this taxon was important for cellulose hydrolysis and contributed to propionate and acetate formation in the microcosms at 5°C and 15°C.

The Acidobacteria belong to the dominant phyla in peatlands, are metabolically highly versatile, and are repeatedly characterized as well adapted to cold and acidic environments [84, 176, 468]. Relative abundances of Acidobacteriaceae in the different clone libraries were more balanced and did not show a clear labeling from [¹³C]cellulose (Figure 12). However, within the Acidobacteriaceae, there are two subfamily-level OTUs (OTU3b and 3e) that are enriched in ‘heavy’ fractions compared to ‘light’ fractions at the end of the incubation at 15°C and 5°C (Table A2). OTU3e was closely related to the slowgrowing microaerophil Telmatobacter bradus (98% maximum identity), which was isolated from a peatland and was the first cultured member of the Acidobacteria that could grow anaerobically on cellulose [324].

OTU3b was was distantly related to Koribacter versatillis (93% maximum identity), which was isolated as an aerobe (anerobic growth was not tested) and harbors cellulase genes in its genome [468]. The agricultural soil isolate KBS 83, is another cellulolytic facultative aerobe of the Acidobacteriaceae [112], and A. capsulatum (the type species of the Acidobacteriaceae) is also a facultative aerobe and harbors cellulase genes in its genome but could not grow on cellulose [199, 324, 468]. The collective data suggest that the capability to grow anaerobically on cellulose is spread among the Acidobacteriaceae, and genera within this family are likely to contribute to cellulose hydrolysis in peatlands and might especially adapted to changing redox conditions. However other subfamily OTUs within the Acidobacteriaceae (OTUs 3a and 3c) were abundant but not labled in the [¹³C]cellulose treatments and may be capable of cellulose hydrolysis under aerobic conditions or may contribute to the degradation of polymers like xylan or pectin [112, 322, 323, 468].

The Holophagaceae represent a family that are only distantly related to other members of the Acidobacteria. None of the two currently available isolates (both are strict anaerobes) have been shown to grow on cellulose [57, 245]. However, 16S rRNA gene sequences affiliated to the Holophagaceae were frequently detected in peatlands [84] and this family constituted 11%

of the 16S rRNA sequences in ‘heavy’ fractions of the [¹³C]cellulose treatment at 15°C (OTU8b;

Figure 12). Thus, the Holophagaceae may contribute to the mineralization of cellulose in peatlands at moderate temperatures.

Figure 11 Rarefaction analyses and 95% confidence intervals of bacterial 16S rRNA sequences obtained from cellulose supplemented microcosms.

OTUs were calculated based on 87.5% similarity cutoff (‘family level’ [492]). t0 and t40, 0 d and 40 d of incubation, respectively, after 17 d of preincubation at 15°C; t80, 80 d of incubation after 22 d of preincubation. L and H; ‘light’ and ‘heavy’ fractions, respectively (Figure 6). Modified from ref [387].

Ruminococcaceae are important cellulose degraders in the digestive tracts of animals and humans, agricultural soil, swamp soil, and municipal wastes [48, 244, 281, 372, 449]. This family was also labled in [¹³C]cellulose treatments in this study but the relative abundance was considerably lower than that of other potential cellulose degraders (e.g., Fibrobacter-related unclassified Bacteria and Prolixibacteraceae; Figure 12), indicating that novel hitherto unrecognized rather than well studied hydrolytic fermenters were the drivers of cellulose degradation under the experimental conditions.

Saccharolytic fermenters compete with cellulolytic fermenters for sugars released during cellulose hydrolysis [17, 241] but may also enhance cellulose hydrolysis by keeping the concentrations of soluble sugars low, which prevents product inhibition of the cellulase systems [254, 336]. OTU9, and 12 were labled in [¹³C]cellulose treatments (Figure 12) and were closely related to saccharolytic fermenters of the Porphyromonadaceae, and Spirochaetaceae, respectively, indicating that saccharolytic fermenters contribute to the degradation of cellulose derived sugars. OTU9 had 99% maximum identity to the propionate

No. of Clones

0 20 40 60 80 100 120 140 160 180

No. of OTUs

0 10 20 30 40 50 60

15ºC t0 L 15ºC t0 H 15ºC t40 L 15ºC t40 H 5ºC t80 L 5ºC t80 H

producing strictely anaerobic non-hydrolyzing fermenter Paludibacter propionicigenes (isolated from rice paddy soils) [446] and therefore, might have contributed to the high amounts of propionate detected during cellulose degradation (Figure 9). The closest cultured relative of OTU12 (96% maximum identity) was Spirochaeta zuelzerae, which was isolated from freshwater mud and ferments glucose to H2, CO2, acetate lactate and succinate [458].

Saccharolytic fermenters of the genus Spirochaeta are known to enhance the rate of cellulose hydrolysis when grown in coculture with hydrolytic fermenters [336] and might fulfill a similar role in peatlands.

Figure 12 Phylogenetic tree of bacterial 16S rRNA sequences retrieved from [¹³C]cellulose treatments (bold) and reference sequences.

Shown are potentially labeled OTUs that displayed increased relative abundances in ‘heavy’ (H) compared to ‘light’ (L) fractions at the end of the incubation. See Table A2 for the sequence descriptor code and a complete list of all bacterial family-level OTUs. The phylogenetic tree was calculated as described in (2.6.4). Branch length are based on the neighbor-joining tree. Filled circles at nodes indicate congruent nodes in the maximum-likelihood, maximum parsimony, and neighbor-joining tree. Open circles indicate congruent nodes in two of the three trees. The bar indicates 0.1 change per nucleotide.

Methanosarcina mazei (AE008384) was used as outgroup. ‘t0’ was after 17 or 22 days of anoxic preincubation at 15°C and 5°C, respectively (2.1.2.1). L and H; ‘light’ and ‘heavy’ fractions, respectively (Figure 6). Modified from ref [387].

Rel. abundance [%]

Sequences within OTU6b were closely related to Clostridium acidisoli (Table A2). C.

acidisoli was isolated from a bog site close to the Fen Schlöppnerbrunnen, ferments a broad range of soluble sugars to acetate, butyrate, lactate, formate, H2, and CO2, growths well under cold and acidic conditions, and is characterized by fast growth rates (at least at 30°C) [218].

Unfortunately, hydrolytic growth on cellulose, xylan, and pectin was not tested. However, the outstanding feature of this microbe is the capability to fix N2 at a pH as low as 3.7, which is not known from other acid tolerant Clostridia. OTU6b was especially abundant at 5°C and might have cooperated with cellulose hydrolyzers in a synergistic relationship in which the hydrolyzer provides soluble sugars and the C. acidisoli-related OTU6b provides the nitrogen source for growth.

OTU6a was not as abundant as OTU6b and was closely related to C. puniceum (Table A2). C. puniceum is pectinolytic, acido- and psychrotolerant, and produces butyrate, acetate, and butanol during fermentation [165, 266]. C. puniceum was the dominant consumer of [¹³C]glucose and [¹³C]xylose in microcosms of the Fen Schlöppnerbrunnen [151]. This suggests that C. puniceum might be more competitive for high concentrations of soluble sugars whereas C. acidisoli might be more competitive for the low sugar concentrations released during cellulose hydrolysis.