Class P dye-decolorizing peroxidase gene: Degenerated primers design and phylogenetic analysis.

Dye-decolorizing peroxidases (DyPs) were classified as a new family of heme peroxidase in 2007. Produced by various bacteria, they are the first bacterial enzymes known able to degrade lignin and dyes, for which their application in waste treatment and pretreatment of lignocellulosic biomass could be envisaged. In this work, a PCR primer pair was created and tested that enabled the detection and quantification of a wide range of bacterial genes of P class DyP in complex matrices. In addition, a phylogenetic tree was built with all available sequences of DyP genes available, offering a first overview of their presence in the bacteria kingdom.

Isolation of bacterial strains able to metabolize lignin and lignin-related compounds.

UNLABELLED: In this study, we identified five strains isolated from soil and sediments able to degrade kraft lignin, aromatic dyes and lignin derivatives. Using 16S rRNA gene sequencing, the isolates were identified as Serratia sp. JHT01, Serratia liquefacien PT01, Pseudomonas chlororaphis PT02, Stenotrophomonas maltophilia PT03 and Mesorhizobium sp. PT04. All the isolates showed significant growth on lignin with no water-extractable compounds. Synthetic aromatic dyes were used to assess the presence of oxidative enzymes. All the isolates were able to use the thiazine dye Methylene blue and the anthraquinone dye Remazol Brilliant Blue R as the sole carbon source. Guaiacol, veratryl alcohol and biphenyl were also mineralized by all the strains isolated. These results suggest they could be used for the treatment of aromatic pollutants and for the degradation of the lignocellulosic biomass. SIGNIFICANCE AND IMPACT OF THE STUDY: The valorization of waste lignin and lignocellulosic biomass by biocatalysis opens up new possibilities for the production of value-added substituted aromatics, biofuel and for the treatment of aromatic pollutants. Bacteria with ligninolytic potential could be a source of novel enzymes for controlled lignin depolymerization. In this work, five soil bacteria were isolated and studied. Every isolate showed significant growth on lignin and was able to degrade several lignin monomers and ligninolytic indicator dyes. They could thus be a source of novel ligninolytic enzymes as well as candidates for a bacterial consortium for the delignification of lignocellulosic biomass.

Effect of land application of manure from enrofloxacin-treated chickens on ciprofloxacin resistance of Enterobacteriaceae in soil.

A field plot experiment was carried out to evaluate the impact of spreading chicken manure containing enrofloxacin (ENR) and its metabolite ciprofloxacin (CIP), on the levels of CIP-resistant Enterobacteriaceae in soil. The manures from chickens treated with ENR and from untreated control chickens were applied on six plots. Total and CIP-resistant Enterobacteriaceae were counted on Violet Red Bile Glucose medium containing 0 to 16mg L(-1) of CIP. A total of 145 isolates were genotyped by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). The minimum inhibitory concentration (MIC) of CIP for the isolates of each ERIC-PCR profile was determined. The most frequently isolated Enterobacteriaceae included Escherichia coli, and to a lesser extent, Enterobacter and 5 other genera from environmental origin. The composition of the E. coli community differed between manure and manured soil suggesting that the E. coli genotypes determined by ERIC-PCR varied significantly in their ability to survive in soil. One of these genotypes, including both susceptible and resistant isolates, was detected up to 89 days after the manure was applied. Most of the E. coli isolated in soil amended with manure from treated chickens was resistant to CIP (with a MIC ranging between 2 and 32mg L(-1)). In contrast, despite the presence of ENR in soil at concentrations ranging from 13-518µg kg(-1), the environmental Enterobacteriaceae isolates had a CIP MIC≤0.064mg L(-1), except one isolate which had a MIC of 0.25mg L(-1), These results showed that spreading manure from ENR-treated chickens enabled CIP-resistant E. coli to persist for at least three months in the soil. However, neither the presence of fluoroquinolones, nor the persistence of CIP-resistant E. coli, increased the CIP-susceptibility of environmental Enterobacteriaceae.

The impact of fouling on the process performance of the thermal treatment of pig slurry using tubular heat exchangers.

The aim of this study was to determine the kinetics of fouling and their influence on the performance of a thermal treatment process used for sanitisation of pig slurry. Two temperatures (55 °C and 80 °C) were investigated. One trial was carried out at 55 °C and 80 °C in which the slurry was not re-circulated and one trial at 80 °C in which 100% or 50% of the slurry was re-circulated. Fouling of the heat exchangers was assessed by on-line monitoring of the drop in pressure, changes in treatment temperature, heat transfer coefficients, heat recycling rate, and energy consumption. Similar energy consumption of around 38 kWh m(-3) of effluent was observed at the two temperatures. The operating periods prior to excessive fouling or blockage were 18 days at 55 °C and four days at 80 °C. Recycling treated manure to obtain 50% dilution of the raw feed increased the viable operating period to 14 days at 80 °C but doubled energy consumption. At 55 °C, the significant drop in the target temperature (>7 °C) with fouling severely jeopardised the process. The nature of the decline in performance suggests that the main fouling mechanisms were bio-fouling at 55 °C and organic/mineral deposits at 80 °C. Recycling treated manure enabled the operating period to be extended but increased the total cost of heating. One hundred percent recycling showed that the fouling potential of the manure was largely eliminated after one thermal treatment, suggesting a pretreatment may be advantageous.

Characteristics and composition of fouling caused by pig slurry in a tubular heat exchanger--recommended cleaning systems.

The structure and composition of the fouling deposits caused by pig slurry heated in a tubular heat exchanger were characterized to understand their formation and thus be able to minimize fouling and define effective routine cleaning methods. Two temperatures (55 °C and 80 °C) were investigated. Two types of fouling were identified: organic/mineral and biofilm. The first only formed at temperatures above 50 °C, often during the heating phase, and was the main problem encountered in treatments at 80 °C. Organic/mineral deposits formed a thin compact sub-layer and a thick porous top layer composed of 67-76% minerals, 9-15% proteins, 8-20% carbohydrates and 0-5% fats. Biofilms formed at temperatures between 25 °C and 70 °C in both the cooling and heating sections of the exchanger. This type of fouling predominated at temperatures below 55 °C. The biofilm covered a thin mineral base layer. Strongly acidic or alkaline washing cycle are recommended to clean Type I deposits, while in-line gas-rumbling is recommended for Type II fouling.

Origin of fecal contamination in waters from contrasted areas: stanols as Microbial Source Tracking markers.

Improving the microbiological quality of coastal and river waters relies on the development of reliable markers that are capable of determining sources of fecal pollution. Recently, a principal component analysis (PCA) method based on six stanol compounds (i.e. 5ß-cholestan-3ß-ol (coprostanol), 5ß-cholestan-3α-ol (epicoprostanol), 24-methyl-5α-cholestan-3ß-ol (campestanol), 24-ethyl-5α-cholestan-3ß-ol (sitostanol), 24-ethyl-5ß-cholestan-3ß-ol (24-ethylcoprostanol) and 24-ethyl-5ß-cholestan-3α-ol (24-ethylepicoprostanol)) was shown to be suitable for distinguishing between porcine and bovine feces. In this study, we tested if this PCA method, using the above six stanols, could be used as a tool in "Microbial Source Tracking (MST)" methods in water from areas of intensive agriculture where diffuse fecal contamination is often marked by the co-existence of human and animal sources. In particular, well-defined and stable clusters were found in PCA score plots clustering samples of "pure" human, bovine and porcine feces along with runoff and diluted waters in which the source of contamination is known. A good consistency was also observed between the source assignments made by the 6-stanol-based PCA method and the microbial markers for river waters contaminated by fecal matter of unknown origin. More generally, the tests conducted in this study argue for the addition of the PCA method based on six stanols in the MST toolbox to help identify fecal contamination sources. The data presented in this study show that this addition would improve the determination of fecal contamination sources when the contamination levels are low to moderate.

Relative decay of fecal indicator bacteria and human-associated markers: a microcosm study simulating wastewater input into seawater and freshwater.

Fecal contaminations of inland and coastal waters induce risks to human health and economic losses. To improve water management, specific markers have been developed to differentiate between sources of contamination. This study investigates the relative decay of fecal indicator bacteria (FIB, Escherichia coli and enterococci) and six human-associated markers (two bacterial markers: Bacteroidales HF183 (HF183) and Bifidobacterium adolescentis (BifAd); one viral marker: genogroup II F-specific RNA bacteriophages (FRNAPH II); three chemical markers: caffeine and two fecal stanol ratios) in freshwater and seawater microcosms seeded with human wastewater. These experiments were performed in darkness, at 20 °C and under aerobic conditions. The modeling of the decay curves allows us (i) to compare FIB and markers and (ii) to classify markers according to their persistence in seawater (FRNAPH II < HF183, stanol ratios < BifAd, caffeine) and in freshwater (HF183, stanol ratios < FRNAPH II < BifAd < caffeine). Although those results depend on the experimental conditions, this study represents a necessary step to develop and validate an interdisciplinary toolbox for the investigation of the sources of fecal contaminations.

Using temperature and time criteria to control the effectiveness of continuous thermal sanitation of piggery effluent in terms of set microbial indicators.

AIM: To determine the minimal conditions (temperature-time), necessary to achieve set sanitation targets for selected microbial indicators during the continuous thermal treatment of pig slurry. METHODS AND RESULTS: The effectiveness of thermal treatment between 55 and 96°C was studied using Escherichia coli, enterococci, sulfite-reducing Clostridia (SRC), mesophilic culturable bacteria (MCB), F+-specific and somatic phages. Identification of SRC and MCB was performed using 16S rRNA gene analysis. Ten minutes at 70°C or 1 h at 60°C was sufficient to reduce the vegetative bacteria by 4-5 log(10), but it had little effect on somatic phages nor on spore formers, dominated by Clostridium sp. At 96°C, somatic phages were still detected, but there was a reduction of 3.1 log(10) for SRC and of 1.4 log(10) for MCB. At 96°C, Clostridium botulinum was identified among the thermotolerant MCB. CONCLUSION: Only those hygienic risks relating to mesophilic vegetative bacteria can be totally eliminated from pig slurry treated at 60°C (60 min) or 70°C (<10 min). SIGNIFICANCE AND IMPACT OF THE STUDY: Hygiene standards based on the removal of the indicators E. coli and enterococci can easily be met by treatment as low as 60°C (enabling, a low-cost treatment using heat recovery). However, even at 96°C, certain pathogens may persist.

Effect of oxygen and temperature on the dynamic of the dominant bacterial populations of pig manure and on the persistence of pig-associated genetic markers, assessed in river water microcosms.

AIMS: The aim is to evaluate the dynamic of Bacteroides-Prevotella and Bacillus-Streptococcus-Lactobacillus populations originating from pig manure and the persistence of pig-associated markers belonging to these groups according to temperature and oxygen. METHODS AND RESULTS: River water was inoculated with pig manure and incubated under microaerophilic and aerobic conditions, at 4 and 20°C over 43 days. The diversity of bacterial populations was analysed by capillary electrophoresis-single-strand conformation polymorphism. The persistence of the pig-associated markers was measured by real-time PCR and compared with the survival of Escherichia coli and enterococci. Decay was characterized by the estimation of the time needed to produce a 1-log reduction (T90). The greatest changes were observed at 20°C under aerobic conditions, leading to a reduction in the diversity of the bacterial populations and in the concentrations of the Pig-1-Bac, Pig-2-Bac and Lactobacillus amylovorus markers with a T90 of 10·5, 8·1 and 17·2 days, respectively. CONCLUSIONS: Oxygen and temperature were found to have a combined effect on the persistence of the pig-associated markers in river waters. SIGNIFICANCE AND IMPACT OF THE STUDY: The persistence profiles of the Pig-1-Bac, Pig-2-Bac and Lact. amylovorus markers in addition to their high specificity and sensitivity support their use as relevant markers to identify pig faecal contamination in river waters.

Persistence of microbial and chemical pig manure markers as compared to faecal indicator bacteria survival in freshwater and seawater microcosms.

Natural seawater and freshwater microcosms inoculated with pig manure were set up to determine the persistence of pig faecal microbial and chemical markers in these two types of surface water. The concentrations of Lactobacillus amylovorus, the Bacteroidales Pig-2-Bac 16S rRNA genetic marker, five stanols and the evolution of two ratios of stanols, R1 (coprostanol to the sum of coprostanol and 24-ethylcoprostanol) and R2 (sitostanol to coprostanol) were analyzed during two months along with the concentration of Faecal Indicator Bacteria (FIB). Pig manure was inoculated to unfiltered water microcosms incubated aerobically at 18 °C in the dark. The faecal contamination load represented by the concentrations of culturable Escherichia coli and/or enterococci remained for two months in the freshwater and seawater microcosms water column. These concentrations followed a biphasic decay pattern with a 97% reduction of the initial amount during a first rapid phase (<6 days) and a remaining proportion undergoing a slower or null second decline. The L. amylovorus marker and five stanols persisted as long as the indicators in both treatments. The Pig-2-Bac marker persisted 20 and 27 days in seawater and freshwater, respectively. The ratios R1 and R2 were in the range specific to pig manure until day 6 in both types of water. These results indicate that Pig-2-Bac, L. amylovorus and stanol ratios might be used in combination to complement FIB testing to determine the pig source of fecal pollution. However, stanol ratios are to be used when the time point of the discharge is known.

Development of microbial and chemical MST tools to identify the origin of the faecal pollution in bathing and shellfish harvesting waters in France.

The microbiological quality of coastal or river waters can be affected by faecal pollution from human or animal sources. An efficient MST (Microbial Source Tracking) toolbox consisting of several host-specific markers would therefore be valuable for identifying the origin of the faecal pollution in the environment and thus for effective resource management and remediation. In this multidisciplinary study, after having tested some MST markers on faecal samples, we compared a selection of 17 parameters corresponding to chemical (steroid ratios, caffeine, and synthetic compounds), bacterial (host-specific Bacteroidales, Lactobacillus amylovorus and Bifidobacterium adolescentis) and viral (genotypes I-IV of F-specific bacteriophages, FRNAPH) markers on environmental water samples (n = 33; wastewater, runoff and river waters) with variable Escherichia coli concentrations. Eleven microbial and chemical parameters were finally chosen for our MST toolbox, based on their specificity for particular pollution sources represented by our samples and their detection in river waters impacted by human or animal pollution; these were: the human-specific chemical compounds caffeine, TCEP (tri(2-chloroethyl)phosphate) and benzophenone; the ratios of sitostanol/coprostanol and coprostanol/(coprostanol+24-ethylcopstanol); real-time PCR (Polymerase Chain Reaction) human-specific (HF183 and B. adolescentis), pig-specific (Pig-2-Bac and L. amylovorus) and ruminant-specific (Rum-2-Bac) markers; and human FRNAPH genogroup II.

Characterization of isolates of Listeria monocytogenes from sludge using pulsed-field gel electrophoresis and virulence assays.

AIMS: To study the diversity and virulence of Listeria monocytogenes isolated from sludge. METHODS AND RESULTS: A total of 60 isolates of L. monocytogenes from sludge were characterized by serotyping, PFGE typing and using in vitro and in vivo virulence assays. The PFGE patterns were compared with those of food and human isolates to determine whether specific group clones are associated with environmental samples. The 60 isolates gave 44 different combined ApaI/AscI PFGE patterns. The PFGE patterns of most isolates were similar or very similar to those of epidemic isolates. The majority (93%) of isolates were found to be virulent by plaque-forming assay and by mouse virulence assay. CONCLUSIONS: Our findings suggest that L. monocytogenes strains found in non-sanitized sludge are virulent and represent a potential health hazard. Although no case of listeriosis related to sludge spread onto agricultural land has been reported, particular attention to this pathogen is needed. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study dealing with the characterization of L. monocytogenes isolates from non-sanitized sludge samples by molecular typing methods and in vitro and in vivo virulence assays. Our findings provide relevant information for evaluating the health risks associated with spreading sludge onto agricultural land.

The efficiency of biological aerobic treatment of piggery wastewater to control nitrogen, phosphorus, pathogen and gas emissions.

Due to the water pollution and in order to reduce the nitrogen load applied on soils, biological nitrogen removal treatment of piggery wastewaters was developed in Brittany (France), with 250-300 units running. Four types of treatment processes were built including a biological reactor allowing to remove about 60-70% of the nitrogen content as gas by nitrification/denitrification. The addition of different mechanical separators (screw-press, centrifuge decanter ...) led to concentration of phosphorus in an exportable solid phase, allowing a reduction up to 80% of the phosphorus applied locally on soils. Moreover, a reduction of the gaseous emissions was observed using this management process as compared to conventional management (storage + land spreading) including ammonia (up to 68%) and greenhouse gases (55%). Finally, the level of enteric and pathogenic bacteria was also decreased with the treatment process as compared to conventional management systems. However, in spite of these results, the significant cost of the treatment must be underlined and alternative systems including anaerobic digestion will have to be studied.

Survival of Listeria monocytogenes and Enterococcus faecium in sludge evaluated by real-time PCR and culture methods.

AIMS: This study evaluates the behaviour in spiked sludge of a pathogenic bacteria, Listeria monocytogenes, by cultural and molecular techniques, and compares its survival with the one of a faecal indicator, Enterococcus faecium. METHODS AND RESULTS: Listeria monocytogenes strain Scott A and E. faecium(T) were followed for 17 days after inoculation in sludge. Kinetics of survival depended on the bacteria and on the technique used [most probable number method, direct plate count or real-time quantitative PCR (qPCR)]. The concentration of L. monocytogenes decreased rapidly regardless of the technique, but the decrease was much more dramatic with culture techniques than with qPCR. On the contrary, the concentrations of culturable E. faecium(T) were stable. CONCLUSIONS: The results suggest that the cells of L. monocytogenes strain Scott A might have entered a viable, but nonculturable (VBNC) status, whereas cells of the indicator bacteria, E. faecium(T), maintained themselves better and stayed culturable. SIGNIFICANCE AND IMPACT OF THE STUDY: The difference of survival kinetics in the sludge of a faecal indicator (E. faecium) and a pathogenic bacterium (L. monocytogenes) may be linked to the fact that they either enter or do not enter into a VBNC status.

Decrease of enteric micro-organisms from rural sewage sludge during their composting in straw mixture.

AIMS: To study the decrease of enteric micro-organisms including viable nematode eggs, enteroviruses, faecal indicators (Escherichia coli and enterococci) and pathogenic bacteria (Listeria monocytogenes, Salmonella sp. and Clostridium perfringens) of a rural sewage sludge when it is composted for 7 months in mixture with straw. METHODS AND RESULTS: Numbers of the test organisms and the physico-chemical parameters were measured on a monthly basis on the mixture, on the compost after being turned, and on the pile in three positions representing the part by which air is incoming, the bottom of the pile and the part through which air is outgoing. The lowest temperature in the pile was observed at the bottom, where it did not exceed 50 degrees C against 66 degrees C in the two other areas. There were no significant differences between the three areas in terms of micro-organism survival. Infectious enteroviruses were inactivated rapidly and were not found after the first turning whereas some genomes were detected until after the third turning. Escherichia coli and enterococci presented a similar survival rate and their number decreased by 4 log(10) whereas Salmonella decayed at a greater rate than L. monocytogenes. The numbers of C. perfringens decreased gradually to reach a final concentration in the mature compost of about 10(2) CFU g(-1) dry matter (d.m.), which was similar to that of the faecal indicators. CONCLUSIONS: The hygienic effect of sludge composting in mixture with straw results in a significant reduction of enteric micro-organisms, the concentration of the faecal indicators in the final product being < 64 most probable number g(-1) d.m. The concentrations of Salmonella, enteroviruses and viable nematode eggs in the final product were not detectable which is in accordance with the French legislation. SIGNIFICANCE AND IMPACT OF THE STUDY: The results which pointed out the different behaviour of the test micro-organisms reflect the difficulty to propose a relevant indicator of hygienization. Otherwise, they show that composting is an efficient means for hygienization of sludge of rural wastewater treatment, where the straw is available close to their place of production.

Comparison of a cultural method with ListerScreen plus Rapid'L.mono or PCR-ELISA methods for the enumeration of L. monocytogenes in naturally contaminated sewage sludge.

Cultural methods used to count Listeria monocytogenes in sewage sludge are laborious and time consuming, and alternative methods are needed to reduce analysis time and improve detection limits. In this study, a survey of L. monocytogenes in sewage sludge is presented with a comparative study between a cultural method and immunomagnetic separation using a ListerScreen test followed by identification of L. monocytogenes with Rapid'L.mono agar or PCR-ELISA. These two alternative methods improved the detection of L. monocytogenes in different types of sludge, irrespective of their physical and chemical characteristics. The ListerScreen method coupled with detection of L. monocytogenes on Rapid'L.mono offers the advantage of being less sophisticated than the molecular method and allows isolation of the organism, which may be useful in epidemiological studies. However, ListerScreen coupled with PCR-ELISA proved best for high-sensitivity detection of L. monocytogenes in sewage samples.

Heteroduplex mobility assay for the identification of Listeria sp and Listeria monocytogenes strains: application to characterisation of strains from sludge and food samples.

One hundred and ten Listeria sp. isolates from sewage sludge were identified according to phenotypic and genotypic methods. The Listeria sp. strains isolated from five types of sludge from three sewage treatment plants in Angers (France) and the surrounding area included L. monocytogenes (55.5%), L. innocua (29.1%), L. seeligeri (13.6%) and L. welshimeri (1.8%). The majority of L. monocytogenes strains belonged to serotypes 4b, 1/2b and 1/2a. Moreover, a heteroduplex mobility assay based on the 16S rRNA sequences was tested for its ability to identify the six species of the genus Listeria. This study, performed on 283 Listeria sp. strains from human, food and sewage sludge samples, showed that all the species were distinguishable from one another. L. innocua and L. seeligeri showed respectively three and two distinct banding patterns. Within L. monocytogenes, four groups (I-IV) were defined. The majority of food and environmental isolates were clustered in group I and it is noteworthy that group IV clustered epidemiologic isolates and strains belonging to serotypes 4b, 1/2a and 1/2b.

Occurrence of Listeria sp and L monocytogenes in sewage sludge used for land application: effect of dewatering, liming and storage in tank on survival of Listeria species.

The application of sewage sludge to agricultural land is widely used in France. To determine the impact of sludge treatments, concentrations of Listeria sp., Listeria monocytogenes and faecal indicators were monitored in five types of sludge from three sewage treatment plants in Angers (France) and its suburbs over a 1-year period. On the whole, bacteria were reduced in numbers through sludge treatments. Apart from liming, which leads to reduced levels of bacteria below detection limits, other sludge treatments did not eliminate Listeria sp. and faecal indicators. Listeria sp. and L. monocytogenes were found respectively in 87% and 73% of dewatered sludges and in 96% and 80% of sludges stored in tanks. Concentrations of L. monocytogenes, ranging from 0.15 to 20 MPN g(-1) dry matter in dewatered sludge and from 1 to 240 MPN g(-1) dry matter in sludge stored in tanks, did not show seasonal variations. Spreading of sanitised sludge onto agricultural land results in the addition of 10(6)-10(8) L. monocytogenes per hectare per year, which may contribute to the increase in the dissemination of this pathogenic species in the environment.

Detection of a whitening fluorescent agent as an indicator of white paper biodegradation: a new approach to study the kinetics of cellulose hydrolysis by mixed cultures.

A simple and reliable method to estimate paper degradation by cellulolytic bacteria is described. This method is based on the detection in the culture medium of a fluorescent whitening agent (FWA) added to white paper during the manufacturing process. Preliminary results using a Cellulomonas strain cultivated in a liquid medium containing FWA, indicated that this component is non-toxic at a final concentration of 0.01 per thousand (v/v) and that the fluorescence decreased during the first 24 h of incubation, i.e. during exponential growth phase, suggesting an adsorption of FWA on bacterial cells. Consequently, all experiments have been performed with a liquid medium containing FWA (0.01 per thousand v/v) and white paper (8.0 g/l) as cellulose source. Mixed bacterial populations (MBPs) were prepared from refuse samples. These MBPs, which mainly consisted of bacterial rod cells, were used as inocula and fluorescence was measured after 30 h of incubation, i.e. after the stationary phase was reached. A high linear correlation (R(2) = 0.979) was found between the percentages of degraded paper (%P) deduced from residual paper weight and the fluorescence values (F) of the culture medium and the following equation between %P and F was determined: %P = 8.71x10(-5) x F. An additional experiment using a second MBP showed a strong correlation (R(2) = 0.990) between the measured %P and the %P estimated from F values, confirming the reproducibility of the method. Moreover, the time course of paper degradation by five replicate flasks from a unique MBP was set up. Paper degradation was detected 3 to 5 days after the beginning of the stationary phase. The average degradation rate between the 7th and the 11th day of incubation was 11.4% per day. Rates of paper degradation ranged from 31 to 60% after 10 days and from 77 to 88% after 3 weeks of incubation, depending on the inoculum.

Phenotypic differentiation of bifidobacteria of human and animal origins.

The phenotypes of 153 strains belonging or related to the genus Bifidobacterium were studied. These organisms included 38 collection strains and 115 wild strains (41 strains of human origin, 56 strains of animal origin, and 18 strains obtained from rivers or sewage). Our phenotypic analysis revealed seven main groups that were subdivided into 20 subgroups. Seven subgroups contained no type or collection strain. Among the human strains, the type strains of Bifidobacterium pseudocatenulatum and B. catenulatum fell into group I, which contained the type strains of B. adolescentis (subgroup Ib), B. dentium (subgroup Ic), and B. angulatum (ungrouped). The type strain of B. breve belonged to subgroup IIIa1, and the type strains of B. infantis and B. longum fell into subgroup IIIb1. Group VII comprised only wild strains that were isolated from human infant feces. Among the animal strains, group II consisted mainly of bifidobacteria that were isolated from pig feces and contained the type strains of B. suis (subgroup IIb), B. thermophilum (subgroup IIf), B. choerinum, and B. boum (ungrouped). Wild strains belonging to group V were isolated from pig, calf, cow, and chicken feces; this included the type strains of B. animalis (subgroup Va), B. magnum (subgroup Vb), B. pseudolongum, and B. globosum (subgroup Vc). The strains of human origin (groups I, III, and VII) were well separated from the animal strains (groups II, IV, and V). It was not surprising that the wild strains isolated from surface water or sewage were distributed in the animal groups as well as the human groups. Thus, bifidobacteria can be considered to be successful indicators of human or animal fecal pollution when they are correctly classified. The acidification patterns were not adequate to differentiate Bifidobacterium species, as determined previously by Mitsuoka (Bifidobacteria Microflora 3:11-28, 1984) and Scardovi (p. 1418-1434, in P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt, ed., Bergey's Manual of Systematic Bacteriology, vol. 2, 1986). However, enzymatic tests furnished new taxonomic criteria for the genus.