In Situ Identification of Keratinhydrolyzing Organisms In

RESEARCH ARTICLE In situ identiﬁcation of keratin-hydrolyzing organisms in swine manure inoculated anaerobic digesters Yun Xia1, Daniel I. Masse´ 1, Tim A. McAllister2, Carole Beaulieu3, Guylaine Talbot1, Yunhong Kong2 & Robert Seviour4

1Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, Sherbrooke, QC, Canada; 2Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, AB, Canada; 3De´ partement de biologie, Universite´ de Sherbrooke, Sherbrooke, QC, Canada; and 4Biotechnology Research Centre, La Trobe University, Bendigo, Vic., Australia Downloaded from https://academic.oup.com/femsec/article/78/3/451/600873 by guest on 23 September 2021

Correspondence: Daniel I. Masse´ , Dairy and Abstract Swine Research and Development Centre, Agriculture and Agri-Food Canada, 2000 Feathers, a poultry byproduct, are composed of > 90% keratin which is resis- College Street, Sherbrooke, QC, Canada, tant to degradation during anaerobic digestion. In this study, four 42-L anaero- J1M 0C8. Tel.: +1 819 565 9171; fax: +1 bic digesters inoculated with adapted swine manure were used to investigate 819 564 5507; e-mail: daniel. [email protected] feather digestion. Ground feathers were added into two anaerobic digesters for biogas production, whereas another two without feathers were used as negative Received 10 February 2011; revised 9 May control. Feather degradation and enhanced methane production were recorded. 2011; accepted 21 July 2011. Final version published online 15 September Keratin-hydrolyzing organisms (KHOs) were visualized in the feather bag fluids 2011. after boron-dipyrromethene (BODIPY) fluorescence casein staining. Their abundances correlated (R2 = 0.96) to feather digestion rates. A 16S rRNA clone DOI: 10.1111/j.1574-6941.2011.01188.x library was constructed for the bacterial populations attached to the feather particles. Ninety-three clones (> 1300 bp) were retrieved and 57 (61%) Editor: Alfons Stams belonged to class Clostridia in the phylum Firmicutes, while 34 (37%) belonged to class Bacteroidia in the phylum Bacteroidetes. Four oligonucleotide FISH Keywords probes were designed for the major Clostridia clusters and used with other anaerobic digesters; keratin-hydrolyzing FISH probes to identify the KHOs. Probe FIMs1029 hybridized with most organisms; BODIPY FL protease staining; > fluorescence in situ hybridization; full-cycle ( 80%) of the KHOs. Its targeted sequence perfectly matches that possessed rRNA approach. by 10 Clostridia 16S rRNA gene clones belonging to a previously uncharacter- ized new genus closely related to Alkaliphilus in the subfamily Clostridiaceae 2 of family Clostridiaceae.

biotechnologically more attractive alternatives. For exam- Introduction ple, feathers have been recycled after cooking under high Feathers are a poultry byproduct composed of > 90% pressure and temperature to make a feather meal suitable keratin (Astbury & Beighton, 1961), mostly as b-keratin for use as an animal protein supplement (Odetallah et al., (Sawyer et al., 2000). b-keratin is recalcitrant to degra- 2003) or as a slow-releasing nitrogen fertilizer (Choi & dation by many proteases because of the presence of Nelson, 1996). However, hydrothermal treatment of extensive disulﬁde bonds and cross-linkages (Papado- feather keratin is expensive and destroys several valuable poulos et al., 1986). Because feathers are generated in amino acids. The resulting product is poor in digestibility millions of tons annually, their disposal is an important and has a variable nutrient quality (Wang & Parsons, part of solid waste management (Onifade et al., 1998). 1997). Traditionally, feathers are treated as an organic solid Feathers have been used in anaerobic digestion systems waste, being incinerated or disposed at waste disposal to produce methane biogas but are poorly degraded

MICROBIOLOGY ECOLOGY MICROBIOLOGY sites. These treatments are wasteful because feather kera- under anaerobic conditions (Salminen & Rintala, 2002). tin is rich in useful amino acids and its disposal by such Pretreatments including thermal, chemical, and enzymatic methods generates greenhouse gases (Salminen & Rintala, methods have been used in attempts to increase their 2002). Consequently, efforts have been made to develop digestion rates (Onifade et al., 1998). When Williams &

Shih (1989) enriched a mixture of bacteria using ball- bags and washed (delicate cycle) in a washing machine milled feather agar, the resultant bacterial community (Frigidaire, Martinez, GA) with tap water. Feather sam- showed keratinase activity capable of degrading auto- ples were then dried at 45 °C in a Unithern dryer (Con- claved whole feathers. Feather degradation has been struction CQLTD, England) until a constant weight was reported in anaerobic digesters (ADs) treating poultry reached (after about 8 weeks). The samples were ground waste including manure and/or mixed fractions of bone, through a 4-mm screen (Thomas-Wiley Laboratory Mill) trimmings and offal under thermophilic (Williams & and divided into portions of 33 g each and placed in Shih, 1989) or mesophilic (Salminen & Rintala, 2002) nitrogen-free polyester bags with a pore size of 50 lm condition. However, the chemical transformation and the (ANKOM Technology, Macedon, NY). Each bag was organisms responsible for this degradation have not been sealed with a plastic tie wrap and washed in the washing

characterized in detail. machine to remove any remaining residual particles with Downloaded from https://academic.oup.com/femsec/article/78/3/451/600873 by guest on 23 September 2021 Keratin-hydrolyzing bacteria and keratinase enzyme a diameter < 50 lm. Finally, the bags were dried at 45 °C mixtures have also been reported to digest prions respon- until a constant weight ( 31 g each) was achieved. As sible for transmissible spongiform encephalopathies or required, 14 bags were then attached onto a steel stick prion diseases including bovine spongiform encephalopa- and inserted into an AD. thy (BSE), sheep scrapie, deer chronic waste disease, and human Creutzfeldt-Jakob disease (Gupta & Ramnani, Anaerobic digester set up 2006). Keratinases secreted by thermophilic bacterial strains VC13, VC15, S290 and Streptomyces sp. S6 (Tsi- Four 42-L Plexiglas ADs and one 7-m3 semi-industrial roulnikov et al., 2004), Bacillus licheniformis strain PWD-1 AD, as described by Masse´ et al. (2001), were used in this (Langeveld et al., 2003) and Bacillus MSK103 (Yoshioka study. The 7-m3 semi-industrial AD was used to adapt et al., 2007) were shown to inactivate prions, because the fresh swine manure collected from a commercial pig farm structures of prions and feather keratin share many char- (Sherbrooke, Quebec) that processes about 4000 pigs per acteristics. Both are fibrous and insoluble proteins rich in year, which was then used to inoculate (representing b-sheet (Fraser et al., 1972). BSE is the most notorious 100% of the volume) the 42-L ADs. The semi-industrial prion disease, which has cost the Canadian cattle industry AD was fed with fresh swine manure with a retention approximately $6.3 billion since 2003, as a result of closed time of 14 days. At the time of inoculation of the markets and consumer concerns over food safety issues 42-L ADs, the semi-industrial 7 m3 AD had been operat- related to it (http://www1.agric.gov.ab.ca). Therefore, the ing at 25 °C for more than 2 years. control and inactivation of prions from cattle carcasses Feather bags were added to two 42-L ADs (feather ADs during processing have been major public and animal with duplicate) at different times. Fourteen bags (No.1 to health concerns (Hill et al., 1997). No.14) were added to each AD on day 0 [initial load 1 In this study, ground feathers were added in bags to 0.88g g VSS (volatile suspended solid) manure, repre- ADs inoculated with adapted swine manure for methane senting 30% of the total Chemical oxygen demand production. The keratin-hydrolyzing organisms (KHOs) (COD) in each AD]. Three bags (from bags No.15 to responsible for the feather digestion were detected micro- No. 26) were added at day 22, day 55, day 85, and day scopically by BODIPY fluorescent protease staining 113. The other two ADs (control ADs with duplicate) (BODIPY FL casein staining) and further identified using were operated without addition of feather bags serving as the full-cycle rRNA approach (Amann et al., 1995). A 16S negative controls. The volume of the inoculum for these rRNA clone library was constructed from the bacterial four ADs was 35 L. All ADs were operated in a closed populations attached to feather particles inside feather room at 25 °C for 153 days. bags. Oligonucleotide FISH probes were designed to target the major clusters of the 16S rRNA gene clones Physiochemical characterization of anaerobic retrieved and used to identify the KHOs in situ. digesters Total solids, volatile solids, total suspended solids (TSS), Materials and methods and volatile suspended solids were determined according to standard methods (APHA, 1998). Soluble COD was Source and preparation of chicken feathers determined according to the closed reflux colorimetric Freshly plucked white chicken feathers were collected method described in the standard methods. Methane from a slaughterhouse (Saint-Anselme, QC, Canada) and (CH4), total Kjeldahl nitrogen (TKN), ammonia nitrogen transferred to the laboratory within 4 h. The feathers (NH3-N), and liquid volatile fatty acids (VFAs) were were divided into 2 kg portions, placed in clean cotton analyzed following the procedures described by Masse´

ª 2011 Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada FEMS Microbiol Ecol 78 (2011) 451–462 Published by Blackwell Publishing Ltd. Keratin-hydrolyzing bacteria in anaerobic digesters 453 et al. (2001). COD of raw feathers was determined fol- 1995), respectively. Another portion was transferred into lowing standard methods. At different time intervals (22– 2 mL Eppendorf tubes that were immediately frozen in 33 days), three feather bags (No.1 to No.12) were taken liquid nitrogen and stored at 80 °C for DNA extraction. out, and washed with deionized water, dried for 48 h at The remainder was centrifuged at 800 g for 15 min to 45 °C, and weighed to determine feather degradation. remove large particles before being used for BODIPY FL Bags (No.15 to No.26) were kept until the end of the casein staining. experimental trial. Bags (No.13 and 14) were used to collect fresh samples for BODIPY FL casein staining; DNA BODIPY FL casein staining combined with DAPI extraction and FISH (see later sections for details). These staining and enumeration two bags were put back immediately after sampling.

The BODIPY FL casein staining was performed following Downloaded from https://academic.oup.com/femsec/article/78/3/451/600873 by guest on 23 September 2021 the procedure described previously (Xia et al., 2007). To Mass balancing, digestion rate calculations stain microorganisms attached to feather particles, briefly, The extent of feather digestion and rates of feather diges- a 200 lL aliquot of the supernatant was mixed with the tion were estimated using a mass balance over COD as same volume of freshly prepared 2 9 Tris-HCl buffer described by O’Sullivan & Burrell (2007). The CH4,H2, (10 mM, pH 7.8) before 200 lL BODIPY FL casein VFAs, and biomass data were all converted to units of working solution was added. The mixture was placed in a grams of COD equivalents for application to the mass 10-mL serum bottle wrapped in aluminum foil and incu- balance. The extent of feather digestion was estimated bated at 25 °C for 30 min on a rotating disk (220 r.p.m) according to the following equation: before microscopic examination. Samples after incubation with BODIPY FL casein were spread evenly on three-well Extent of digestion ¼ðSCOD þ SCOD þ SCOD CH4 H2 VFA (10 lL in each well) gelatin-coated Teflon printed slides þ Þ= SCODbiomass CODinitial (Electron Microscopy Sciences) and dried in a dark room before being mounted with antifade reagent CITI fluor where COD initial is feather COD added to an AD, SCOD (Electron Microscopy Sciences) and examined microscop- CH4, SCOD H2, SCODVFA,and SCODbiomass were the ically. COD of CH4,H2, VFAs, and biomass correspondently To stain the bacteria in the mixed liquor, fresh mixed due to feather digestion. The feather digestion rate was liquor samples were collected directly from each AD, a calculated from the slope of the extent of digestion curve. 400 lL aliquot then centrifuged at 4500 g for 10 min, and It was equal to the SCOD at time [t + Dt] minus the the supernatant discarded. The biomass pellet was resus- SCOD at time [t], divided by the time step. pended in 1 9 Tris-HCl, and BODIPY FL casein working 1 solution was added to a final concentration of 0.5 mg g TSS with a final reaction volume of 600 lL. The incuba- Sample preparation for microbiological tion and microscopic examination conditions were the analyses same as those used for staining and examination of bacte- To collect any bacteria attached to the feather particles, ria attached to the feather particles described above. fresh feather samples (5–10 g) collected from the feather The BODIPY FL casein staining combined with DAPI bags (No. 13–14) were drained for 3–5 min on a sieve (4′,6-diamidino-2-phenylindole) staining was also per- and resuspended in bacteria-free AD filtrate (50–100 mL). formed according to the procedure described previously The filtrate was prepared from the mixed liquor of the (Xia et al., 2007, 2008). Briefly, cells staining positively same AD by centrifugation at 16 000 g for 30 min and with BODIPY FL casein were detected microscopically filtration through a series of filters (Whatman Nuclepore and their positions on the slides recorded. Then, the CITI track-etched membranes) with pore sizes of 3, 1, 0.45, fluor was washed away by gently rinsing slides (for and 0.2 lm, used sequentially. The mixture was trans- 1 min) with 70% ethanol. After being air-dried DAPI 1 ferred to a thick-walled polyethylene bag (180 mm 9 (100 lL, 0.003 mg mL ) was spread over the slides and 300 mm) and subjected to vigorous mechanical pummel- kept at room temperature for 10 min. Then, the slides ling for 2 min in a Colworth Stomacher 400 (A. J. Sew- were dipped in distilled water three to five times before ard & Co. Ltd, London). This suspension was then being air-dried and examined microscopically. The abun- filtered through a sterilized eight-layer cheesecloth. A por- dances of the KHO cells stained with BODIPY FL casein tion of filtrated bag fluids was fixed in ice-cold ethanol were estimated as a percentage of total cell numbers (50% final concentration) or paraformaldehyde (PFA) stained by DAPI in the same microscopic field. At least (4% final concentration) for FISH probing gram-positive 60 sets of images taken with the 100 9 objective lens (Roller et al., 1994) and gram-negative bacteria (Amann, from three different wells (20 images from each) were

FEMS Microbiol Ecol 78 (2011) 451–462 ª 2011 Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada Published by Blackwell Publishing Ltd. 454 Y. Xia et al. counted for each sample. All cell number estimations Silva (Schloss, 2009) against their most closely related 16S were performed in ImageJ (Abramoff et al., 2004) using rRNA gene sequences. The database generated was digital images. retrieved into ARB (Ludwig et al., 2004) and used to build phylogenetic (parsimony) trees. Bootstrap values were calculated with Mega4 (Tamura et al., 2007) using DNA extraction the model Kumura 2-Parameter. Using “CLASSIFIER” The feather bag ﬂuid samples stored at 80 °C were used and “SEQMATCH”, 16S rRNA gene based identiﬁcations for DNA extraction. Samples collected from day 60-113 and similarity comparisons were performed, respectively, when a high abundance of KHOs was detected, were provided in RDP. Operational taxonomic units (OTUs, ® used. DNA was extracted with a FastPrep -24 System & determined using the furthest neighbor algorithm) and

Kit following the protocol provided by the provider (MP Chao 1 estimations were calculated using Mothur (version Downloaded from https://academic.oup.com/femsec/article/78/3/451/600873 by guest on 23 September 2021 Biomedicals, 29525 Fountain Pkwy, Solon, OH). DNA 1.10.2, http://www.mothur.org). extraction was performed as described by Kong et al. (2010). Briefly, 500-lL aliquots of feather bag fluids were Design and specification of oligonucleotide centrifuged for 10 min at 10 000 g. The pellet obtained probes was re-suspended in 465 lL potassium phosphate buffer (0.4 M) which was supplemented with 33.2 lL of lyso- Oligonucleotide probes targeting 16S rRNA were designed 1 zyme (100 mg mL ) and 3.32 lL of mutanolysin (2.5 U using ARB software. Their names, specificities, and form- 1 lL ) to a final volume of about 500 lL. The suspension amide concentrations for FISH are listed in Fig. 4. The was incubated at 37 °C for 30 min before 6.65 lL pro- optimal formamide concentrations of these probes were 1 teinase-K (20 mg mL ) was added and further incubated first determined using fixed cells in feather bag fluids. at 37 °C for 1 h. The DNA extracted individually from The highest formamide concentration (tested in 5% step- each sample was mixed in equal amounts and used as wise increases) at which a clear fluorescent signal was PCR templates. observed was selected as the putative optimal FA concentration for each gene probe. The formamide concentration of probe FIMs1029 hybridizing with the Clone library construction rod-shaped KHOs was finally determined using Clone- The bacterial universal primer pair 63F (5′-AGGCC- FISH technique before being used to identify the KHOs. TAACACATGCAAGTC-3′) and 1392R (5′-GGGCGG- Clone-FISH was performed according to Schramm et al. WGTGTACAAGGC-3′) (Marchesi et al., 1998) were used (2002). The plasmid and host cell pair used was pGEM-T to amplify bacterial 16S rRNA genes. The PCR cycle was and JM103 (DE3), respectively (Promega, Fisher Scientific). as follows: 25 cycles of denaturation (1 min at 95 °C), Briefly, the swine manure digester clones (Fig. 5) with zero annealing (1 min at 55 °C), and extension (1.5 min at (clone SPAD28) or one mismatch (clone SPAD22) with 72 °C) before a final extension at 72 °C for 8 min. To probe FIMs1029 were inserted into the plasmid pGEMT ensure products were of the expected size, the PCR equipped with a T7 RNA polymerase, and cloned into amplicons were run on 1% agarose gels. Then, they were JM109 (DE3) competent cells. These cells were incubated purified using a QIAquick PCR purification kit (Invitro- at 37 °C with IPTG (1 mM) and chloramphenicol 1 gen, Burlington, Ontario, Canada) according to manu- (170 mg L ) before being fixed in paraformaldehyde facturer’s instructions, before being ligated into the (4%) for FISH probing. The formamide concentration pCRII-TOPO vector provided in the TOPO TA cloning chosen for these probes was determined as that used previ- kit (Invitrogen). Clones with the correct inserts were ously with the fixed cells in feather bag fluids. sequenced at Laval University (http://www.bioinfo.ulaval. ca/seq) with an ABI 3130xl Genetic Analyzer (Applied BODIPY FL casein staining combined with FISH Biosystems-Hitachi, Foster City, CA). and enumeration BODIPY FL casein staining combined with FISH was per- Phylogenetic analysis formed following the procedure described previously (Xia Partial 16S rRNA gene sequences obtained from sequenc- et al., 2007). FISH was performed on the slides after ing were assembled using SEQUENCHER 4.5 (Gene Code BODIPY FL casein staining. FISH was performed accord- Corperation) and evaluated using the Chimera check pro- ing to Amann (1995). All oligonucleotide probes were gram provided in RDP (Cole et al., 2005) and Bellero- purchased from Opron (Huntsville, AL). Probe BAC303 phon (Huber et al., 2004). After removing any putative (Manz et al., 1996) targeting most Bacteroidaceae and chimera sequences, they were aligned using “Aligner” in Prevotellaceae was used to target all the Bacteroidetes

ª 2011 Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada FEMS Microbiol Ecol 78 (2011) 451–462 Published by Blackwell Publishing Ltd. Keratin-hydrolyzing bacteria in anaerobic digesters 455 clones (Fig. 4) retrieved in this study. Probe Clost I Nucleotide sequence accession numbers (Ku¨sel et al., 1999) targeting subgroup of Clostridia clusters I and II was used to target the ﬁve clones belonging The 16S rRNA gene sequences obtained in this study have to subfamily Clostridiaceae 1 (Fig. 3). Probe NONEUB been deposited in the GenBank database under accession (Wallner et al., 1993) was used as a negative control for numbers HQ698156-HQ698248. detecting any general autoﬂuorescence. If necessary the pretreatment used in CARD-FISH (Ferrari et al., 2006) to Results improve the penetration ability of gene probes was also adopted in this study. The percentages of the KHO cells AD performance hybridizing with probe FIMs1029 were estimated as a

percentage of total cell numbers stained by BODIPY FL Feather mass in the bags was gradually degraded over the Downloaded from https://academic.oup.com/femsec/article/78/3/451/600873 by guest on 23 September 2021 casein staining in the same microscopic ﬁeld. At least 60 153-day period of the experiment. VFAs (measured sets of images taken with the 100 9 objective lens from weekly) including acetic acid, propionic acid, and isobu- three different slide wells (20 images from each) were tyric acid were detected in the feather ADs and control counted for each sample. All countings were performed ADs but all at a low level. In the control ADs without in IMAGEJ using digital images. feathers, NH3-N and TKN ﬂuctuated between 5.5 and 1 1 6.1 g L and between 6.2 and 6.9 g L , respectively,

throughout the experiment. In contrast, NH3-N and TKN Microscopy 1 in the feather ADs fluctuated between 5.5 and 6.1 g L 1 Images of bacterial samples stained with BODIPY FL and between 6.7 and 6.9 g L , respectively, in the first 1 casein, DAPI, and FISH were captured randomly using an 60 days and then increased up to 7.5 and 8.4 g L , epifluorescence microscope (Image-Pro Plus, Olympus. respectively, at the end of experiment. In both the feather 1x70) equipped with a CCD digital camera SC500J (Cool- ADs and control ADs, the pH remained relatively con- SNAP-Procf, Media Cybernetic). stant at between seven and eight throughout the

(a) (b)

Fig. 1. Fluorescent images of bacteria present in the feather bag fluid and mixed liquor of the anaerobic digesters inoculated with adapted swine manure. (a) A BODIPY fluorescence-labeled casein staining image showing that most of the keratin-hydrolyzing organisms with cell-surface associated keratinase are rods. (b) and (c) are color-combined FISH images from feather bag fluid samples and the mixed liquor samples, respectively using probe FIMs1029 (Cy3 labeled, set as red) and EUBmix (Fluorescein labeled, set as green) showing the yellow-colored rods (as a combination of red and green) hybridized with probe FIMs1029. (d) A color-combined fluorescent image using FISH image with probe FIMs1029 (green-colored), a BODIPY fluorescence-labeled casein staining image (red-colored) and a DAPI staining image (blue-colored) showing that the rod-shaped keratin-hydrolyzing organisms (light-blue colored cells, e.g. those labeled by arrows) stained positively with BODIPY fluorescence- labeled casein hybridized with probe FIMs1029 and stained by DAPI staining. The bar in each image represents 10 lm.

DAPI) increased from 1.7% at day 22 to 8.2% at day 55, and then to 14.3% at day 85 before decreasing to 11.6% at day 113, which correlated (R2 = 0.96) to the feather digestion rates detected, indicating that these were the KHOs responsible for the hydrolysis of the feather keratin (Fig. 2a). BODIPY FL staining was also applied to the mixed liquor samples from the feather and control ADs. Proteo- lytic organisms were found in all samples, and their cell morphologies were the same as those attached to the

feather particles. Their relative abundances in communi- Downloaded from https://academic.oup.com/femsec/article/78/3/451/600873 by guest on 23 September 2021 ties from the feather ADs remained quite constant (between 3.1% and 3.5% of total cell number) for the ﬁrst 60 days, and then gradually increased to 4.3% at day 85 before decreasing to 3.0% at day 113 (Fig. 2b). In contrast, their relative abundances in the control ADs gradually decreased from 3.4% of the total cell number at the beginning of the experiment to 1.4% at day 113 (Fig. 2b).

Identification of the KHOs using clone library construction and FISH probing A 16S rRNA gene clone library was constructed from bac- Fig. 2. (a) Correlation of the abundance of KHOs (square with black terial populations attached to the feather particles inside line) inside the feather bags to the feather digestion rate (circle with the feather bags. One hundred 16S rRNA clones of near broken line) in the anaerobic digesters with feather addition. (b) The abundance of the KHOs in the mixed liquor of anaerobic digesters complete length (c. 1300 bp) were sequenced. Seven were with (square with black line) and without feather addition. (circle with identified putatively as chimeras and removed. Of the 93 broken line). Standard deviations of the KHO percentage values were remaining sequences, 72 belonged to previously unde- calculated based on at least six countings. scribed bacterial species sharing < 97% similarity with all other available 16S rRNA gene sequences held in the RDP digestion. At the end of the experiment the mean cumu- database (date checked 28 July 2010). Most (57 clones) of lative methane (measured weekly) produced in the feather the 93 clones belonged to members of the low G+C ADs (219.39 L) was 1.84 times higher than that produced Firmicutes and the remainder (36 clones), with the excep- in the control ADs (119.15 L). Mass balance calculations tion of two clones, one of which was Gammaproteobacte- show that a total of 94% of the feathers was degraded ria and the other a member of Synergistetes (Synergistia), after 89 days at an average removal rate of 4.8 mg were all members of the Bacteroidetes (Table 1). 1 COD g feather per day. Statistical analysis of repeated All the Firmicutes identified (Table 1) were members of batch digestions of feather has revealed a variability coef- the Clostridia, consisting of members from the genera ficient of ± 1.10% (calculated as the 95% confidence limit Syntrophomonas in the Syntrophomonadaceae, Anaerovir- of the mean). gula from the family Incertae Sedis XIV, Tissierella, Anaer- osphaera and Peptoniphilus all from the family Incertae Sedis XI, Cryptanaerobacter from the Peptococcaceae, Clos- Correlation of abundance of proteolytic tridium and Alkaliphilus from the Clostridaceae, Lutispora organisms in feather bags to feather digestion from the Gradilibacteraceae. One clone could be identified rates only as an unclassified Clostridia (class) and one as an The proteolytic organisms responsible for the feather unclassified Clostridiales (order). Many could be identified digestion inside the feather bags were detected using only at the family level, and these included members of BODIPY FL staining. Most proteolytic cells were rod the Porphyromonadaceae, Incertae Sedis XI, Ruminococca- shaped, although a few cocci of different sizes were also ceae, Peptostreptococcaceae, and Clostridiaceae. Similarly, seen (Fig. 1a). BODIPY FL casein staining combined with all the Bacteroidetes clones identified belong to the Bacter- DAPI staining showed that their relative abundance inside oidia consisting of members from the genera Porphyro- the feather bags (against the total bacteria stained with monas and Petrimonas in the Porphyromonadaceae. One

Table 1. Classiﬁcation (> 80 conﬁdence level) of 16S rRNA gene clones in the feather bags fed into the anaerobic digester inoculated adapted swine manure using CLASSIFIER

Phylogenetic classiﬁcation

Phylum Class Order Family Genus Number of clones

Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Porphyromonas 1 Petrimonas 18 Ua- Porphyromonadaceae N/Ab 1 U-Bacteroidales N/A N/A 11 U-Bacteroidetes N/A N/A N/A N/A 2 Firmicutes Clostridia Clostriales Syntrophomonadaceae Syntrophomonas 1

Incertae Sedis XIV Anaerovirgula 1 Downloaded from https://academic.oup.com/femsec/article/78/3/451/600873 by guest on 23 September 2021 Incertae Sedis XI Tissierella 5 Anaerosphaera 1 Peptoniphilus 1 U-Incertae Sedis XI N/A 11 Peptococcaceae Cryptanaerobacter 1 U-Ruminococcaceae N/A 6 U-Peptostreptococcaceae N/A 1 Clostridiaceae 1 Clostridium 5 Clostridiaceae 2 Alkaliphilus 5 U-Clostridiaceae N/A 5 Gracilibacteraceae Lutispora 10 U-Clostridiales N/A N/A 1 U-Clostridia N/A N/A N/A 4 Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae Azomonas 1 Synergistetes Synergistia Synergistales Synergistaceae Cloacibacillus 1 aU, represents unclassiﬁed. bN/A, indicates not applicable.

Bacteroidetes was identified as an unclassified member of possessed by the five clones belonging to the Clostridia- the Porphyromonadaceae. ceae 1 (Fig. 3). No currently available probe target sites Figures 3 and 4 show the phylogenetic relationships of were detected in other Firmicutes clones. Consequently, these clones to their closest relatives. Agreeing with data four new probes (Fig. 5) were designed to target the from analyses based on the RDP database, the Firmicutes sequences retrieved in this study, including those of clones (Fig. 3) identified are phylogenetically diverse, members of Firmicutes clusters (Fig. 3) of Incertae Sedis clustering mostly at different distances with sequences XI (probe CLOSs146), Clostridiaceae 2 (probe FIMs1029), from uncultured bacteria. In contrast, most Bacteroidetes Grocilibacteraceae (probe CLOSs654), and Ruminococca- clones (Fig. 4) form two closely related clusters with one ceae (probe RUMs278). After optimization of their strin- belonging to the Porphyromonadaceae and the other to gencies (see Materials and methods for more details) members of unclassified Bacteroidales. MOTHUR analysis these probes were used to FISH. showed that the 93 clones represented 39 OTUs at a dis- In all samples examined, probe BAC303 hybridized tance of 0.03. Thirty-five OTUs belonged to new bacterial with a few cocci (data not shown), whereas probes species, sharing < 97% 16S rRNA sequence similarity CLOSs146, CLOSs654, and RUMs278 each hybridized with all other available sequences. Twenty-eight OTUs are with a few rod-shaped cells (data not shown). In con- Firmicutes and five are Bacteroidetes. The Proteobacteria trast, probe FIMs1029 hybridized with many rods inside and Synergisteres are represented by only one OTU. Spe- the feather bags (Fig. 1b) and in the mixed liquor cies richness estimated by Chao 1 estimation at a 0.03 (Fig. 1c). No cells were detected hybridizing with the distance for the bacterial populations inside the feather probe Clost I in any of the samples examined. Pretreat- bags is 60. ment using HCl and different enzymes did not change Before designing probes, 16S rRNA sequences were the FISH results. Probes FIMs1029, CLOSs146, matched to all available SSU rRNA targeted probes in CLOSs654, RUMs278, and BAC303 were used to identify probeBase (Loy et al., 2003) using ARB. Probe BAC303 the KHOs present in the feather bags using BODIPY FL perfectly matches the target site of all the Bacteroidetes casein staining combined with FISH. Only probe clones (Fig. 4). The target site of probe Clost I is FIMs1029 hybridized KHO cells (Fig. 1d), and subjective

Fig. 3. A distance tree (neighbor-joining) built with the Firmicutes 16S rRNA gene sequences obtained in this study (those with names in bold). The oligonucleotide probes designed or chosen to perfectly match with these clones are also shown. The bootstrap values (only those > 50% are shown) were calculated in MEGA 4 based on 1000 resamplings. Scale bar represents one substitution per 10 nucleotides.

counting showed that about 80–90% of KHO cells in CLASSIFIER as members of genus Alkaliphilus in the sub- feather bag fluids collected at different times hybridized family Clostridiaceae 2. Clones SPAD 6, 23, 24, 28, 32 with it. This probe targets clones SPAD 6, 23, 24, 28, 32, were identified at a higher (> 80%) confidence level than 37, 38, 75, 81, and 107 (Fig. 3). All were identified by others (70–79%).

ª 2011 Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada FEMS Microbiol Ecol 78 (2011) 451–462 Published by Blackwell Publishing Ltd. Keratin-hydrolyzing bacteria in anaerobic digesters 459 Downloaded from https://academic.oup.com/femsec/article/78/3/451/600873 by guest on 23 September 2021

Fig. 4. A distance tree (neighbor-joining) built with the Bacteroidetes 16S rRNA gene sequences retrieved in this study (those with names in bold, the two sequences belonging to Proteobacteria and Synergistetes, respectively, are also shown in this tree). The coverage of probe BAC303 is also shown. The bootstrap values (only those > 50% are shown) were calculated in MEGA 4 based on 1000 resamplings. Scale bar represents 1 substitution per 10 nucleotides.

Fig. 5. Name, sequence, speciﬁcity, and formamide concentration of the new oligonucleotide probes designed in this study. The names in the parentheses are name based on the nomenclature of Alm et al. (1996). aTM represents melting temperature (ºC) of the probes. bFA represents formamide concentration (%) of each probe used in FISH. The target sites of Clones SPAD22 and SPAD28 by probe FIMs1029 are also shown.

feathers added were degraded after 89 days. No similar Discussion studies have been reported previously, but the data pre- In this article chicken feather degradation is reported in sented here suggest that digestion of feathers in swine AD microbial communities inoculated with adapted swine manure inoculated ADs is feasible for methane biofuel manure at 25 °C. The only pretreatment used was to production. In addition, as the chemical structure of the grind the feathers into smaller particles (diame- feather keratin is similar to that of prions (Fraser et al., ter < 4 mm). Mass balance data suggest that 94% of the 1972), the results from this study suggested that AD

FEMS Microbiol Ecol 78 (2011) 451–462 ª 2011 Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada Published by Blackwell Publishing Ltd. 460 Y. Xia et al. systems adapted to feather digestion could be used to anaerobic ecosystems. A. transvaalensis isolated from a study prion degradation. South African gold mine is a strictly anaerobic chemoorg-

In the feather ADs, NH4-N levels increased substan- anotroph capable of utilizing proteinaceous substrates tially with feather digestion. Methanogens are known to including yeast extract, peptone, tryptone, and casein (Ta- have very low tolerances to elevated NH4-N levels (Kayh- kai et al., 2001). Similarly, A. crotonatoxidans isolated anian, 1994), and loss of their activity (~ 56.5%) has been from a methanogenic environment is a strictly anaerobic reported at NH4-N concentrations ranging from 4.1 to bacterium capable of utilizing yeast extract, peptone, and 5.7 g L 1 (Chen et al., 2008). However, in the current tryptone (Cao et al., 2003). study, CH4 generation could be detected throughout the Proteolytic bacteria positively stained with BODIPY FL operation of the feather ADs. Therefore, it appears that casein were also detected in the inocula and the mixed

the NH4-N levels measured here did not affect markedly liquor of the control ADs without added feathers. Most Downloaded from https://academic.oup.com/femsec/article/78/3/451/600873 by guest on 23 September 2021 biogas production, which may result from the selection of them also hybridized with the FISH probe FIMs1029 and adaption of methanogen populations able to survive (data not shown). This indicates that the KHOs responsi- under these conditions (Jarrell et al., 1987). ble for feather degradation may also use proteins other BODIPY FL casein staining was used to label the KHOs than keratin, which may be present in the adapted swine in this study. This is because enzymes in anaerobic diges- manure used here, thus allowing these KHOs to grow tion are mainly cell-associated (Angelidaki & Sanders, there. The gradual decrease in their relative abundance in 2004). Its principle is that once quenched BODIPY dye- these control ADs suggests these other protein may be labeled casein is hydrolyzed by any proteases, fluorescent metabolized and rapidly disappear. precipitates attached onto the surfaces of bacteria excret- All that was known previously about KHOs had been ing these enzymes thus enabling single cell tagging of pro- derived from pure culture studies. The KHO isolates cur- teolytic bacteria. BODIPY FL casein has mainly been used rently described are mainly members of the genus Bacillus, to study protease activity of purified enzymes (e.g. Welder but other representatives are found among Fervidobacteri- et al., 2002). Only quite recently has it been applied to um, Thermoanaerobacter, and Streptomyces (Gupta & study protease activity in situ in bacterial cells (Yoshioka Ramnani, 2006). Thus, our knowledge about their identity et al., 2003) and in uncultured bacterial populations in and ecophysiology in complex natural ecosystems like communities of activated sludge wastewater treatment ADs remains limited. This study has shown for the first plants (Xia et al., 2007; Xia et al., 2008). BODIPY FL time that bacteria other than those cultured isolates may casein detects serine, metallo-, acid, and sulfhydryl prote- play important roles there in keratin degradation. ases (Molecular Probes). Microbial keratinases are mainly metallo- or serine proteases (Lin et al., 1996; Riffel et al., Acknowledgements 2003; Thys & Brandelli, 2006) and thus can be detected with this substrate at a single cell level of resolution. Financial support for Y.X was through a peer review pro- Most (80–90%) of the KHOs in the feather bags ject and the specified risk material disposal project of hybridized with FISH probe FIMs1029 designed in this Agriculture and Agri-Food Canada. We thank Denis Des- study. Their relative abundances correlated well with lauriers, Frederic Beaudoin, and Gilles Grondin for their feather digestion rates. Consequently, they are considered excellent technical support. to be the main KHOs responsible for the feather digestion in this study. The target site of probe FIMs1029 is pos- References sessed by 10 16S rRNA cloned sequences retrieved in this study (Fig. 3). These are only distantly related to the type Abramoff MD, Magalhaes PJ & Ram SJ (2004) Image strains of Alkaliphilus species, sharing < 75% similarity processing with ImageJ. Biophotonics Int 11:36–41. with A. oremlandii their closest relative, and other Alkal- Alm EW, Oerther DB, Larsen N, Stahl DA & Raskin L (1996) iphilus species. However, these clone sequences share The oligonucleotide probe database (OPD). Appl Environ 62 – > 94% similarity with each other (data not shown). Microbiol : 3557 3559. Therefore, they almost certainly represent members of a Amann RI(1995) In situ hybridization of micro-organisms by new genus in the subfamily Clostridiaceae 2. These clones whole cell hybridization with rRNA-targeted nucleic acid probes, part 3.3.6. Molecular Microbial Ecological Manual constitute three OTUs indicating that the KHOs may be (Akkermans ADL, van Elsas JD & de Bruijn FJ, eds), diverse and consist of at least three different species. This – – pp. 1 15. Kluwer Academic Publications, London. view is supported by the observation that 10 20% of Amann RI, Ludwig W & Schleifer KH (1995) Phylogenetic the KHO cells staining with BODIPY FL casein did identification and in situ detection of individual microbial not hybridize with the probe FIMs1029. Interestingly, cells without cultivation. Microbiol Rev 59: 143–169. some Alkaliphilus are known to be proteolytic bacteria in

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ª 2011 Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada FEMS Microbiol Ecol 78 (2011) 451–462 Published by Blackwell Publishing Ltd.