Investigation of keratinolytic and non-keratinolytic fungi grown above or below a 1-cm sewage sludge blanket

This study investigated the incidence of keratinolytic and non-keratinolytic fungi grown above or below a 1-cm sewage sludge blanket. The hair baiting method was used. Incubation was carried out at 23 and 37  °C. The number of keratinolytic fungi occurrences below a sludge blanket (anoxic conditions) was almost two times smaller than the number of fungal occurrences above this blanket (oxic conditions). The anoxic conditions did not significantly affect the number of non-keratinolytic fungi. Qualitative differences were also observed. Trichophyton ajelloi with its teleomorph Arthroderma uncinatum and some other fungi were found to prefer oxic conditions. In the case of non-keratinolytic fungi, the most evident differences were observed at 37  °C. Aspergillus fumigatus prevailed above a sludge blanket, while Pseudallescheria boydii was the predominating species below this blanket. The incidence of keratinolytic fungi was dependent on sludge physico-chemical characteristics: mainly on sludge organic matter stabilization (total nitrogen and ammonium nitrogen contents, proteolytic activity and C:N ratio) and hygienization (total and fecal coliforms) factors.     

Nitrogen Fixation (Acetylene Reduction) in a Salt Marsh Amended with Sewage Sludge and Organic Carbon and Nitrogen Compounds

Seasonal distribution of nitrogen fixation by Spartina alterniflora epiphytes and in surface and soil samples was investigated in a Georgia salt marsh which was amended with sewage sludge or with glucose and/or ammonium nitrate. There was no significant difference between the rates of fixation in the unamended and sewage sludge plots. Additional perturbation experiments suggested that nitrogen addition indirectly stimulates nitrogen fixation by enhancing Spartina production and root exudation. Glucose additions, on the other hand, suppressed nitrogen fixation on a long-term basis. It is suggested that the microbial population in the soil out-competed the plants for the available nitrogen and in turn suppressed plant production and possibly root exudation. A comparison of nitrogen fixation in clipped and unclipped Spartina plots substantiated the suggestion that root exudation probably supports nitrogen fixation. Fixation in the clipped plots was significantly lower (P < 0.05) than the rates in the unclipped plots.     

Keratinolytic fungi in sewage sludge

Sewage sludge from the Upper Silesia Region of Poland were surveyed for keratinolytic fungi. Out of 100 Petri dishes examined, 89 were positive for these micro-organisms. Altogether, 185 fungal appearances belonging to 10 species were observed. Trichophyton terrestre with its teleomorph Arthroderma quadrifidum, T. ajelloi with A. uncinatum, Microsporum gypseum with Arthroderma sp., and Chrysosporium keratinophilum with Aphanoascus keratinophilus prevailed in the sludges. The sewage treatment technologies together with the sludge structure, humidity and pH were found to be critical factors determining the occurrence of keratinolytic fungi in the sludge environment. The qualitative and quantitative composition of keratinolytic fungi could be a useful tool in evaluation of sludge treatment processes.     

Morphological characterization of in-vitro human hair keratinolysis,produced by identified wild strains of Chrysosporium species

Chrysosporium species were isolated from soil and keratinized material. Primary isolation was performed following the general method of hair baiting on modified Czapek-agar media with washed, defated and sterilized human hair fragments added. Strains were maintained in test tubes of potato dextrose agar at 29 °C and cultivated on phytone yeast extract agar at 28 °C for 14 days for identification. Isolates were characterized using Van Oorschot's key. Keratinolytic activity was expressed following a subjective scale representing degree/severity of attack upon hair surface and presence of fungal structures observed in substrate. Culture results and characterization methods were effective for soil Chrysosporium strain isolation. A new hair attack mode is described. Of 71 keratinolytic fungal isolates, eight (12%) Chrysosporium species were identified. One keratinolytic Chrysosporium sp. isolate is yet to be identified.This revised version was published online in October 2005 with corrections to the Cover Date.     

Community Analysis of Ammonia and Nitrite Oxidizers during Start-Up of Nitritation Reactors

Partial nitrification of ammonium to nitrite under oxic conditions (nitritation) is a critical process for the effective use of alternative nitrogen removal technologies from wastewater. Here we investigated the conditions which promote establishment of a suitable microbial community for performing nitritation when starting from regular sewage sludge. Reactors were operated in duplicate under different conditions (pH, temperature, and dilution rate) and were fed with 50 mM ammonium either as synthetic medium or as sludge digester supernatant. In all cases, stable nitritation could be achieved within 10 to 20 days after inoculation. Quantitative in situ hybridization analysis with group-specific fluorescent rRNA-targeted oligonucleotides (FISH) in the different reactors showed that nitrite-oxidizing bacteria of the genus Nitrospira were only active directly after inoculation with sewage sludge (up to 4 days and detectable up to 10 days). As demonstrated by quantitative FISH and restriction fragment length polymorphism (RFLP) analyses of the amoA gene (encoding the active-site subunit of the ammonium monooxygenase), the community of ammonia-oxidizing bacteria changed within the first 15 to 20 days from a more diverse set of populations consisting of members of the Nitrosomonas communis and Nitrosomonas oligotropha sublineages and the Nitrosomonas europaea-Nitrosomonas eutropha subgroup in the inoculated sludge to a smaller subset in the reactors. Reactors operated at 30°C and pH 7.5 contained reproducibly homogeneous communities dominated by one amoA RFLP type from the N. europaea-N. eutropha group. Duplicate reactors at pH 7.0 developed into diverse communities and showed transient population changes even within the ammonia oxidizer community. Reactors at pH 7.5 and 25°C formed communities that were indistinguishable by the applied FISH probes but differing in amoA RFLP types. Communities in reactors fed with sludge digester supernatant exhibited a higher diversity and were constantly reinoculated with ammonium oxidizers from the supernatant. Therefore, such systems could be maintained at a higher dilution rate (0.75 day⁻¹ compared to 0.2 day⁻¹ for the synthetic wastewater reactors). Despite similar reactor performance with respect to chemical parameters, the underlying community structures were different, which may have an influence on stability during perturbations.     

Dynamics of the composition of a microbial consortium during start-up of a single-stage constant flow laboratory nitritation/anammox setup

Dynamics of the composition of the microbial community was studied during start-up of a single-stage completely mixed constant flow laboratory setup for ammonium removal by the nitritation/anammox process from the filtrate of digested sludge of the Kuryanovo wastewater treatment plant (KWTP), Moscow. To decrease the period of the start-up, the setup was initially inoculated with two types of activated sludge (nitrifying sludge from a KWTP aeration tank and sludge from a sequencing batch reactor enriched with anammox bacteria). The start-up and adjustment stage was therefore decreased to 35–40 days, and nitrogen removal efficiency reached 80% after 120 days of the setup operation. Taxonomic analysis of the composition of the microbial community was carried out by pyrosequencing of the 16S rRNA fragments obtained using the universal and planctomycetes-specific primers. In the course of adaptation of activated sludge to increasing nitrogen load, microbial community of the setup became less diverse and more specialized. The contribution of anammox bacteria of the family Brocadiaceae, closely related to Candidatus “Brocadia caroliniensis,” increased gradually. Members of the order Nitrosomonadales were involved in ammonium oxidation to nitrite. While nitrite-oxidizing bacteria of the genus Nitrospira were also detected, their share decreased with accumulation of the activated sludge. The contribution of other bacteria varied as well: the shares of the phyla Ignavibacteria, Chloroflexi, and Acidobacteria increased significantly (up to 13, 12, and 10%, respectively of the total number of reads), while relative abundance of the Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes, Synergistetes, Aminicenantes, Thermotogae, and Cloacimonetes decreased. Thus, application of pyrosequencing made it possible to monitor succession of the bacterial community involved in nitrogen removal by nitritation/anammox process.     

Isolation and characterization of a novel heterotrophic nitrifying and aerobic denitrifying bacterium Pseudomonas stutzeri KTB for bioremediation of wastewater

A novel heterotrophic nitrifying and aerobic denitrifying bacterium, KTB, was isolated from activated sludge flocci collected from a biological aerated filter according to the modified Takaya method and identified as Pseudomonas stutzeri by 16S rDNA gene sequence analysis. When shaking-cultured in the presence of 4.331 mmol/L of nitrate, 4.511 mmol/L of nitrite and 4.438 mmol/L of ammonium, the strain grew fast, with μ_max being 0.42, 0.45, and 0.56/h, and displayed high nitrogen removal efficiency, with nitrogen removal rate being 0.239, 0.362, and 0.361 mmol/L/h and nitrogen removal ratio being 99.1, 100.0, and 100.0% in 18 h, respectively. The removal mainly occurred in the logarithmic phase. Nitrite accumulation did not affect denitrification performance. Nitrate concentration was below the detectable limit during the whole growth cycle when ammonium was used as sole nitrogen source. It tolerated high DO level and exhibited excellent aggregation ability. A possible pathway involved in the nitrogen removal process, which demonstrated a full nitrification and denitrification route, was speculated. The strain might be a great candidate for biological removal of nitrogen compounds from wastewater.     

Nitrogen and Phosphorus Removal from Combined Sewage Components by Microbial Activity

When primary domestic sewage sludge was combined with settled sewage or secondary-treatment plant effluent, synergism resulted. The activity (measured by oxygen uptake, and the removal of Kjeldahl nitrogen and orthophosphate from solution) which resulted from incubating sludge together with settled sewage exceeded the sum of the activities when these components were incubated separately. A similar synergistic effect occurred with sludge and effluent. The sewage sludges were deficient in readily available nitrogen, but no shortage of phosphorus was demonstrated. The addition of ammonium and orthophosphate salts to sludge, in concentrations equivalent to those found in settled sewage and effluent, stimulated sludge oxygen uptake at least 80% as much as settled sewage or effluent. It is suggested that the synergism reflects increased microbial activity resulting from widened carbon-nitrogen and carbon-phosphorus ratios achieved by combining sludge with nutrient-rich settled sewage or effluent.     

Activity and Composition of the Denitrifying Bacterial Community Respond Differently to Long-Term Fertilization

The objective of this study was to explore the long-term effects of different organic and inorganic fertilizers on activity and composition of the denitrifying and total bacterial communities in arable soil. Soil from the following six treatments was analyzed in an experimental field site established in 1956: cattle manure, sewage sludge, Ca(NO₃)₂, (NH₄)₂SO₄, and unfertilized and unfertilized bare fallow. All plots but the fallow were planted with corn. The activity was measured in terms of potential denitrification rate and basal soil respiration. The nosZ and narG genes were used as functional markers of the denitrifying community, and the composition was analyzed using denaturing gradient gel electrophoresis of nosZ and restriction fragment length polymorphism of narG, together with cloning and sequencing. A fingerprint of the total bacterial community was assessed by ribosomal intergenic spacer region analysis (RISA). The potential denitrification rates were higher in plots treated with organic fertilizer than in those with only mineral fertilizer. The basal soil respiration rates were positively correlated to soil carbon content, and the highest rates were found in the plots with the addition of sewage sludge. Fingerprints of the nosZ and narG genes, as well as the RISA, showed significant differences in the corresponding communities in the plots treated with (NH₄)₂SO₄ and sewage sludge, which exhibited the lowest pH. In contrast, similar patterns were observed among the other four treatments, unfertilized plots with and without crops and the plots treated with Ca(NO₃)₂ or with manure. This study shows that the addition of different fertilizers affects both the activity and the composition of the denitrifying communities in arable soil on a long-term basis. However, the treatments in which the denitrifying and bacterial community composition differed the most did not correspond to treatments with the most different activities, showing that potential activity was uncoupled to community composition.     

城市污泥土地利用研究

通过培养试验、盆栽试验和田间试验系统地研究了污泥的组分特征、性质及其农地和城市园林绿化地利用对作物或绿化灌木、土壤肥力及其环境的影响,结果表明,污泥富含有机质和氮磷养分,且养分当季有效性介于化肥农家肥之间。施用污泥将明显提高土壤肥力,表现改善土壤物理性质;增加土壤肥机质和氮磷水平,并增加土壤生物活性,因此施用污泥的处理作物产量较高,并有利于后茬作物的稳健生长。但污泥施用也存在重金属、病原物等污染     

Can Organic Amendments Be Useful in Transforming a Mediterranean Shrubland into a Dehesa?

Transforming a shrubland into a dehesa system may be useful for recovering certain productive and regulatory functions of ecosystems such as grazing potential, soil erosion control, and also for reducing the risk of wildfire. However, the productivity of the herbaceous cover and tree development in the transformed system may be limited by soil fertility, especially after wildfire events. Previous studies have shown that adequate doses of sewage sludge may improve soil fertility and facilitate plant recovery, but few studies have focused on plant biodiversity assessment. Here, we compare the effects of sewage sludge that has undergone different post‐treatments (dewatering, composting, or thermal drying) as a soil amendment used to transform a fire‐affected shrubland into a dehesa, on tree growth and pasture composition (vegetation cover, species richness, and diversity). In the short term, sewage sludge causes changes in both pasture cover and tree growth. Although no major differences in vegetation species richness and composition have been detected, fertilization using sewage sludge was shown to modify the functional diversity of the vegetation community. Rapid replacement of shrubs by herbaceous cover and ruderal plants (e.g. Bromus hordeaceus and Leontodon taraxacoides) and of the three grass species sown (Festuca arundinacea, Lolium perenne, and Dactylis glomerata) was observed, whereas N‐fixing species (leguminous) tended to be more abundant in nonfertilized soils and soils amended with composted sludge. These results indicate that sewage sludge modifies the functionality of vegetation when applied to soils, and that the response varies according to the treatment that the sludge has undergone.     

Comparative analysis of growth and physio-biochemical responses of Hydrilla verticillata to different sediments in freshwater microcosms

The long-term effect of Hydrilla verticillata to different dredged sediments was investigated in laboratory aquatic microcosms. Two kinds of lake sediments, i.e., fertile sludge and brown clay, which were acquired by deep or slight dredging and differed in their nutrients levels and particle sizes, were used as growth substrate. One year after H. verticillata seedlings were transplanted into the microcosms, growth and physio-biochemical characters of the plants were estimated. In our study, sludge sediment eventually promoted the increase of the biomass, shoot length, bifurcation, nitrogen, phosphorus, soluble protein content of H. verticillata, but not root number and root mass ratio. Additionally, the soluble sugar content was significantly lower in summer (August) in the sludge sediment. Sediment nutrients also increased photosynthetic pigment content, while POD and SOD activities were not affected. The fertile sludge and brown clay formed by different dredging are quite different, but both sediments could meet the requirement of H. verticillata during early growth stage. The fertile sediment after moderate dredge-up fit the growth of H. verticillata. The brown clay formed after excessive dredging showed a lack of nutrients and therefore led to the weak growth of the aquatic plants. It is concluded that moderate dredging was suitable for H. verticillata growth and for absorbing nitrogen and phosphorus from eutrophic fresh water. The experiment also suggested that submersed aquatic plant restoration in freshwater can be promoted by human assistance.     

Properties of a new strain of Nitrosomonas isolated from an aerobic biofilm in a domestic sewage-treatment system

A new ammonia-oxidizing strain, isolated from an aerobic biofilm in a domestic sewage-treatment system, was identified as a species of Nitrosomonas different from Nitrosomonas europaea. This strain had morphological features and growth characteristics typical of members of the genus Nitrosomonas. The G+C content of the DNA of this strain was 48.5 mol%, being lower than that of known strains of N. europaea. The extent of the homology between the DNA of this strain and that of other strains of N. europaea was less than 30%. After cells of this isolate, immobilized in a polyacrylamide gel, had been added to the aerobic reactor of a laboratory-scale sewage-treatment system, the concentration of ammonium nitrogen in the effluent decreased to 2 mg/l without the accumulation of nitrite, and removal of more than 70% of the nitrogen from the input sewage was achieved.     

Fungi in sediments of the sea of Japan and their biologically active metabolites

The most abundant marine fungi encountered in various regions of the Sea of Japan belong to the generaPenicillium, Aspergillus, Wardomyces, Trichoderma, Chrysosporium, andChaetomium. Facultative marine fungi of the generaScytalidium, Verticillium, andOidiodendron and obligate marine fungi of the genusDendryphiella are much less abundant. The composition of marine sediments and the anthropogenic load on them were found to influence the abundance and species diversity of fungi, as well as the occurrence of fungal strains producing hemolytically active substances. The biodiversity of mycobiota and the abundance of hemotoxin-producing fungi in marine sediments may be used to evaluate the anthropogenic load on marine biocenoses. Hemolytic compounds were produced by 57% of the fungi isolated from marine sediments. The hemolytic activity ofChaetomium spiculipilium was revealed in the fraction of the culture liquid containing extracellular fatty acids and pigments. The fatty acid composition of this marine fungus was determined.     

The effects of applying sewage sludge into Jiangxi red soil on the growth of vegetables and the migration and enrichment of Cu and Zn

Jiangxi red soil was used as the tested soil and water spinach (Ipomoea aquatic) and Chinese chive (Allium tuberosum) were used as the tested vegetables in this study to investigate the effects of different amounts of sewage-sludge application on the growth of vegetables and the migration and enrichment patterns of Cu and Zn in vegetables using the potted method. The results indicated that the application of sewage sludge could improve the properties of red soil and promote vegetable growth. The dry weight of water spinach and Chinese chive reached the maximal levels when treated with the amount of sewage sludge at 4% and 10%, which was 4.38 ± 0.82 g and 1.56 ± 0.31 g, respectively. The dry weights after the application of sewage sludge were all larger than control treatment (CK) without sludge application. With increases in the applied amount of sewage sludge, the concentrations of Cu and Zu in red soil continued to increase, and the peak value was not reached. After the two vegetables were planted, the concentrations of Cu and Zn in red soil decreased by different degrees. The degrees of decrease of Zn were generally higher than those of Cu. The enrichment coefficient of water spinach on Cu showed a trend of increase followed by a decrease and reached the peak value of 1.04 ± 0.38 when the applied amount was 4%. The enrichment coefficient of Chinese chive on Cu overall showed a decreasing trend and did not reach the peak value under the treatment levels used in this experiment. The enrichment pattern of Chinese chive on Zn was not obvious, and the differences among all treatment levels were not significant (p < 0.05). However, the enrichment coefficient after the application of sewage sludge was significantly lower than that without the application of sludge.     

Production of keratinolytic enzyme by a newly isolated feather-degrading Stenotrophomonas maltophilia that produces plant growth-promoting activity

A novel feather-degrading Stenotrophomonas maltophilia R13 was isolated from rhizospheric soil of reed. The strain R13 produces keratinolytic enzyme using chicken feather as the sole carbon and nitrogen source. Addition of 0.1% glucose and 0.12% polypeptone to the feather medium increased the enzyme production. The optimum temperature and initial pH for the enzyme production were 30 °C and 7.0. The maximum yield of the enzyme was 82.3 ± 1.0 U/ml in the optimal feather medium; this value was about 5.5-fold higher than the yield in the basal feather medium. S. maltophilia R13 possessed disulfide reductase activity along with keratinolytic activity. As a result of feather degradation, 18 free amino acids were produced in the culture; the concentration of total amino acid was 2298.8 μM. The strain R13 produced IAA in the optimal feather medium without l-tryptophan supplementation, indicating simultaneous production of keratinolytic activity and IAA by S. maltophilia R13. The strain R13 grown in the optimal feather medium also inhibited mycelial growth of some phytopathogenic fungi. This result suggests that antifungal activity of the strain R13 could be produced in the same conditions observed for keratinolytic activity. Thus, S. maltophilia R13 could be not only used to enhance the nutritional value of feather meal but is also a potential bioinoculant in agricultural environments.     

The effect of sewage sludge flooding on growth and morphometric parameters of Phragmites australis (Cav.) Trin. ex Steudel

Phragmites australis is the most frequently used species in constructed wetlands (CW) for dewatering and mineralization of sludge. Such CW create a very hostile environment for plants because their roots are exposed to permanently anaerobic conditions. Our working hypothesis assumed a simple relationship between the success of reed in colonising sludge flood stands and maximalisation of shoot oxygen production and transport to rhizomes and roots. Morphometric parameters and density of common reed shoots growing in reed bed systems inundated with sewage sludge were investigated. It was proved that flooding with sewage sludge significantly influenced characteristics of common reed populations. In comparison with natural stands, on stands treated with sewage sludge, maximal density of shoots was higher, biomass was twice as high, shoot diameter was significantly greater, and leaf weight ratio of an individual shoot was also significantly higher. Tendency to shorten shoots was noted. Growing period was longer for reed shoots treated with sewage sludge than on natural stands.     

Watering, Fertilization, and Slurry Inoculation Promote Recovery of Biological Crust Function in Degraded Soils

Biological soil crusts are very sensitive to human-induced disturbances and are in a degraded state in many areas throughout their range. Given their importance in the functioning of arid and semiarid ecosystems, restoring these crusts may contribute to the recovery of ecosystem functionality in degraded areas. We conducted a factorial microcosm experiment to evaluate the effects of inoculation type (discrete fragments vs slurry), fertilization (control vs addition of composted sewage sludge), and watering frequency (two vs five times per week) on the cyanobacterial composition, nitrogen fixation, chlorophyll content, and net CO₂ exchange rate of biological soil crusts inoculated on a semiarid degraded soil from SE Spain. Six months after the inoculation, the highest rates of nitrogen fixation and chlorophyll a content were found when the biological crusts were inoculated as slurry, composted sewage sludge was added, and the microcosms were watered five times per week. Net CO₂ exchange rate increased when biological crusts were inoculated as slurry and the microcosms were watered five times per week. Denaturing gradient gel electrophoresis fingerprints and phylogenetic analyses indicated that most of the cyanobacterial species already present in the inoculated crust had the capability to spread and colonize the surface of the surrounding soil. These analyses showed that cyanobacterial communities were less diverse when the microcosms were watered five times per week, and that watering frequency (followed in importance by the addition of composted sewage sludge and inoculation type) was the treatment that most strongly influenced their composition. Our results suggest that the inoculation of biological soil crusts in the form of slurry combined with the addition of composted sewage sludge could be a suitable technique to accelerate the recovery of the composition and functioning of biological soil crusts in drylands.     

Co-digestion of intermediate landfill leachate and sewage sludge as a method of leachate utilization

This study examines the co-digestion of intermediate landfill leachate and sewage sludge from a municipal wastewater treatment plant. Application of leachate as a co-fermentation component increased the concentrations of soluble organic compounds (expressed as total organic carbon), ammonium nitrogen, and alkalinity in the digester influents.The biogas yield obtained from the co-fermentation of a 20:1 sewage sludge: intermediate leachate mixture was 1.30 m³ per kg of removed volatile solids (VS), while that from a 10:1 mixture was 1.24 m³ per kg of removed VS. These values exceeded the biogas yield for the sludge alone by 13% and 8%, respectively. The leachate addition influenced the proportion of methane to a minor extent. Increased methane yields of 16.9% and 6.2% per kg of removed VS were found for the two sewage sluge:intermediate leachate mixtures, respectively.     

Substrate uptake,loss, and reserve in ammonia-oxidizing bacteria (AOB) under different substrate availabilities

The controversial arguments on the true substrate in nitritation kinetics might be due to the cells' dual substrate-transport system. Our experiments revealed that, under ammonia-rich environments, it diffused into the membrane (ammonia was the direct substrate); but, under oligotrophic, ammonium ion was actively transported (ammonium was the direct substrate). Facilitating this change-over, the bacterial composition in the sludge was altered, although the predominant was Nitrosomonas eutropha in most of the six chemostats. Also, the substrate affinity constant (K_s) fell resulting in partial compensation for the reduced availability of substrate. When the environmental ammonia concentration was lower than the cytoplasmic one, a backward diffusion appeared to take place, which probably had the cells accelerate its energy-consuming ammonium transport. The % ammonium oxidizing bacteria (AOB) to the total number of bacteria in the sludge remarkably decreased when cells were grown under oligotrophic environments. This could be evidence of the cellular energy dissipation caused by ammonia loss and recovery. Intracellular total ammonium nitrogen (TAN) accumulations were observed, which gradually increased from a basal value of ∼1 M (for AOB grown under copious environments) to much higher values (grown under oligotrophic environment). It did not affect the reaction kinetics but potentially served as a reserve against famine.