Lethal and sub-lethal activity of Brevibacillus laterosporus on the mosquito Aedes albopictus and side effects on non-target water-dwelling invertebrates.

The biocidal potential of Brevibacillus laterosporus against mosquitoes of major medical importance has been widely documented, but its effects on non-target invertebrates are still poorly known. In this study, we determined the lethal and sub-lethal effects of B. laterosporus strain UNISS 18, an entomopathogenic bacterium known for its effectiveness against synanthropic Diptera, on the larvae of the Asian tiger mosquito Aedes albopictus, a vector of several pathogens to humans. Moreover, we compared the larvicidal activity with the lethal action on the invasive snail Physella acuta and on two non-target water-dwelling species: the mayfly Cloeon dipterum, and the harlequin fly Chironomus riparius. B. laterosporus exhibited significant lethal effects on all the tested species with a concentration-dependent activity. However, the susceptibility varied among species, with a higher susceptibility of Ae. albopictus (LC50 = 0.16 × 107 spores mL-1) than the other species (LC50 = 0.31, 0.33, and 0.30 × 107 spores mL-1 for C. dipterum, C. riparius, and P. acuta, respectively). While 1st instar mosquito larvae were very susceptible to the bacterial infection, no effects on preimaginal development stages and adult emergence were observed at sub-lethal spores' concentrations. Even if the efficacy of B. laterosporus against Ae. albopictus and the invasive freshwater snail P. acuta is promising for their control, the susceptibility of non-target beneficial aquatic insects, highlights the need of accurate evaluations before applying B. laterosporus for pest management in water environments.

Safety evaluation of the entomopathogenic bacterium Brevibacillus laterosporus for the green lacewing Chrysoperla agilis (Neuroptera: Chrysopidae).

The safety of the entomopathogenic bacterium Brevibacillus laterosporus for the natural insect predator Chrysoperla agilis was evaluated in this study. For this purpose, laboratory bioassays were conducted exposing different larval instars and adults of the chrysopid to bacterial spore preparations, in order to evaluate the possible effects on survival, longevity, immature development, and adult reproductive performance. The sub-lethal effects were investigated by feeding the bacterium directly to adults and larvae of C. agilis or to mealworm beetles (Tenebrio molitor) used as hosts for chrysopids (tritrophic interaction). Direct feeding of B. laterosporus spores to different lacewing larvae instars and to adults did not cause mean mortality levels significantly different from untreated control, and slight though not significant effects of treatments were generally observed on insect longevity, development, fecundity and egg hatching. In the case of lacewing larvae feeding on treated mealworm beetles, adult emergence percentage was reduced approximately 12%, in comparison with untreated control. Based on these results, the use of B. laterosporus for pest management in the agroecosystem, appears to be compatible with chrysopids.

Insights of organic fertilizer micro flora of bovine manure and their useful potentials in sustainable agriculture.

Exploration of diverse environmental samples for plant growth-promoting microbes to fulfill the increasing demand for sustainable agriculture resulted in increased use of bacterial biofertilizer. We aimed for the isolation of plant growth-promoting as well as antibiotic sensitive bacteria from bovine manure samples. The basic theme of our study is to highlight potentials of bacteria in manure and the unchecked risk associated with the application of manure i.e. introducing antibiotic-resistant microbial flora, as fertilizer. Fifty-two, morphologically distinct isolates; from eight different manure samples, were subjected to plant growth-promoting parametric tests along with antibiotic resistance. Thirteen antibiotic sensitive bacterial strains with potentials of plant growth promotion further characterized by 16S rRNA ribotyping and the identified genera were Stenotrophomonas, Achromobacter, Pseudomonas, and Brevibacillus. Successful radish seeds germination under sterile in-vitro conditions showed the potential of selected bacterial isolates as plant growth-promoting bacteria. The results of this study confirmed plant growth-promoting characteristics of bovine manures' bacterial strains along with an alarming antibiotic resistance load which comprises 75% of bacterial isolated population. Our study showed distinct results of un-explored manure bacterial isolates for plant growth promotion and flagged ways associated with unchecked manure application in agriculture soil through high load of antibiotic resistant bacteria.

Microbial coexistence through chemical-mediated interactions.

Many microbial functions happen within communities of interacting species. Explaining how species with disparate growth rates can coexist is important for applications such as manipulating host-associated microbiota or engineering industrial communities. Here, we ask how microbes interacting through their chemical environment can achieve coexistence in a continuous growth setup (similar to an industrial bioreactor or gut microbiota) where external resources are being supplied. We formulate and experimentally constrain a model in which mediators of interactions (e.g. metabolites or waste-products) are explicitly incorporated. Our model highlights facilitation and self-restraint as interactions that contribute to coexistence, consistent with our intuition. When interactions are strong, we observe that coexistence is determined primarily by the topology of facilitation and inhibition influences not their strengths. Importantly, we show that consumption or degradation of chemical mediators moderates interaction strengths and promotes coexistence. Our results offer insights into how to build or restructure microbial communities of interest.

Bioremediation of triphenyl phosphate by Brevibacillus brevis: Degradation characteristics and role of cytochrome P450 monooxygenase.

Triphenyl phosphate (TPHP) has been detected with increasing frequency in environmental samples, which has aroused great attention regarding its potential adverse effects. In this study, biodegradation of TPHP by Brevibacillus brevis was investigated. The results revealed that the highest degradation efficiency of 3µmol/L TPHP by B. brevis reached 92.1% at pH7 and 30°C. The major metabolites of TPHP, diphenyl phosphate and phenyl phosphate were detected within 5days of incubation with the maximum concentrations at 308.2 and 11.8nmol/L, respectively. The activities of superoxide dismutase and catalase along with malondialdehyde content also increased significantly, indicating that TPHP caused a severe oxidative stress on B. brevis. Meanwhile, the addition of cytochrome P450 (CYP) inhibitor piperonyl butoxide markedly decreased the degradation of TPHP by B. brevis. Further transcription studies using quantitative real-time RT-PCR confirmed that the expression of CYP gene in B. brevis were significantly down-regulated. These findings demonstrated the important role of CYP in the degradation of TPHP. To our best knowledge, this is the first report about the biodegradation of TPHP by B. brevis. Overall, this study provides new insights into the potential mechanisms of TPHP biodegradation by microorganisms.

Rhizosphere-associated Alcaligenes and Bacillus strains that induce resistance against blast and sheath blight diseases, enhance plant growth and improve mineral content in rice.

AIMS: To examine the biocontrol activities of five rhizobacterial strains (i.e. Alcaligenes faecalis strains Bk1 and P1, Bacillus amyloliquefaciens strain Bk7 and Brevibacillus laterosporus stains B4 and S5), to control the rice blast and sheath blight diseases in greenhouse and to study their possible modes of action. METHODS AND RESULTS: Five potential plant growth-promoting rhizobacterial (PGPR) strains isolated from rice rhizospheres were tested for in vitro antifungal activities against Magnaporthe oryzae, Rhizoctonia solani, Botrytis cinerea and Fusarium graminearum. In vitro trials showed that three strains, Bk1, P1 and Bk7, were able to unanimously suppress the mycelial growth of the target pathogens. In greenhouse, the application of these three PGPR strains significantly suppressed the incidences of rice blast and sheath blight diseases. At 2 weeks after pathogen inoculation, the highest percentages of disease suppression were noted for Alc. faecalis strain Bk1 (72%) for rice blast, Alc. faecalis strain P1 (71%) for sheath blight, followed by B. amyloliquefaciens strain Bk7. Moreover, these strains significantly improved the plant growth, enriched the content of mineral nutrients in seedlings and increased the expression of major defence-related rice genes. All three strains were marked positive for phosphate solubilization, the production of indoleacetic acid, ammonia and siderophores and catalase activity. In addition, these strains were able to form biofilms and carried multiple lipopeptide biosynthetic genes as revealed by multiplex PCR. CONCLUSION: This study reports new potential biocontrol agents for blast and sheath blight diseases of rice. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to better understanding of the mechanisms involved in interaction between beneficial rhizobacteria, fungal pathogens and host plants.

The Dual Carboxymethyl Cellulase and Gelatinase Activities of a Newly Isolated Protein from Brevibacillus agri ST15c10 Confer Reciprocal Regulations in Substrate Utilization.

A protein showing endoglucanase-peptidase activity was prepared from a newly isolated bacterium (ST15c10). We identified ST15c10 as Brevibacillus agri based on electron-microscopic images and its 16S-rDNA sequence (GenBank accession No. HM446043), which exhibits 98.9% sequence identity to B. agri (KZ17)/B. formosus (DSM-9885T)/B. brevis. The enzyme was purified to homogeneity and gave a single peak during high-performance liquid chromatography on a Seralose 6B-150 gel-matrix/C-18 column. MALDI-TOF mass-spectrometry and bioinformatics studies revealed significant similarity to M42-aminopeptidases/endoglucanases of the CelM family. These enzymes are found in all Brevibacillus strains for which the genome sequence is known. ST15c10 grows optimally on carboxymethyl cellulose (CMC)-gelatin (40°C/pH 8-9), and also shows strong growth/carboxymethyl cellulase (CMCase) activity in submerged bagasse fermentation. The purified enzyme also functions as endoglucanase with solid bagasse/rice straw. Its CMCase activity (optimal at pH 5.6 and 60°C/Km = 35.5 µM/Vmax = 1,024U) was visualized by zymography on a CMC-polyacrylamide gel, which provided a strong band of approximately 70 kDa. The purified enzyme also showed strong peptidase (gelatinase) activity (pH 7.2/40°C during zymography on 6-12% gelatin/1% gelatin-PAGE (at approx. 70 kDa). The CMCase activity is inhibited by the metal ions Mn/Cu/Fe/Co (50%), Hg/KMnO4 (100%), and by glucose or lactose (50-75%; all at 1 mM). The observed dose/time-dependent inhibition by Hg ions could be prevented with 2-mercaptoethanol. A comparison of the B. agri endoglucanase-aminopeptidase (ELK43520; 350 aa) with other members of the M42-family revealed the conservation of active-site residues Cys256/Cys260, which were previously identified as metal-binding sites. Regulation of the endoglucanase activity probably occurs via metal binding-triggered changes in the redox state of the enzyme. Studies on this type of enzyme are of high importance for basic scientific and industrial research.

Assessing the bioremediation potential of arsenic tolerant bacterial strains in rice rhizosphere interface.

The arsenic tolerant bacterial strains Staphylococcus arlettae (NBRIEAG-6), Staphylococcus sp. (NBRIEAG-8) and Brevibacillus sp. (NBRIEAG-9) were tested for their roles in enhancing plant growth and induction of stress-related enzymes in rice (Oryza sativa L. cv. NDR-359) plants at two different concentrations, 30 and 15mg/kg of As(V) and As(III), respectively. An experiment was conducted to test the effect of these strains on plant growth promotion and arsenic uptake. We found 30%-40% reduction in total As uptake in bacteria-inoculated plants, with increased plant growth parameters compared to non-inoculated plants. Moreover, the bacteria-inoculated plants showed reduced activity of total glutathione (GSH) and glutathione reductase (GR) compared to their respective controls, which suggests the bacteria-mediated reduction of oxidative stress in plants. Thus, these strains were found to be beneficial in terms of the biochemical and physiological status of the plants under arsenic stress conditions. Furthermore, one-way ANOVA and principal component analysis (PCA) on enzymatic and non-enzymatic assays also revealed clear variations. The results support the distinction between control and treatments in both shoots and roots. Therefore, this study demonstrates the potential of rhizobacteria in alleviating arsenic stress in rice plants.

A consortium of rhizobacterial strains and biochemical growth elicitors improve cold and drought stress tolerance in rice (Oryza sativa L.).

In the present study, a consortium of two rhizobacteria Bacillus amyloliquefaciens Bk7 and Brevibacillus laterosporus B4, termed 'BB', biochemical elicitors salicylic acid and ß-aminobutyric acid (SB) and their mixture (BBSB) were investigated for cold and drought stress tolerance in rice plants. After withholding water for 16 days, rice plants treated with BBSB showed 100% survival, improved seedling height (35.4 cm), shoot number (6.12), and showed minimum symptoms of chlorosis (19%), wilting (4%), necrosis (6%) and rolling of leaves. Similarly, BB inoculation enhanced plant growth and reduced overall symptoms in rice seedlings subjected to 0 ± 5 °C for 24 h. Our results imply several mechanisms underlying BB- and BBSB-elicited stress tolerance. In contrast to the control, both treatments significantly decreased leaf monodehydroascorbate (MDA) content and electrolyte leakage, and increased leaf proline and cholorophyll content. Moreover, activities of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT) increased 3.0- and 3.6-fold, respectively. Moreover, expression of OsMYB3R-2, OsDIL, OsDREB1A and OsCDPK13 genes was significantly up-regulated, suggesting that these genes play important roles in abiotic stress tolerance of rice. In addition, bacterial strains Bk7 and B4 were able to produce high amounts of IAA and siderophores, and colonise the plant roots, while only strain Bk7 exhibited the capability to form biofilms and solubilise inorganic phosphate. This study indicates that the BB and BBSB bio-formulations can be used to confer induced systematic tolerance and improve the health of rice plants subject to chilling and drought stress.

Antifungal activity of Brevibacillus laterosporus JX-5 and characterization of its antifungal components.

The establishment of safe and effective methods for controlling fungal disease is an urgent issue in agriculture and forestry. Microbiological control of plant disease is expected to achieve better results than use of chemically derived fungicides. This study aimed to establish Brevibacillus laterosporus JX-5 as a potential microbiological control agent of poplar canker. The bacterium was isolated from the poplar rhizosphere and demonstrated significant growth inhibition of several pathogenic fungi in vitro. The antifungal components of Br. laterosporus JX-5 were isolated and identified. The fermentation broth of Br. laterosporus JX-5 and its main antifungal component, designated as component B, reduced Botryosphaeria dothidea associated canker of the excised poplar branch by 70 and 90%, respectively. Component B is considerably heat-stable, adaptable to a broad pH range, and UV-resistant. It could inhibit Bo. dothidea by permeating the fungal membrane, fracturing the nuclei, damaging the cell wall, and eventually killing the pathogenic fungus. The antifungal activity exhibited by Br. laterosporus JX-5 and its bioactive metabolic products indicate its feasibility as a potential biocontrol agent for plant diseases.

Preliminary screening of the larvicidal effect of Brevibacillus laterosporus strains against the blowfly Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae).

INTRODUCTION: This study evaluated whether different strains of Brevibacillus laterosporus could be used to control larvae of the blowfly Chrysomya megacephala, a pest that affects both human and animal health. METHODS: Mortality rates were recorded after 1-mL suspensions of sporulated cells of 14 different strains of B. laterosporus were added to 2.5g of premixed diet consisting of rotting ground beef fed to first instar larvae of C. megacephala. All bioassays were performed using 10 larvae per strain, with a minimum of three replicates for each bioassay. Larval mortality was recorded daily up to seven days. RESULTS: Strains Bon 707, IGM 16-92, and Shi 3 showed the highest toxicity toward the larvae producing 70.5%, 64.5%, and 51.6% of larval mortality, respectively, which was significantly higher than that in the control group (p < 0.05). In contrast, strains NRS 1642, NRS 661, NRS 590 BL 856, NRS 342, ATCC 6457, Bon 712, and NRS 1247 showed limited or no pathogenic activity against the target larvae. CONCLUSIONS: Our preliminary data indicated that B. laterosporus could be used to develop bioinsecticides against C. megacephala.

Preliminary screening of the larvicidal effect of Brevibacillus laterosporus strains against the blowfly Chrysomya megacephala(Fabricius, 1794) (Diptera: Calliphoridae)

: This study evaluated whether different strains of Brevibacillus laterosporus could be used to control larvae of the blowfly Chrysomya megacephala , a pest that affects both human and animal health.

Biological control as a strategy to reduce the impact of mycotoxins in peanuts, grapes and cereals in Argentina.

Mycotoxins including aflatoxins, deoxynivalenol, fumonisins and ochratoxin A are among the main fungal secondary metabolites detected as natural contaminants in South America in different commodities such as peanuts (aflatoxins), cereals (deoxynivalenol and fumonisins) or grapes (ochratoxin A). Different strategies including crop rotation, tillage practices, fungicide application and planting less susceptible cultivars are used in order to reduce the impact of these mycotoxins in both food and feed chains. The development of fungicide resistance in many fungal pathogens as well as rising of public concern on the risks associated with pesticide use led to the search for alternative environmentally friendly methods. Biological control of plant pathogens and toxigenic fungi offers an alternative that can complement chemical control in the frame of an integrated pest management to reduce the impact of mycotoxins in the food and feed chains. The advances made in Argentina on reducing the impact of toxigenic fungi and mycotoxins in peanut, grapes and cereals using the biocontrol strategy are summarised. Native bacteria, yeasts and filamentous fungi have been selected to evaluate them as potential biocontrol agents. Field trials showed that Bacillus subtilis RC 218 and Brevibacillus sp. RC 263 were effective at reducing deoxynivalenol accumulation in wheat. The application of Clonostachys rosea isolates on wheat stubble reduced Fusarium colonisation on the stubble. Bacillus amyloliquefaciens and Microbacterium oleovorans showed good activity to control both Fusarium verticillioides growth and the accumulation of fumonisins at pre-harvest stage in maize. Control of toxigenic Aspergillus flavus and aflatoxin accumulation in peanuts was achieved using a native atoxigenic Aspergillus flavus strain based on competitive exclusion of the toxigenic strains. Kluyveromyces thermotolerans strains were used as biocontrol agents to reduce the impact of Aspergillus section Nigri and ochratoxin A accumulation in grapes.

Brevibacillus sp. KUMAs2, a bacterial isolate for possible bioremediation of arsenic in rhizosphere.

Arsenic (As) contamination of soil and water has been considered as a major global environmental issue during last few decades. Among the various methods so far reported for reclamation of As contaminated rhizosphere soil, bioremediation using bacteria has been found to be most promising. An As resistant bacterial isolate Brevibacillus sp. KUMAs2 was obtained from As contaminated soil of Nadia, West Bengal, India, which could resist As(V) and As(III) a maximum of 265mM and 17mM, respectively. The strain could remove ~40 percent As under aerobic culture conditions. As resistant property in KUMAs2 was found to be plasmid-borne, which carried both As oxidizing and reducing genes. The strain could promote chilli plant growth under As contaminated soil environment by decreasing As accumulation in plant upon successful colonization in the rhizosphere, which suggests the possibility of using this isolate for successful bioremediation of As in the crop field.

Characterizing the mode of action of Brevibacillus laterosporus B4 for control of bacterial brown strip of rice caused by A. avenae subsp. avenae RS-1.

Biological control efficacy of Brevibacillus laterosporus B4 associated with rice rhizosphere was assessed against bacterial brown stripe of rice caused by Acidovorex avenae subsp. avenae. A biochemical bactericide (chitosan) was used as positive control in this experiment. Result of in vitro analysis indicated that B. laterosporus B4 and its culture filtrates (70%; v/v) exhibited low inhibitory effects than chitosan (5 mg/ml). However, culture suspension of B. laterosporus B4 prepared in 1% saline solution presented significant ability to control bacterial brown stripe in vivo. Bacterization of rice seeds for 24 h yielded a greater response (71.9%) for controlling brown stripe in vivo than chitosan (56%). Studies on mechanisms revealed that B. laterosporus B4 suppressed the biofilm formation and severely disrupted cell membrane integrity of A. avenae subsp. avenae, causing the leakage of intracellular substances. In addition, the expression level of virulence-related genes in pathogen recovered from biocontrol-agent-treated plants showed that the genes responsible for biofilm formation, motility, niche adaptation, membrane functionality and virulence of A. avenae subsp. avenae were down-regulated by B. laterosporus B4 treatment. The biocontrol activity of B. laterosporus B4 was attributed to a substance with protein nature. This protein nature was shown by using ammonium sulfate precipitation and subsequent treatment with protease. The results obtained from this study showed the potential effectiveness of B. laterosporus B4 as biocontrol agent in control of bacterial brown stripe of rice.

Efficacy of entomopathogenic bacteria for control of Musca domestica.

The aim of this study was to evaluate the larvicidal activity, and sub lethal effects of entomopathogenic bacteria Brevibacillus laterosporus, Bacillus thuringiensis var. israelensis, B. thuringiensis var. kurstaki, and a commercial formulation of Bacillus sphaericus on Musca domestica. Bacterial suspensions were prepared in different concentrations and added to the diet of newly-hatched larvae which were monitored until the adult stage. The larvae were susceptible to the B. laterosporus, B. thuringiensis var. israelensis, and B. thuringiensis var. kurstaki bacteria in varied concentration levels. These bacteria have larvicidal and sub lethal effects on the development of flies, reducing both adult size, and impairing the reproductive performance of the species.

Abiotic and microbiotic factors controlling biofilm formation by thermophilic sporeformers.

One of the major concerns in the production of dairy concentrates is the risk of contamination by heat-resistant spores from thermophilic bacteria. In order to acquire more insight in the composition of microbial communities occurring in the dairy concentrate industry, a bar-coded 16S amplicon sequencing analysis was carried out on milk, final products, and fouling samples taken from dairy concentrate production lines. The analysis of these samples revealed the presence of DNA from a broad range of bacterial taxa, including a majority of mesophiles and a minority of (thermophilic) spore-forming bacteria. Enrichments of fouling samples at 55°C showed the accumulation of predominantly Brevibacillus and Bacillus, whereas enrichments at 65°C led to the accumulation of Anoxybacillus and Geobacillus species. Bacterial population analysis of biofilms grown using fouling samples as an inoculum indicated that both Anoxybacillus and Geobacillus preferentially form biofilms on surfaces at air-liquid interfaces rather than on submerged surfaces. Three of the most potent biofilm-forming strains isolated from the dairy factory industrial samples, including Geobacillus thermoglucosidans, Geobacillus stearothermophilus, and Anoxybacillus flavithermus, have been characterized in detail with respect to their growth conditions and spore resistance. Strikingly, Geobacillus thermoglucosidans, which forms the most thermostable spores of these three species, is not able to grow in dairy intermediates as a pure culture but appears to be dependent for growth on other spoilage organisms present, probably as a result of their proteolytic activity. These results underscore the importance of abiotic and microbiotic factors in niche colonization in dairy factories, where the presence of thermophilic sporeformers can affect the quality of end products.

Observations on house fly larvae midgut ultrastructure after Brevibacillus laterosporus ingestion.

The pathological and histopathological course caused by Brevibacillus laterosporus on house fly larvae has been investigated conducting observations on insect behavior and midgut ultrastructure. After dissection and fixation, gut tissues were analyzed under transmission electron microscopy (TEM) in order to compare in vivo-treated and non-treated (control) fly specimens. Treated larvae showed extensively reduced feeding and growth rate, then became sluggish and died within 72 h. A progressive midgut epithelium deterioration was observed in treated larvae, compared to the control. Ultrastructural changes consisted of microvilli disruption, cytoplasm vacuolization and general disorganization, endoplasmic reticulum deformation, mitochondria alteration. Deterioration became progressively more dramatic until the infected cells released their content into the gut lumen. Disruption was associated also with midgut muscular sheath and connective tissue. These ultrastructural changes are similar to those widely described for other entomopathogenic bacteria, such as Bacillus thuringiensis, against different insect species. The rapid disruption of cellular fine structure supports a hypothesis based on an interaction of toxins with the epithelial cell membranes reminiscent of the specific B. thuringiensis δ-endotoxins mechanism of action on other insect targets.

[Diversity of antagonistic bacteria isolated from rhizosphere of several cash crops].

By adopting antimicrobial spectrum test, BOXAIR-PCR, physiological and biochemical, and 16S rDNA sequencing analysis, this paper analyzed the diversity of 55 antagonistic bacterial strains isolated from the rhizosphere of 10 cash crops. There was a high diversity of the antagonism of the strains. Based on BOXAIR-PCR, all the strains were clustered into 7 groups at the similarity level of 72.1%, and divided into 25 groups at the similarity level of 85.0%. All the strains belonged to Bacillus, Paenibacillus, Brevibacillus, Pseudomonas and Alcaligenes, respectively. The antagonistic bacteria isolated from the rhizosphere had high genetic diversity and high diversity in antagonistic activity.

Purification and characterization of a serine protease secreted by Brevibacillus sp. KH3 for reducing waste activated sludge and biofilm formation.

A novel protease secreted by Brevibacillus sp. KH3 isolated from excess sludge at 50 °C and used as a sludge-lysing strain was investigated in this study. Sludge reduction was minimized by protease inhibitors and a 40-kDa protease, which significantly contributed to this sludge-reducing activity, was purified as the target protein. The final purified protease demonstrated 92-fold higher specific activity than the initial crude extracts. The sludge-reducing efficiency deteriorated relative to decreased protease activity triggered by EDTA; thus, the purified protease was a causative agent in reducing excess sludge. The 40-kDa protease was a serine metalloprotease and showed the highest activity at 50 °C and pH 8.0, and the activity was enhanced in the presence of calcium ions, indicating that the purified protease contained calcium ion. Furthermore, this 40-kDa protease inhibited biofilm formation in excess sludge. These results imply that sludge reduction is because of reduction of biofilm formation in excess sludge.