Competitive ability of amylolytic bacteria in activated sludge

Shifts were induced into the microbial community of activated sludge by the pulse addition of soluble starch. The subsequent changes of amylolytic and proteolytic microbial populations were recorded. Four amylolytic strains were isolated and characterized with regard to carrying capacity, specific surface and growth kinetics. The competitive ability of these strains was studied by means of two-member competition experiments. These experiments were analysed according to the Lotka-Volterra model and the de Wit method. The different results obtained suggest that the dominance of the amylolytic Pseudomonas sp. (code 01) is based on a combined occurrence of high amylolytic activity, large relative cell surface, high maximum specific growth rate and reduced sensitivity towards associated proteolytic populations.     

Microorganisms from composting leaves: Ability to produce extracellular degradative enzymes

Mixed populations of bacteria, fungi, and actinomycetes in a leaf compost pile were examined over a 100-day test period for their ability to produce extracellular proteolytic, lipolytic, amylolytic, cellulolytic, pectolytic, and ureolytic enzymes and ability to utilize alkanes. Urea was added to the leaves to adjust the carbon to nitrogen ratio but was of little value in maintaining the proper ratio since it was degraded within the first few days. The degradative enzymes excreted by microorganisms was dependent on the temperature of the pile. In many cases organisms able to produce specific extracellular enzymes at medium temperatures were able to grow at high temperatures, but either did not excrete the specific enzymes or the enzymes were inactivated by the high temperature.     

Beneficial Protective Role of Endogenous Lactic Acid Bacteria Against Mycotic Contamination of Honeybee Beebread

The purpose of this article is to reveal the role of the lactic acid bacteria (LAB) in the beebread transformation/preservation, biochemical properties of 25 honeybee endogenous LAB strains, particularly: antifungal, proteolytic, and amylolytic activities putatively expressed in the beebread environment have been studied. Seventeen fungal strains isolated from beebread samples were identified and checked for their ability to grow on simulated beebread substrate (SBS) and then used to study mycotic propagation in the presence of LAB. Fungal strains identified as Aspergillus niger (Po1), Candida sp. (BB01), and Z. rouxii (BB02) were able to grow on SBS. Their growth was partly inhibited when co-cultured with the endogenous honeybee LAB strains studied. No proteolytic or amylolytic activities of the studied LAB were detected using pollen, casein starch based media as substrates. These findings suggest that some honeybee LAB symbionts are involved in maintaining a safe microbiological state in the host honeybee colonies by inhibiting beebread mycotic contaminations, starch, and protein predigestion in beebread by LAB is less probable. Honeybee endogenous LAB use pollen as a growth substrate and in the same time restricts fungal propagation, thus showing host beneficial action preserving larval food. This study also can have an impact on development of novel methods of pollen preservation and its processing as a food ingredient.     

Bacterial contaminants in liquid packaging boards: assessment of potential for food spoilage

Liquid packaging boards and blanks were examined for microbial contaminants. A total of 218 strains were identified and representatives of the most frequent species were characterized for their potential for food spoilage. Contaminants found were aerobic spore-forming bacteria, mostly Bacillus megaterium, B. licheniformis, B. cereus group, B. pumilus, Paenibacillus macerans, P. polymyxa, P. pabuli and B. flexus. Production of amylolytic, proteolytic, lipolytic and phospholipolytic enzymes was common. Approximately 50% of the B. cereus group strains were positive in the diarrhoeal enterotoxin immunoassay test or in the enterotoxin reversed passive latex agglutination test. Strains capable of growth at 6°C were found among B. cereus group, P. pabuli, P. validus, B. megaterium and P. polymyxa. All B. licheniformis strains grew at 55°C. The spores of B. licheniformis were most resistant to hydrogen peroxide. The B. cereus group strains were recognizable by fatty acid components not present in any of the other paperboard strains, 11-methyldodecanoic acid (13:0 iso) and trans-9-hexadecenoic acid (16:1 ω 7 trans), each contributing 7% or more to the total cellular fatty acids.     

High local substrate availability stabilizes a cooperative trait

Cooperative behavior is widely spread in microbial populations. An example is the expression of an extracellular protease by the lactic acid bacterium Lactococcus lactis, which degrades milk proteins into free utilizable peptides that are essential to allow growth to high cell densities in milk. Cheating, protease-negative strains can invade the population and drive the protease-positive strain to extinction. By using multiple experimental approaches, as well as modeling population dynamics, we demonstrate that the persistence of the proteolytic trait is determined by the fraction of the generated peptides that can be captured by the cell before diffusing away from it. The mechanism described is likely to be relevant for the evolutionary stability of many extracellular substrate-degrading enzymes.     

Virulence of mutants and revertants of Metarhizium anisopliae var. anisopliae toward Rhodnius prolixus

The derivation of mutants of Metarhizium anisopliae var. anisopliae which had lost or showed reduced ability to produce extracellular amylases, lipases, and proteases was studied by the enzymatic index. Reversion induced by ultraviolet light was also studied. The virulence of mutants and revertant strains was evaluated in bioassays against third-instar nymphs of Rhodnius prolixus. The mutant strains for amylase and lipase showed enzymatic indices equal to one, but this was not the case for the proteolytic mutants. The revertant strains were phenotypically similar to the parental strains. The virulence of amylolytic and lipolytic mutants was reduced in relation to the parental strain, whereas the proteolytic mutants did not show any significant reduction. Virulence was restored in all revertant strains.     

Psychrotrophic amylolytic bacteria from deep sea sediment of Prydz Bay, Antarctic: diversity and characterization of amylases

Seventeen psychrotrophic bacteria with cold-adaptive amylolytic, lipolytic or proteolytic activity were isolated from deep sea sediment of Prydz Bay, Antarctic. They were affiliated with γ-Proteobacteria (12 strains) and gram-positive bacteria (5 strains) as determined by 16S rDNA sequencing. The amylase-producing strains belonged to genus Pseudomonas, Rhodococcus, and Nocardiopsis. Two Pseudomonas strains, 7193 and 7197, which showed highest amylolytic activity were chosen for further study. The optimal temperatures for their growth and amylase-producing were between 15 and 20°C. Both of the purified amylases showed highest activity at 40°C and pH 9.0, and retained 50% activity at 5°C. The SDS-PAGE and zymogram activity staining showed that the molecular mass of strain 7193 and 7197 amylases were about 60 and 50 kDa respectively. The Pseudomonas sp. 7193 amylase hydrolyzed soluble starch into glucose, maltose, maltotriose, and maltotetraose, indicating that it had both activities of α-amylase and glucoamylase. The product hydrolyzed by Pseudomonas sp. 7197 amylase was meltotetraose.     

Development of a miniaturized selective counting strategy of lactic acid bacteria for evaluation of mixed starter in a model cassava fermentation

A miniaturized most probable number (MPN) method for the selective enumeration of three bacteria species ( Lactobacillus plantarum A6, Leuconostoc mesenteroides and Lactococcus lactis ) is described. This selective count method, based on specific consumption of carbon substrate and resistance to antibiotics, was used for the quantitative assessment of the three bacteria during mixed cultures in a model cassava fermentation. A typical microbial succession pattern was observed: (i) Lactococcus lactis and Leuc. mesenteroides dominated during the first hours of fermentation as their growth was very rapid ; (ii) from hour 12, Lactobacillus plantarum replaced the two latter strains and Lactococcus lactis disappeared gradually, followed by Leuc. mesenteroides . The growth rates of each strain appeared to be independent of the others, while acidification rates increased strongly in mixed cultures compared with pure cultures. No positive interactions resulting from the amylolytic character of Lactobacillus plantarum A6, and no negative interactions resulting from the Nis⁺ property of Lactococcus lactis , were revealed between the three strains under the model conditions used.     

产淀粉酶益生乳杆菌的筛选及鉴定

目的以健康仔猪肠道及粪便样品为基础,从中筛选产淀粉酶的乳杆菌菌株,并评价其作为益生菌候选菌株潜力。方法用MRS培养基分别从仔猪新鲜粪便和小肠黏膜上分离到乳酸菌,采用改良的产淀粉酶选择性培养基初筛得到能降解淀粉活性的菌株,并研究菌株的淀粉水解活性、抗逆能力、粘附特性及对抗生素的敏感性。结果从备选的485株乳酸菌中筛选得到具有初步淀粉酶活性的菌株25株（占总筛选数量的5.2%）,复筛选育得到具有较强淀粉酶活性的乳杆菌3株。进一步研究了这3株乳杆菌的抗逆能力、粘附特性以及对抗生素的耐药性,并对最终选育得到的菌株进行生理生化及16S rRNA分子鉴定。经选育鉴定的罗伊乳杆菌G8-5淀粉降解能力最强,并能耐受pH 3.0的酸度、1.0%的胆盐浓度,在小肠上皮细胞上的粘附效率超过15个以上,并对常用的抗生素具有较高的敏感率。结论罗伊乳杆菌G8-5符合安全益生菌的要求,可以作为产淀粉酶的益生乳杆菌优良的候选菌株。     

Isoenzyme characterization of proteases and amylases and partial purification of proteases from filamentous fungi causing biodeterioration of industrial paper

Biodeterioration is an undesirable process that can affect cultural heritage and economically important materials. Although several biotic and abiotic conditions can accelerate this process, microorganisms are perhaps its main promoters. Fungi are the most important microbial agents of biodeterioration of industrial paper stored in archives. The high genetic plasticity of these organisms allows them to adapt to different environments, using almost any class of materials as substrate. Fungi produce a wide array of enzymes, including cellulases, amylases, and proteases, which are responsible for their gross biodeterioration activity. Thirty-two morphotypes of filamentous fungi were isolated on different media from industrial paper at an advanced stage of biodeterioration. The isolates showed different degrees of cellulolytic, proteolytic, and amylolytic activities on plate assays. The highest proteolytic and amylolytic activities were selected for isoform characterization, which provided an indication of the biochemical diversity that allowed them to colonize these materials. Eladia sacculum was the morphotype selected for partial purification of basic proteases since it has three basic isoforms, simplifying the purification process. We obtained a protein of 35 kDa with a pI of 8.9.     

Response of bacteria to earthworm surface excreta

Response of bacteria to the surface excreta of the Aporrectodea caliginosa earthworm was studied. The excreta were obtained by a 1 h incubation of the earthworms in petri dishes with subsequent collection of the slime. Both inhibition and stimulation of growth were revealed, as well as suppression of the respiratory activity of some bacterial species treated with A. caliginosa surface excreta. The organisms studied included various taxa of soil bacteria (19 strains), bacteria isolated from A. caliginosa intestine and excrements (82 strain), and 48 Bacillus thuringiensis strains. For the cultures of soil bacteria, the respiratory activity was determined using the formazan color reaction due to the activity of the respiratory cycle enzymes. Earthworm excreta caused a consistent 30–50% decrease of dehydrogenase activity in 13 out of the 19 cultures. Determination of the growth rates (derived from OD₆₂₀ of cell suspensions) after 10 h of incubation revealed growth stimulation in 48 out of the 82 strains isolated from intestines and excrement. Other strains exhibited no reaction to the excreta. For 29 out of 45 B. thuringiensis strains, growth stimulation was observed, while growth of two strains was suppressed; other strains exhibited no reaction to the excreta. No relation was found between bacterial reaction to the excreta and their taxonomic position. These results correlate with the research, demonstrating antibacterial and antifungal activity of the extracts from the earthworm body and digestive tract. Thus, earthworms, apart from their medium-forming function, affect the formation of soil microbial communities by direct stimulation or suppression of specific microbial populations.     

Bacteriocins of ruminal bacteria

Similar sequences of distribution of structural genes encoding enterocin A (isolated from the ruminal strainE. faecium BC25) and enterolysin A (isolated from the ruminal amylolytic strainS. bovis II/I) were demonstrated by PCR using oligonucleotide primers specific for these bacteriocins within the ruminal enterococcal and streptococcal strains. Variable occurrence of these bacteriocins was found within the populations of Gram-positive ruminal cocci. An erratum to this article is available at .     

Aerobic treatment of pig slurry: microbial aspects

During the treatment of raw pig slurry by batch aerobic fermentation, the presence of surfaces for bacterial attachment resulted in a 1,000-fold increase in the microbial population. In the absence of such surfaces, a 65% reduction of the chemical oxygen demand (COD) of the manure was obtained after 168 h of aeration, whereas a value of 90% was observed in their presence. In the early stages of the treatment, during which the redox potential passed from a value characteristic of anaerobic conditions (-305 mV) to a stable value typical of aerobic conditions (+125 mV) some 120 h later, an amylolytic population dominated by Gram-negative bacteria (chiefly Acinetobacter calcoaceticus) developed; this was followed by a phase in which a proteolytic population tended to become dominant. Populations of some potentially pathogenic bacterial strains characteristic of the porcine intestinal flora declined noticeably during the treatment.     

Identification and characterization of yeast isolated from indonesian fermented food

Yeast strains with amylolytic activity were isolated from cassavatapé and its precursor,ragi. they were divided into two groups based on their characteristics: group 1, possessing high amylolytic activity and low ethanol yield; and group 2, possessing low amylolytic activity and high ethanol yield. The major strains of the group 1 were identified asEndomyces fibuliger, and those of group 2 were identified asPichia anomala. Based on 18S rDNA analysis, an isolate fromragi that had a high amylolytic activity was thought to be an undescribed species that was related to the basidiomycetous genera.     

Optimization of growth and hydrolytic enzymes production by Fusarium culmorum using response surface method

Production of hydrolytic enzymes by a phytopathogenic fungus Fusarium culmorum was investigated. The proteolytic activity was observed when the fungus was grown in the medium containing starch or soybean meal as a carbon source. The amylolytic and lipolytic activities were not found. Response surface modeling was applied to shake-flask culture of the fungus to determine the optimum concentration of carbon source and optimal culture time for growth and protease production. The results indicated that the maximum yield of protease production corresponded to the concentration of soybean meal of 1.4?g/ml and culture time of 4.5?days. The fungus growth depends on the concentration of carbon source in the medium whereas the enzyme production was also influenced by the culture time and interaction between these two variables.     

Effect of growth temperature on reserve mobilization and hydrolytic enzyme activities in Vigna mungo cotyledons

Seeds of Vigna mungo were allowed to germinate at 27, 18 and15°C, and time-course changes of hydrolytic enzyme activitiesand the mobilization rate of reserve components in cotyledonswere studied. The seeds germinated at 27 and 18°C grew normally,whereas the growth at 15°C was markedly retarded. In cotyledonsof seedlings grown at 27 and 18°C, amylolytic and proteolyticenzyme activities increased at early stages of growth and therates of starch and protein mobilization changed correspondingto the hydrolytic enzyme activities. At 15°C the enzymeactivities increased gradually during the experimental periodof 16 days, but the reserves in cotyledons remained almost unchangeduntil the end of the experimental period. Changes of zymogram patterns of amylolytic and proteolytic activitiesin cotyledons of seedlings grownat 27, 18 and 15°C wereexamined using polyacrylamide gel electrophoresis. The intensitiesof a main band of a-amylase and at least two bands of protease(gelatin-hydrolyzing activity) increased concurrently with invitro activities of amylolytic and proteolytic enzymes. At leastthree bands of starch phosphorylase were present in cotyledonsat early stages of germination and their intensities decreasedduring the growth of seedlings at 27, 18 and 15°C. (Received June 4, 1980; )     

Protease activity of Klebsiella pneumoniae of different virulence

The study of substrate specificity and activity of proteolytic enzymes secreted by K. pneumoniae strains with different virulence was carried out. The strains were cultivated in a liquid semi-synthetic medium. The biomass was inactivated, and the supernatant fluid was separated from microbial cells by centrifuging. In the supernatant thus obtained and in the fractions isolated by gel filtration with the subsequent purification on DEAE Sepharose elastase-like, trypsin-like and chemotrypsin-like proteolytic activity was determined. In K. pneumoniae strains with different virulence only a single proteolytic enzyme--elastase with a mol. wt. of 21 kD--was detected. The protease activity of the supernatant culture fluid did not depend on the virulence of the strain and was equal to 5,416-7,476 I.U./ml. The activity of the purified enzyme was 100% of the elastase-like activity of the supernatant culture fluid. The most virulent K. pneumoniae strain K2, whose LD50 for white mice was less than 10 microbial cells, was characterized by lower elastase-like activity. The absence of correlation between protease activity and K. pneumoniae virulence may be explained by the fact that surface glycoproteins of eukaryotic cells are glycosilated and thus slightly accessible for proteases.     

Mode of Association,Enzyme Producing Ability and Identification of Autochthonous Bacteria in the Gastrointestinal Tract of Two Indian Air-Breathing Fish,Murrel (<Emphasis Type="Italic">Channa punctatus</Emphasis>) and Stinging Catfish (<Emphasis Type="Italic">Heteropneustes fossilis</Emphasis>)

The mode of association of epithelium-associated bacteria in the gastrointestinal (GI) tract of two Indian air-breathing fish species, the murrel, Channa punctatus and the stinging catfish, Heteropneustes fossilis was demonstrated through scanning and transmission electron microscopy (SEM and TEM). The SEM examination revealed substantial numbers of rod shaped bacterial cells associated with the microvillus brush borders of enterocytes in proximal (PI) and distal regions (DI) of the GI tract of both the fish species. The TEM investigation indicated endocytosis and translocation of bacteria in the microvilli. The isolated bacterial strains (two each from the PI and DI of murrel and stinging catfish) were quantitatively evaluated for their extracellular amylase, cellulase and protease production. All the bacterial strains exhibited high cellulolytic activity than that of amylolytic and proteolytic enzymes. Only two strains, CPF1 and CPF2, isolated from the PI of murrel exhibited high proteolytic activity. Maximum amylase activity was exhibited by the strain, HFH5, isolated from the DI of stinging catfish. Totally six most promising enzyme-producing autochthonous bacterial strains were identified based on partial 16S rRNA gene sequence analytical results. All the strains showed close (92–99 %) similarity to Bacillus licheniformis.     

Artificially selecting bacterial communities using propagule strategies†

Artificial selection is a promising approach to manipulate microbial communities. Here, we report the outcome of two artificial selection experiments at the microbial community level. Both used “propagule” selection strategies, whereby the best-performing communities are used as the inocula to form a new generation of communities. Both experiments were contrasted to a random selection control. The first experiment used a defined set of strains as the starting inoculum, and the function under selection was the amylolytic activity of the consortia. The second experiment used multiple soil communities as the starting inocula, and the function under selection was the communities’ cross-feeding potential. In both experiments, the selected communities reached a higher mean function than the control. In the first experiment, this was caused by a decline in function of the control, rather than an improvement of the selected line. In the second experiment, this response was fueled by the large initial variance in function across communities, and stopped when the top-performing community “fixed” in the metacommunity. Our results are in agreement with basic expectations from breeding theory, pointing to some of the limitations of community-level selection experiments that can inform the design of future studies.     

Effect of inoculum composition and low KCl supplementation on the biological and rheological stability of an immobilized-cell system for mixed mesophilic lactic starter production.

Two strains of Lactococcus lactis subsp. lactis (L. lactis KB and KBP) and one of L. lactis subsp. lactis biovar. diacetylactis (L. diacetylactis MD) were immobilized separately in kappa-carrageenan-locust bean gum gel beads. Continuous fermentations were carried out in supplemented whey permeate in a 1-L pH-controlled stirred tank reactor inoculated with a 30% (v/v) bead inoculum and a bead ratio of 55:30:15 for KB, KBP, and MD, respectively. The process demonstrated a high productivity and microbial stability during the 7-week continuous culture. Compared with previous experiments carried out with an inoculum bead ratio of 33:33:33 for KB, KBP, and MD beads, respectively, the modification of the inoculum bead ratio had apparently little effect on free and immobilized, total and specific populations. A dominant behavior of L. diacetylactis MD over the other strains of the mixed culture was observed both with free-cell populations in the effluent and with immobilized-cell populations. Additional experiments were carried out with other strain combinations for continuous inoculation-prefermentation of milk. The data also confirmed the dominance of L. diacetylactis during long-term continuous immobilized-cell fermentations. This dominance may be tentatively explained by the local competition involved in the development of the bead cross-contamination and in citrate utilization by L. diacetylactis strains. The gel beads demonstrated a high rheological stability during the 7-week continuous fermentation even at low KCl supplementation of the broth medium (25 mM KCl).