Utilization of wild Cressa cretica biomass for pectinase production from a halo-thermotolerant bacterium.

Halophytes are the native inhabitants of saline environment. Their biomass can be considered as a potential substrate for the production of microbial enzymes. This study was intended at feasible utilization of a halophytic biomass, Cressia cretica, for pectinase production using a halo- and thermo-tolerant bacterium, Bacillus vallismortis MH 10. The data from fractionation of the C. cretica biomass revealed presence of 17% pectin in this wild biomass. Seven different factors (temperature, agitation, pH, inoculum size, peptone concentration, substrate concentration, and incubation time) affecting pectinase production using C. cretica were assessed through a statistical tool, Plackett-Burman design. Consequently, two significant factors (incubation time and peptone concentration) were optimized using the central composite design. The strain produced 20 IU mL-1 of pectinase after 24 h under optimized conditions. The enzyme production kinetics data also confirmed that 24 h is the most suitable cultivation period for pectinase production. Fourier transform infrared spectroscopy and scanning electron microscopy of C. cretica biomass ascertained utilization of pectin and structural changes after fermentation. The purification of pectinase by using DEAE column yielded specific activity and purification fold of 88.26 IU mg-1 and 3.2, respectively. The purified pectinase had a molecular weight of >65 kDa. This study offers prospects of large-scale production of pectinase by halotolerant strain in the presence of economical and locally grown substrate that makes the enzyme valuable for various industrial operations.

Effect of the type of acetic fermentation process on the chemical composition of prickly pear vinegar (Opuntia ficus-indica).

BACKGROUND: In several countries, the cactus plant (Opuntia ficus-indica (L). Mill.) has received renewed attention because of its ecological, socio-economic and environmental role. In this study, prickly pear vinegar was produced employing two types of acetification processes: surface and submerged culture. Both acetification processes were performed at different temperatures (30, 37, 40 °C) by using two different species of thermotolerant acetic acid bacteria (Acetobacter malorum and Gluconobacter oxydans). Polyphenols and volatile compounds analyzed by ultra-performance liquid chromatography with diode array detection and stir bar sorptive extraction-gas chromatography-mass spectrometry, respectively, were considered as the main variables to determine the effect of the acetification process on the quality of the vinegar. RESULTS: As a result, 15 polyphenols and 70 volatile compounds were identified and quantified in the vinegar samples produced by both acetification processes. The results showed that the surface acetification method led to an increase in the concentration of phenolic components, which was higher than that in the submerged process. However, a significant increase in volatile compounds predominated by esters and acids was observed when submerged culture acetification was employed, whereas alcohols were predominant in surface culture vinegars. Moreover, multivariate statistical analysis showed that the components that mostly contributed to the differentiation between all vinegar samples were the volatile compounds. CONCLUSION: It has been proved that prickly pear vinegar could be successfully produced at higher temperatures than usual, by employing thermotolerant bacteria, and that the type of acetification method significantly affects the final quality of the vinegar produced. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Isolation and application of a thermotolerant nitrifying bacterium Gordonia paraffinivorans N52 in sewage sludge composting for reducing nitrogen loss.

A thermotolerant strain with heterotrophic nitrification capability obtained from sludge composting was identified as Gordonia paraffinivorans N52. Strain N52 utilized 51.8% of ammonium nitrogen (NH4+-N) at 60℃, and the nitrogen balance results indicated that 25.5% of the consumed NH4+-N was changed into nitrification intermediates, 53.0% to intracellular nitrogen, and only 5.2% was lost. The successful detection of enzymes related to nitrification and PCR amplification of functional genes further demonstrated nitrification ability of the isolated strain. Moreover, orthogonal test indicated that conditions for the optimal nitrification performance were C/N 15, 50℃, 150 rpm and pH 8. Compared with the control group, the addition of Gordonia paraffinivorans N52 to sewage sludge composting reduced 27.6% of ammonia emissions, accelerated the conversion from NH4+-N to nitrate nitrogen and decreased the total nitrogen loss. These results suggested that inoculation of Gordonia paraffinivorans N52 effectively controlled ammonia emissions and reduced nitrogen loss in composting.

Characterization of heterotrophic nitrification by a thermotolerant Brevibacillus Agri N2 isolated from sewage sludge composting.

A thermotolerant strain isolated from sewage sludge (SS) composting was identified as Brevibacillus Agri N2, which showed the efficient capability for heterotrophic nitrification under high-temperature conditions. Incubation at 60 °C, strain N2 could utilize 45.47% of ammonium nitrogen (99.64 mg/L), 68.89% of hydroxylamine nitrogen (51.14 mg/L) and 76.77% of nitrite nitrogen (55.20 mg/L), with a minor part of nitrogen loss for 1.64%, 2.82% and 5.01%, respectively. The successful detection of ammonia monooxygenase, hydroxylamine oxidase, and nitrate oxidoreductase and PCR amplification of amoA, hao and nxrA genes provided evidence of nitrification ability by strain N2. Furthermore, single-factor experiments indicated that the optimal conditions for efficient nitrification performance by strain N2 were succinate as carbon source, 50 °C, C/N 12, pH 8 and 200 r/min. Strain N2 could perform the complete nitrification process, with minimal nitrogen loss at high temperature conditions, which indicated it had the potential for practical application for reducing nitrogen loss of SS composting.

Effects of Stepwise Temperature Shifts in Anaerobic Digestion for Treating Municipal Wastewater Sludge: A Genomic Study.

In wastewater treatment plants (WWTP), anaerobic digester (AD) units are commonly operated under mesophilic and thermophilic conditions. In some cases, during the dry season, maintaining a stable temperature in the digester requires additional power to operate a conditioning system. Without proper conditioning systems, methanogens are vulnerable to temperature shifts. This study investigated the effects of temperature shifts on CH4 gas production and microbial diversity during anaerobic digestion of anaerobic sewage sludge using a metagenomic approach. The research was conducted in lab-scale AD under stepwise upshifted temperature from 42 to 48 °C. The results showed that significant methanogen population reduction during the temperature shift affected the CH4 production. With 70 days of incubation each, CH4 production decreased from 4.55 L·g-1-chemical oxygen demand (COD) at 42 °C with methanogen/total population (M·TP-1) ratio of 0.041 to 1.52 L·g-1 COD (M·TP-1 ratio 0.027) and then to 0.94 L·g-1 COD ( M·TP-1 ratio 0.026) after the temperature was shifted to 45 °C and 48 °C, respectively. Methanosaeta was the most prevalent methanogen during the thermal change. This finding suggests that the Methanosaeta genus was a thermotolerant archaea. Anaerobaculum, Fervidobacterium, and Tepidanaerobacter were bacterial genera and grew well in shifted-up temperatures, implying heat-resistant characteristics.

Linking Microbial Community Succession With Substance Transformation in a Thermophilic Ectopic Fermentation System.

Ectopic fermentation system (EFS) is an effective technology for treating mass livestock manure. However, the associations between microbial communities and substance transformation remain controversial. This study aimed to investigate chicken manure EFS lasting 170 days using 16S rRNA sequencing and electrochemical, spectroscopic, and chromatographic analyses. The results showed a noticeable transformation of protein-like substances into humus-like substances. Meanwhile, the electron-accepting capacity increased persistently, effectively reflecting the humification of organic substances. The contents of phenols that promoted electron transfer continued to increase from 2.80 to 6.00%, which could be used as a maturity indicator for EFS. During the heating period, the dominant microbial communities were Chloroflexi and Proteobacteria, whereas thermotolerant bacteria Cyanobacteria and Planctomycetes were significantly enriched from 1.64 to 50.15% during the continuous thermophilic period of EFS. The correlation analysis manifested that these thermotolerant bacteria were the major functional bacteria for the formation of phenols and the key to driving the humification of organic substances. This study provides insights into understanding the humification mechanisms and implementing regulatory strategies in EFS.

Influence of Different Bacteria Inocula and Temperature Levels on the Chemical Composition and Antioxidant Activity of Prickly Pear Vinegar Produced by Surface Culture.

This work intends to determine the effect on the aroma profile, phenolic content and antioxidant activity of prickly pear vinegars produced by the surface culture at two different fermentation temperatures and using different acetic acid bacteria (AAB) inocula. Prickly pear wine was fermented at two temperature levels (30 and 37 °C) by using bacteria inocula containing Acetobacter, Gluconobacter or a mixture of bacteria isolated from Sherry vinegars. Eighty-five individual volatile compounds from different families and sixteen polyphenolic compounds have been identified. It was confirmed that the highest temperature tested (37 °C) resulted in a lower concentration of volatile compounds, while no significant effect on the vinegars' volatile composition could be associated with the AAB inoculum used. Contrariwise, the highest content of polyphenolic compounds was detected in those vinegars produced at 37 °C and their concentration was also affected by the type of AAB inoculum used. Prickly pear wine displayed greater antioxidant activity than juices or vinegars, while the vinegars obtained through the mixture of AAB from Sherry vinegar showed higher antiradical activity than those obtained through either of the two AAB genera used in this study. It can be therefore concluded that, although the volatile content of vinegars decreased when fermented at a higher temperature, vinegars with a higher content in polyphenols could be obtained by means of partial fermentations at 37 °C, as long as thermotolerant bacteria were employed.

Isolation, Biochemical Characterisation and Identification of Thermotolerant and Cellulolytic Paenibacillus lactis and Bacillus licheniformis.

RESEARCH BACKGROUND: Cellulose is an ingredient of waste materials that can be converted to other valuable substances. This is possible provided that the polymer molecule is degraded to smaller particles and used as a carbon source by microorganisms. Because of the frequently applied methods of pretreatment of lignocellulosic materials, the cellulases derived from thermophilic microorganisms are particularly desirable. EXPERIMENTAL APPROACH: We were looking for cellulolytic microorganisms able to grow at 50 °C and we described their morphological features and biochemical characteristics based on carboxymethyl cellulase (CMCase) activity and the API® ZYM system. The growth curves during incubation at 50 °C were examined using the BioLector® microbioreactor. RESULTS AND CONCLUSIONS: Forty bacterial strains were isolated from fermenting hay, geothermal karst spring, hot spring and geothermal pond at 50 °C. The vast majority of the bacteria were Gram-positive and rod-shaped with the maximum growth temperature of at least 50 °C. We also demonstrated a large diversity of biochemical characteristics among the microorganisms. The CMCase activity was confirmed in 27 strains. Hydrolysis capacities were significant in bacterial strains: BBLN1, BSO6, BSO10, BSO13 and BSO14, and reached 2.74, 1.62, 1.30, 1.38 and 8.02 respectively. Rapid and stable growth was observed, among others, for BBLN1, BSO10, BSO13 and BSO14. The strains fulfilled the selection conditions and were identified based on the 16S rDNA sequences. BBLN1, BSO10, BSO13 were classified as Bacillus licheniformis, whereas BSO14 as Paenibacillus lactis. NOVELTY AND SCIENTIFIC CONTRIBUTION: We described cellulolytic activity and biochemical characteristics of many bacteria isolated from hot environments. We are also the first to report the cellulolytic activity of thermotolerant P. lactis. Described strains can be a source of new thermostable cellulases, which are extremely desirable in various branches of circular bioeconomy.

The keratinolytic bacteria Bacillus cytotoxicus as a source of novel proteases and feather protein hydrolysates with antioxidant activities.

BACKGROUND: Argentina's geothermal areas are niches of a rich microbial diversity. In 2020, species of Bacillus cytotoxicus were isolated for the first time from these types of pristine natural areas. Bacillus cytotoxicus strains demonstrated the capability to grow and degrade chicken feathers with the concomitant production of proteases with keratinolytic activity, enzymes that have multitude of industrial applications. The aim of this research was to study the production of the proteolytic enzymes and its characterization. Also, feather protein hydrolysates produced during fermentation were characterized. RESULTS: Among the thermotolerant strains isolated from the Domuyo geothermal area (Neuquén province, Argentina), Bacillus cytotoxicus LT-1 and Oll-15 were selected and put through submerged cultures using feather wastes as sole carbon, nitrogen, and energy source in order to obtain proteolytic enzymes and protein hydrolysates. Complete degradation of feathers was achieved after 48 h. Zymograms demonstrated the presence of several proteolytic enzymes with an estimated molecular weight between 50 and > 120 kDa. Optimum pH and temperatures of Bacillus cytotoxicus LT-1 crude extract were 7.0 and 40 °C, meanwhile for Oll-15 were 7.0 and 50 °C. Crude extracts were inhibited by EDTA and 1,10 phenanthroline indicating the presence of metalloproteases. Feather protein hydrolysates showed an interesting antioxidant potential measured through radical-scavenging and Fe3+-reducing activities. CONCLUSION: This work represents an initial approach on the study of the biotechnological potential of proteases produced by Bacillus cytotoxicus. The results demonstrated the importance of continuous search for new biocatalysts with new characteristics and enzymes to be able to cope with the demands in the market.

Data on the isolation and identification of thermotolerant microorganisms from cow manure promising for organic waste processing.

Thermotolerant microorganisms play an essential role in the composting process of organic waste; they are responsible for the degradation process of organic compounds owing to their enzymatic activity in the high-temperature phase of composting. This article presents data on the isolation and identification of thermotolerant microorganisms from cattle manure. In total, three bacterial strains with the ability to grow at 50°C were isolated on different media. The cultural and morphological characteristics of the strains are presented. By analysis of the 16S rRNA gene sequence, the isolates were assigned to the species Bacillus coagulans and Bacillus licheniformis. Isolated strains were characterized for their enzymatic potential (lipolytic, amylolytic, and proteolytic). These results can be promising for further studies in order to design biological additive for accelerated composting of organic waste.

Characteristics of a red-pigmented thermotolerant bacteria (GSB-001) isolated from Gedong Songo hot spring, Bandungan, Semarang – Indonesia

Aims@#Pigments used as food additives for a long time and people prefer natural pigment than synthetic ones because of their safety. Microorganisms are interesting for pigment production and many of them have been used in food industry. Many research have been conducted to find out for natural pigment sources. The objective of this research was to investigate the characteristics of a red-pigmented bacteria isolated from Gedong Songo hot spring, Bandungan- Indonesia. @*Methodology and results@#Bacterial isolates were grown on Nutrient Agar for 24 h, and the morphology of the colonies and of the cells were identified. Biochemical tests included indole, methyl red, catalase, urease tests and carbohydrate fermentation. Molecular identification was based on 16S rRNA sequence. The isolate was a rod shape and Gram-negative. Biochemical tests showed that the isolate was indole negative, catalase positive, methyl red negative and urease negative. This isolate was glucose, maltose and sucrose positive and negative for lactose. 16S rRNA sequence was BLAST and it matched with Serratia marcescens strain S823. The red pigment antioxidant activity showed the highest DPPH radical scavenging of 49.11% obtained from 48 h of incubation. Functional group of S. marcescens pigment on Fourier Transform InfraRed Spectroscopy (FTIR) displayed specific peak at 1740 cm-1 represented of C═O (carbonyl) stretching group. @*Conclusion, significance and impact of study@#Based on morphological, biochemical and moleculer identifications, it showed that the bacteria isolated from Gedong Songo hot spring Bandungan-Indonesia had 94% homology with Serratia marcescens strain S823. Based on DPPH radical scavenging test, it demonstrated that S. marcescens had potency as an antioxidant.

Introduction of a synthetic Thermococcus-derived α-amlyase gene into barley genome for increased enzyme thermostability in grains

Background: The enzymes utilized in the process of beer production are generally sensitive to higher temperatures. About 60% of them are deactivated in drying the malt that limits the utilization of starting material in the fermentation process. Gene transfer from thermophilic bacteria is a promising tool for producing barley grains harboring thermotolerant enzymes. Results: Gene for α-amylase from hydrothermal Thermococcus, optimally active at 75­85°C and pH between 5.0 and 5.5, was adapted in silico to barley codon usage. The corresponding sequence was put under control of the endosperm-specific promoter 1Dx5 and after synthesis and cloning transferred into barley by biolistics. In addition to model cultivar Golden Promise we transformed three Slovak barley cultivars Pribina, Levan and Nitran, and transgenic plants were obtained. Expression of the ~50 kDa active recombinant enzyme in grains of cvs. Pribina and Nitran resulted in retaining up to 9.39% of enzyme activity upon heating to 75°C, which is more than 4 times higher compared to non-transgenic controls. In the model cv. Golden Promise the grain α-amylase activity upon heating was above 9% either, however, the effects of the introduced enzyme were less pronounced (only 1.22 fold difference compared with non-transgenic barley). Conclusions: Expression of the synthetic gene in barley enhanced the residual α-amylase activity in grains at high temperatures.

Characterization and comparison of phytase production by Bacillus and Paenibacillus strains from Thai soils

Aims: The objective of this research was to isolate, screen and identify phytase-producing bacteria from soils and a potent isolate was selected for its phytase production. Methodology and results: Eight spore-forming bacteria isolated from agricultural soils in Thailand were screened for their phytase production. They were identified as Bacillus and Paenibacillus strains based on their phenotypic characteristics and 16S rRNA gene sequence analyses. The phytase production by Bacillus amyloliquefaciens CH3-1 [Group I(a)] was 20.956 ± 0.099 U/mL, while Bacillus subtilis SR9-3 [Group I(b)] produced 20.588 ± 0.099 U/mL. Five isolates in Group I(c), identified as Bacillus aryabhattai, produced phytase at levels ranging from 2.436 ± 0.116 to 20.910 ± 0.000 U/mL, while Paenibacillus cineris CM5-3 (Group II) produced 1.261 ± 0.111 U/mL. A potent strain, CH3-1, produced the highest phytase when cultivated in Phytate Specific Medium (PSM) supplemented with 1% glucose, at pH 7.0 and incubated at 45 °C. Additionally, wheat bran and sorghum seed (0.5%) substrates were used to induce phytase production by replacing Na-phytate. Conclusion, significance and impact of study: Phytase producing bacteria were isolated from soils in Thailand. Gram-positive spore forming thermotolerant Bacillus strains displayed higher phytase activity than a Paenibacillus strain. A potent strain, CH3-1, could utilize agricultural waste as a substrate, which may be useful for animal feed supplementation.

Adherence of Streptococcus pneumoniae and expression analysis of neuraminidase gene (NanA and NanB) after interaction of A549 human lung epithelial cells with pneumococcal strains of various serotypes

Aims: Streptococcus pneumoniae is the most common bacterial respiratory pathogen that can lead to invasive diseases such as pneumonia, bacteremia, and meningitis. The interaction of S. pneumoniae with host respiratory epithelial cells is crucial in the colonization of human respiratory tract and involve in the virulence. The aim of the study is to investigate the adherence of S. pneumoniae and the effect of serotypic variation on neuraminidase genes (NanA and NanB) after interaction of A549 human lung epithelial cells with S. pneumoniae serotypes. Methodology and results: Six different serotypes of S. pneumoniae were used (1, 3, 5, 19F, 23F, and 14). A549 human lung epithelial cells were inoculated with pneumococcal strains of different serotype for 3 hours. The number of adherent bacteria was determined by serial dilution followed by spread plate technique on tryptic soy agar supplemented with 5% sheep blood. Bacterial RNA was harvested from the infected A549 cells. The differential expression level of neuraminidases was observed by quantitative real-time PCR (qRT-PCR). Based on bacterial adherence assay, serotype 14 showed highest adherence, meanwhile, serotype 23F showed lowest adherence. This suggests that serotype 14 has a better affinity to adhere to A549 cells as compared to serotype 23F. Higher NanA gene expression was observed in serotype 5, 23F and 19F, while lower expression in serotype 14. In contrast, NanB gene shows low-level expression in serotype 23F and 19F, while higher expression in serotype 14. This postulates that NanA and NanB gene may have different functions in the pathogenesis of S. pneumoniae. Conclusion, significance and impact of study: Our finding on differential expression of neuraminidase gene of S. pneumoniae of various serotypes on A549 cells might give a better understanding of host pathogen interaction between bacteria serotypes and host cell.

Water from different sources used for the irrigation of vegetables to be marketed: research on Cryptosporidiumspp, giardiaspp, and Coliforms in Parana, Brazil / Água de diferentes fontes utilizadas na irrigação de hortaliças comercializadas: pesquisa de Cryptosporidiumspp, giardiaspp, e coliformes, Paraná, Brasil

SUMMARYThe aim of this work was to compare, from a parasitological ( Cryptosporidiumspp. and Giardia duodenalis), bacteriological (total and thermotolerants coliforms) and physicochemical perspective, water sources used for drinking and irrigation of vegetables intended to be sold for human consumption. From January 2010 to May 2011, samples of different water sources from vegetable producing properties were collected; 100 liters for parasitological analysis, 200 mL for bacteriological analysis, and five liters for physicochemical analysis. Water samples were filtered under vacuum with a kit containing a cellulose acetate membrane filter, 1.2 µm (Millipore(r), Barueri, SP, Brazil). The material retained on the membrane was mechanically extracted and analyzed by direct immunofluorescence (Merifluor(r)kit). From 20 rural properties investigated, 10 had artesian wells (40 samples), 10 had common wells (40 samples), and one had a mine (four samples), the latter contaminated by Cryptosporidiumspp. In samples from artesian wells, 90 to 130 meters depth, 42.5% were positive for total coliforms and 5.0% were identified to have abnormal coloration. From the samples of common wells, 14 to 37 meters depth, 87.5% were contaminated with total coliforms, 82.5% were positive for thermotolerant coliforms, and 12.5% had color abnormalities. We did not detect the presence of Giardiaspp. or Cryptosporidiumspp. in artesian and common wells. The use of artesian or common wells is an important step in the control of the spreading of zoonoses, particularly Cryptosporidiumspp. and Giardiaspp., as well as artesian wells for coliform control in local production of vegetables to be marketed.

RESUMOO objetivo do estudo foi investigar fontes de água utilizadas para consumo e irrigação de hortaliças a serem comercializadas sob o aspecto parasitológico ( Cryptosporidiumspp. e Giardia duodenalis), bacteriológico (coliformes totais e termotolerantes) e físico-químico. De janeiro de 2010 a maio de 2011 foram coletadas amostras de água de diferentes fontes de abastecimento de propriedades produtoras de hortaliças; 100 litros para análise parasitológica, 200 mL para bacteriológica e cinco litros para análise físico-química. As amostras de água foram filtradas a vácuo com um kit de filtragem contendo uma membrana de acetato de celulose, 1,2 µm, (Millipore(r), Barueri, São Paulo, Brasil). O material retido na membrana foi extraído mecanicamente e analisado por imunofluorescência direta (kit Merifluor(r)). De 20 propriedades rurais, 10 tinham poços artesianos (40 amostras), 10 semi-artesianos (40 amostras) e um possuía uma mina (quatro amostras). Esta última contaminada por Cryptosporidiumspp. Das amostras de poços artesianos com 90 a 130 metros de profundidade, 42,5% foram positivas para coliformes totais e 5,0% apresentavam coloração alterada. Em amostras de poços semi-artesianos com 14 a 37 m de profundidade, 87,5% apresentaram coliformes totais, 82,5% termotolerantes, e 12,5% destas amostras tinham alteração de cor. Não foi detectada a presença de Giardiaspp. e Cryptosporidiumspp. em poços artesianos ou semi-artesianos. A utilização de poços artesianos ou semi artesianos constitui importante medida no controle da disseminação de zoonoses, principalmente Cryptosporidiumspp. e Giardiaspp., assim como de poços artesianos para o controle de coliformes, em locais de produção de hortaliças irrigadas, a serem comercializadas.

Identification and characterization of thermotolerant acetic acid bacteria strains isolated from coconut water vinegar in Sri Lanka.

From the pellicle formed on top of brewing coconut water vinegar in Sri Lanka, three Acetobacter strains (SL13E-2, SL13E-3, and SL13E-4) that grow at 42 °C and four Gluconobacter strains (SL13-5, SL13-6, SL13-7, and SL13-8) grow at 37 °C were identified as Acetobacter pasteurianus and Gluconobacter frateurii, respectively. Acetic acid production by the isolated Acetobacter strains was examined. All three strains gave 4% acetic acid from 6% initial ethanol at 37 °C, and 2.5% acetic acid from 4% initial ethanol at 40 °C. Compared with the two other strains, SL13E-4 showed both slower growth and slower acetic acid production. As well as the thermotolerant SKU1108 strain, the activities of the alcohol dehydrogenase and the aldehyde dehydrogenase of SL13E-2 and SL13E-4 were more stable than those of the mesophilic strain. The isolated strains were used to produce coconut water vinegar at higher temperatures than typically used for vinegar production.

Isolation and Identification of Thermoacidiphilic Bacteria from Apple Juice Concentrate / 微生物学通报

The thermotolerant bacteria which was isolated from the Apple Juice Concentrate(AJC) was investigated by comparison with the standard strain of Aliyclobacillus acidoterrestris DSM3922 using Kirin-medium acidified with malic acid.The results shows the two polluted bacteria isolated from AJC can grow under the temperature of 21?C～55?C and pH of 2.4～6.2,which was corresponded with the characteristics of the thermo-acidiphilic Alicyclobacillus spp..Further more,the cell morphology,colony morphology,cultural characteristics and physiological characteristics tests indicated the two isolated strains of thermotolerant bacteria have obviously similar characteristics with the standard strain of Aliyclobacillus acidoterrestris DSM3922.