Driving the wedge: Understanding an improved Cas9 to better engineer others.

In their recent structural work, Eggers et al.1 rationalize how key mutations in the WED domain of the compact and thermostable Geobacillus stearothermophilus Cas9 bolster its editing efficiency in mammalian cells, and they use these insights to rationally improve another Cas9.

Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9.

Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.

Investigating the mechanism of interfacial tension reduction through the combination of low-salinity water and bacteria.

In the enhanced oil recovery (EOR) process, interfacial tension (IFT) has become a crucial factor because of its impact on the recovery of residual oil. The use of surfactants and biosurfactants can reduce IFT and enhance oil recovery by decreasing it. Asphaltene in crude oil has the structural ability to act as a surface-active material. In microbial-enhanced oil recovery (MEOR), biosurfactant production, even in small amounts, is a significant mechanism that reduces IFT. This study aimed to investigate fluid/fluid interaction by combining low biosurfactant values and low-salinity water using NaCl, MgCl2, and CaCl2 salts at concentrations of 0, 1000, and 5000 ppm, along with Geobacillus stearothermophilus. By evaluating the IFT, this study investigated different percentages of 0, 1, and 5 wt.% of varying asphaltene with aqueous bulk containing low-salinity water and its combination with bacteria. The results indicated G. Stearothermophilus led to the formation of biosurfactants, resulting in a reduction in IFT for both acidic and basic asphaltene. Moreover, the interaction between asphaltene and G. Stearothermophilus with higher asphaltene percentages showed a decrease in IFT under both acidic and basic conditions. Additionally, the study found that the interaction between acidic asphaltene and G. stearothermophilus, in the presence of CaCl2, NaCl, and MgCl2 salts, resulted in a higher formation of biosurfactants and intrinsic surfactants at the interface of the two phases, in contrast to the interaction involving basic asphaltene. These findings emphasize the dependence of the interactions between asphaltene and G. Stearothermophilus, salt, and bacteria on the specific type and concentration of asphaltene.

Genomic mining of Geobacillus stearothermophilus GF16 for xylose production from hemicellulose-rich biomasses using secreted enzymes.

The valorization of lignocellulosic biomass, derived from various bio-waste materials, has received considerable attention as a sustainable approach to improve production chains while reducing environmental impact. Microbial enzymes have emerged as key players in the degradation of polysaccharides, offering versatile applications in biotechnology and industry. Among these enzymes, glycoside hydrolases (GHs) play a central role. Xylanases, in particular, are used in a wide range of applications and are essential for the production of xylose, which can be fermented into bioethanol or find use in many other industries. Currently, fungal secretomes dominate as the main reservoir of lignocellulolytic enzymes, but thermophilic microorganisms offer notable advantages in terms of enzyme stability and production efficiency. Here we present the genomic characterization of Geobacillus stearothermophilus GF16 to identify genes encoding putative enzymes involved in lignocellulose degradation. Thermostable GHs secreted by G. stearothermophilus GF16 were investigated and found to be active on different natural polysaccharides and synthetic substrates, revealing an array of inducible GH activities. In particular, the concentrated secretome possesses significant thermostable xylanase and ß-xylosidase activities (5 ×103 U/L and 1.7 ×105 U/L, respectively), highlighting its potential for application in biomass valorization. We assessed the hemicellulose hydrolysis capabilities of various agri-food wastes using the concentrated secretome of the strain cultivated on xylan. An impressive 300-fold increase in xylose release compared to a commercially available cocktail was obtained with the secretome, underscoring the remarkable efficacy of this approach.

Detection of risk areas in dairy powder processes: The development of thermophilic spore forming bacteria taking into account their growth limits.

Anoxybacillus flavithermus, Geobacillus stearothermophilus and Bacillus licheniformis are the main contaminants found in dairy powders. These spore-forming thermophilic bacteria, rarely detected in raw milk, persist, and grow during the milk powder manufacturing process. Moreover, in the form of spores, these species resist and concentrate in the powders during the processes. The aim of this study was to determine the stages of the dairy powder manufacturing processes that are favorable to the growth of such contaminants. A total of 5 strains were selected for each species as a natural contaminant of dairy pipelines in order to determine the minimum and maximum growth enabling values for temperature, pH, and aw and their optimum growth rates in milk. These growth limits were combined with the environmental conditions of temperature, pH and aw encountered at each step of the manufacture of whole milk, skim milk and milk protein concentrate powders to estimate growth capacities using cardinal models and the Gamma concept. These simulations were used to theoretically calculate the population sizes reached for the different strains studied at each stage in between two successive cleaning in place procedures. This approach highlights the stages at which risk occurs for the development of spore-forming thermophilic bacterial species. During the first stages of production, i.e. pre-treatment, pasteurization, standardization and pre-heating before concentration, physico-chemical conditions encountered are suitable for the development and growth of A. flavithermus, G. stearothermophilus and B. licheniformis. During the pre-heating stage and during the first effects in the evaporators, the temperature conditions appear to be the most favorable for the growth of G. stearothermophilus. The temperatures in the evaporator during the last evaporator effects are favorable for the growth of B. licheniformis. In the evaporation stage, low water activity severely limits the development of A. flavithermus.

Crystal structure and solution scattering of Geobacillus stearothermophilus S9 peptidase reveal structural adaptations for carboxypeptidase activity.

Acylaminoacyl peptidases (AAPs) play a pivotal role in various pathological conditions and are recognized as potential therapeutic targets. AAPs exhibit a wide range of activities, such as acylated amino acid-dependent aminopeptidase, endopeptidase, and less studied carboxypeptidase activity. We have determined the crystal structure of an AAP from Geobacillus stearothermophilus (S9gs) at 2.0 Å resolution. Despite being annotated as an aminopeptidase in the NCBI database, our enzymatic characterization proved S9gs to be a carboxypeptidase. Solution-scattering studies showed that S9gs exists as a tetramer in solution, and crystal structure analysis revealed adaptations responsible for the carboxypeptidase activity of S9gs. The findings present a hypothesis for substrate selection, substrate entry, and product exit from the active site, enriching our understanding of this rare carboxypeptidase.

Enhancing the anti-thixotropic properties of waxy maize starch modified by different α-amylases and its underlying molecular mechanism.

The large thixotropy of the starch-thickened foods is often unfavorable in many applications. This study examined the contribution of the proportion of amylopectin chain length to time-dependence of starch gels. The α-amylase (AM) from Bacillus stearothermophilus and maltogenic α-amylase (MA) from Bacillus subtilis were used to trim amylopectin in different reaction patterns. HPLC, HPAEC and IBC data suggested AM attacked B-chains (DP 12-36), causing an increment in number of the chains with DP 6-12, whereas MA primarily trimmed the short B-chains (DP 12-18) and partial A-chains (DP 9-12) to generate short chains with DP 6-9. Interestingly, the recovery of AM-gels was faster than MA-gels at the same degree of hydrolysis when subjected to shear according to the linear correlation analysis. When releasing the same mass of sugar, shortening of the long internal chains played an important role in reducing time dependence of starch gel rather than the external side chains. Possible models were proposed to illustrate the differences in the mechanism of rapid-recovery caused by different side-chain distributions. The outcome provided a new perspective to regulate the thixotropy behavior of starch through enzyme strategies in the granular state.

Recombinant TP-84 Bacteriophage Glycosylase-Depolymerase Confers Activity against Thermostable Geobacillus stearothermophilus via Capsule Degradation.

The TP-84 bacteriophage, which infects Geobacillus stearothermophilus strain 10 (G. stearothermophilus), has a genome size of 47.7 kilobase pairs (kbps) and contains 81 predicted protein-coding ORFs. One of these, TP84_26 encodes a putative tail fiber protein possessing capsule depolymerase activity. In this study, we cloned the TP84_26 gene into a high-expression Escherichia coli (E. coli) system, modified its N-terminus with His-tag, expressed both the wild type gene and His-tagged variant, purified the recombinant depolymerase variants, and further evaluated their properties. We developed a direct enzymatic assay for the depolymerase activity toward G. stearothermophilus capsules. The recombinant TP84_26 protein variants effectively degraded the existing bacterial capsules and inhibited the formation of new ones. Our results provide insights into the novel TP84_26 depolymerase with specific activity against thermostable G. stearothermophilus and its role in the TP-84 life cycle. The identification and characterization of novel depolymerases, such as TP84_26, hold promise for innovative strategies to combat bacterial infections and improve various industrial processes.

Novel extracellular synthesized silver nanoparticles using thermophilic Anoxybacillus flavithermus and Geobacillus stearothermophilus and their evaluation as nanodrugs.

In this investigation, two new thermophilic bacteria were isolated. The new isolates were characterized by 16S rRNA, biochemical, morphological, and physiological analyzes and the isolates were identified as Geobacillus stearothermophilus strain Gecek20 and thermophilic Anoxybacillus flavithermus strain Gecek19. Various biological activities of extracellular Ag-NPs synthesized from thermophilic G. stearothermophilus strain Gecek20 and thermophilic A. flavithermus strain Gecek19 were evaluated. The produced NPs were analyzed by SEM, SEM-EDX, and XRD analyses. The antioxidant abilities of new synthesized Ag-NPs from thermophilic G. stearothermophilus strain Gecek20 (T1-Ag-NPs) and new synthesized Ag-NPs from thermophilic A. flavithermus strain Gecek19 (T2-Ag-NPs) were studied by DPPH inhibition and metal chelating ability. The highest DPPH and metal chelating abilities of T1-Ag-NPs and T2-Ag-NPs at 200 mg/L concentration were 93.17 and 90.85%, and 75.80 and 83.64%, respectively. The extracellular green synthesized T1-Ag-NPs and T2-AgN-Ps showed DNA nuclease activity at all tested concentrations. Moreover, both new synthesized Ag-NPs had antimicrobial activity against the strains studied, especially on Gram positive bacteria. T1-Ag-NPs and T2-AgNPs also showed powerful Escherichia coli growth inhibition. The highest biofilm inhibition percentages of T1-Ag-NPs and T2-Ag-NPs against Pseudomonas aeruginosa and Staphylococcus aureus were 100.0%, respectively, at 500 mg/L.

Enhanced Thermostability of Geobacillus stearothermophilus α-Amylase by Rational Design of Disulfide Bond and Application in Corn Starch Liquefaction and Bread Quality Improvement.

α-Amylase (EC 3.2.1.1) from Geobacillus stearothermophilus (generally recognized as safe) exhibited thermal inactivation, hampering its further application in starch-based industries. To address this, we performed structural analyses based on molecular dynamics targeting the flexible regions of α-amylase. Subsequently, we rationally designed a thermostable mutant, AmyS1, by introducing disulfide bonds to stabilize the flexible regions. AmyS1 showed excellent thermostability without any stability-activity trade-off, giving a 40-fold longer T1/2 (1359 min) at 90 °C. Thermostability mechanism analysis revealed that the introduction of disulfide bonds in AmyS1 refined weak spots and reconfigured the protein's force network. Moreover, AmyS1 exhibited improved pH compatibility and enhanced corn starch liquefaction at 100 °C with a 5.1-fold increased product concentration. Baking tests confirmed that AmyS1 enhanced bread quality and extended the shelf life. Therefore, mutant AmyS1 is a robust candidate for the starch-based industry.

Trivalent rare earth metal cofactors confer rapid NP-DNA polymerase activity.

A DNA polymerase with a single mutation and a divalent calcium cofactor catalyzes the synthesis of unnatural N3'→P5' phosphoramidate (NP) bonds to form NP-DNA. However, this template-directed phosphoryl transfer activity remains orders of magnitude slower than native phosphodiester synthesis. Here, we used time-resolved x-ray crystallography to show that NP-DNA synthesis proceeds with a single detectable calcium ion in the active site. Using insights from isotopic and elemental effects, we propose that one-metal-ion electrophilic substrate activation is inferior to the native two-metal-ion mechanism. We found that this deficiency in divalent activation could be ameliorated by trivalent rare earth and post-transition metal cations, substantially enhancing NP-DNA synthesis. Scandium(III), in particular, confers highly specific NP activity with kinetics enhanced by more than 100-fold over calcium(II), yielding NP-DNA strands up to 100 nucleotides in length.

The novel sterilization device: the prototype testing.

Currently, there are numerous methods that can be used to neutralize pathogens (i.e., devices, tools, or protective clothing), but the sterilizing agent must be selected so that it does not damage or change the properties of the material to which it is applied. Dry sterilization with hydrogen peroxide gas (VHP) in combination with UV-C radiation is well described and effective method of sterilization. This paper presents the design, construction, and analysis of a novel model of sterilization device. Verification of the sterilization process was performed, using classical microbiological methods and flow cytometry, on samples containing Geobacillus stearothermophilus spores, Bacillus subtilis spores, Escherichia coli, and Candida albicans. Flow cytometry results were in line with the standardized microbiological tests and confirmed the effectiveness of the sterilization process. It was also determined that mobile sterilization stations represent a valuable solution when dedicated to public institutions and businesses in the tourism sector, sports & fitness industry, or other types of services, e.g., cosmetic services. A key feature of this solution is the ability to adapt the device within specific constraints to the user's needs.

Transposon-encoded nucleases use guide RNAs to promote their selfish spread.

Insertion sequences are compact and pervasive transposable elements found in bacteria, which encode only the genes necessary for their mobilization and maintenance1. IS200- and IS605-family transposons undergo 'peel-and-paste' transposition catalysed by a TnpA transposase2, but they also encode diverse, TnpB- and IscB-family proteins that are evolutionarily related to the CRISPR-associated effectors Cas12 and Cas9, respectively3,4. Recent studies have demonstrated that TnpB and IscB function as RNA-guided DNA endonucleases5,6, but the broader biological role of this activity has remained enigmatic. Here we show that TnpB and IscB are essential to prevent permanent transposon loss as a consequence of the TnpA transposition mechanism. We selected a family of related insertion sequences from Geobacillus stearothermophilus that encode several TnpB and IscB orthologues, and showed that a single TnpA transposase was broadly active for transposon mobilization. The donor joints formed upon religation of transposon-flanking sequences were efficiently targeted for cleavage by RNA-guided TnpB and IscB nucleases, and co-expression of TnpB and TnpA led to substantially greater transposon retention relative to conditions in which TnpA was expressed alone. Notably, TnpA and TnpB also stimulated recombination frequencies, surpassing rates observed with TnpB alone. Collectively, this study reveals that RNA-guided DNA cleavage arose as a primal biochemical activity to bias the selfish inheritance and spread of transposable elements, which was later co-opted during the evolution of CRISPR-Cas adaptive immunity for antiviral defence.

Improved thermostability of type I pullulanase from Bacillus thermoliquefaciens by error-prone PCR.

Pullulanase (PulB) is a starch-debranching enzyme. In order to improve its catalytic performance, random mutagenesis was performed on the pullulanase gene derived from Bacillus thermoliquefaciens. Two rounds of error-prone PCR were carried out. Mutant T252S was screened in the first round of error-prone library, which had the highest catalytic activity. During the second round of mutations, mutant enzyme G250P/T252S/G253T/N255K was screened, which had further improved catalytic activity and the best thermostability. Compared with the parent enzyme, the specific activity of mutant enzyme G250P/T252S/G253T/N255K increased by 1.9 times, Km decreased by 22.7 %, kcat increased by 28.7 %, and kcat/Km increased by 68.4 %. The thermostability of the mutant enzyme improved significantly, showing that the half-life at 60 °C was extended to 7.5 h, which was 87.5 % higher than that of the parent enzyme. The mutation sites in these two rounds were concentrated in the 250-255 regions, indicating that this region was an important region affecting the catalytic activity and Thermostability. The reasons for the change of enzymtic properties was also preliminarily analyzed through three-dimensional simulation.

Thermostable homologues of the periplasmic siderophore-binding protein CeuE from Geobacillus stearothermophilus and Parageobacillus thermoglucosidasius.

Siderophore-binding proteins from two thermophilic bacteria, Geobacillus stearothermophilus and Parageobacillus thermoglucosidasius, were identified from a search of sequence databases, cloned and overexpressed. They are homologues of the well characterized protein CjCeuE from Campylobacter jejuni. The iron-binding histidine and tyrosine residues are conserved in both thermophiles. Crystal structures were determined of the apo proteins and of their complexes with iron(III)-azotochelin and its analogue iron(III)-5-LICAM. The thermostability of both homologues was shown to be about 20°C higher than that of CjCeuE. Similarly, the tolerance of the homologues to the organic solvent dimethylformamide (DMF) was enhanced, as reflected by the respective binding constants for these ligands measured in aqueous buffer at pH 7.5 in the absence and presence of 10% and 20% DMF. Consequently, these thermophilic homologues offer advantages in the development of artificial metalloenzymes using the CeuE family.

Polyhydroxyalkanoate biosynthesis and optimisation of thermophilic Geobacillus stearothermophilus strain K4E3_SPR_NPP.

Polyhydroxyalkanoates (PHA) can be used to combat the challenges associated with plastic because it is biodegradable and can be produced from renewable resources. Extremophiles are considered to be potential PHA producers. An initial screening for the PHA synthesizing ability of a thermophilic bacteria Geobacillus stearothermophilus strain K4E3_SPR_NPP was carried out using Sudan black B staining. Nile red viable colony staining was used to further verify that the isolates produced PHA. Crotonic acid assays were used to determine the concentrations of PHA. The bacteria showed 31% PHA accumulation per dry cell weight (PHA/DCW) when glucose was used as a carbon source for growth. The molecule was identified to be medium chain length PHA, A copolymer of PHA containing poly(3-hydroxybutyrate)-poly(3-hydroxyvalerate)-poly(3-hydroxyhexanoate) (PHB-PHV-PHHX) using 1H-NMR. Six carbon sources and four nitrogen sources were screened for the synthesis of maximum PHA content, of which lactose and ammonium nitrate showed 45% and 53% PHA/DCW respectively. The important factors in the experiment are identified using the Plackett-Burman design, and optimization is performed using the response surface method. Response surface methodology was used to optimize the three important factors, and the maximum biomass and PHA productions were discovered. Optimal concentrations yielded a maximum of 0.48 g/l biomass and 0.32 g/l PHA, measuring 66.66% PHA accumulation. Dairy industry effluent was employed for the synthesis of PHA, yielding 0.73 g/l biomass and 0.33 g/l PHA, measuring 45% PHA accumulation. These findings add credibility to the possibility of adopting thermophilic isolates for PHA production using low-cost substrates.

Enhancing 2-O-α-D-glucopyranosyl-L-ascorbic acid synthesis by weakening the acceptor specificity of CGTase toward glucose and maltose.

2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) is a stable derivative of L-ascorbic acid (L-AA), which has been widely used in food and cosmetics industries. Sugar molecules, such as glucose and maltose produced by cyclodextrin glycosyltransferase (CGTase) during AA-2G synthesis may compete with L-AA as the acceptors, resulting in low AA-2G yield. Multiple sequence alignment combined with structural simulation analysis indicated that residues at positions 191 and 255 of CGTase may be responsible for the difference in substrate specificity. To investigate the effect of these two residues on the acceptor preference and the AA-2G yield, five single mutants Bs F191Y, Bs F255Y, Bc Y195F, Pm Y195F and Pm Y260F of three CGTases from Bacillus stearothermophilus NO2 (Bs), Bacillus circulans 251 (Bc) and Paenibacillus macerans (Pm) were designed for AA-2G synthesis. Under optimal conditions, the AA-2G yields of the mutants Bs F191Y and Bs F255Y AA-2G were 34.3% and 7.9% lower than that of Bs CGTase, respectively. The AA-2G yields of mutant Bc Y195F, Pm Y195F and Pm Y260F were 45.8%, 36.9% and 12.6% higher than those of wild-type CGTases, respectively. Kinetic studies revealed that the residues at positions 191 and 255 of the three CGTases were F, which decreased glucose and maltose specificity and increased L-AA specificity. This study not only proposes for the first time that the AA-2G yield can be improved by weakening the acceptor specificity of CGTase toward sugar byproducts, but also provides new insight on the modification of CGTase that catalyze the double-substrate transglycosylation reaction.

Quantitative microbial spoilage risk assessment of plant-based milk alternatives by Geobacillus stearothermophilus in Europe.

Geobacillus stearothermophilus is one of the predominant spoilers of UHT-treated food products, due to its extremely heat-resistant spores. However, the surviving spores should be exposed to temperature higher than their minimum growth temperature for a certain time to germinate and grow to spoilage levels. Considering the projected temperature increase due to climate change, the events of non-sterility during distribution and transportation are expected to escalate. Hence, the aim of this study was to build a quantitative microbial spoilage risk assessment (QMRSA) model to quantify the risk of spoilage of plant-based milk alternatives within Europe. The model consists of four main steps: 1. Initial contamination of raw materials 2. Heat inactivation of spores during UHT treatment 3. Partitioning 4. Germination and outgrowth of spores during distribution and storage. The risk of spoilage was defined as the probability of G. stearothermophilus to reach its maximum concentration (Nmax = 107.5 CFU/mL) at the time of consumption. The assessment was performed for North (Poland) and South (Greece) Europe, and the risk of spoilage was estimated for the current climatic conditions and a climate change scenario. Based on the results, the risk of spoilage was negligible for the North European region, while the risk of spoilage in South Europe was 6.2 × 10-3 95% CI (2.3 × 10-3;1.1 × 10-2) under the current climatic conditions. The risk of spoilage was increased for both tested countries under climate change scenario; from zero to 1.0 × 10-4 in North Europe, risk multiplied 2 or 3 in South Europe depending on air conditioning implementation at consumer's place. Therefore, the heat treatment intensity and the use of insulated trucks during distribution were investigated as mitigation strategies and led to significant reduction of the risk. Overall, the QMRSA model developed in this study can support risk management decisions of these products by quantify the potential risk under current climatic conditions and climate change scenarios.

Comparative Genomic Analysis of a Thermophilic Protease-Producing Strain Geobacillus stearothermophilus H6.

The genus Geobacillus comprises thermophilic gram-positive bacteria which are widely distributed, and their ability to withstand high temperatures makes them suitable for various applications in biotechnology and industrial production. Geobacillus stearothermophilus H6 is an extremely thermophilic Geobacillus strain isolated from hyperthermophilic compost at 80 °C. Through whole-genome sequencing and genome annotation analysis of the strain, the gene functions of G. stearothermophilus H6 were predicted and the thermophilic enzyme in the strain was mined. The G. stearothermophilus H6 draft genome consisted of 3,054,993 bp, with a genome GC content of 51.66%, and it was predicted to contain 3750 coding genes. The analysis showed that strain H6 contained a variety of enzyme-coding genes, including protease, glycoside hydrolase, xylanase, amylase and lipase genes. A skimmed milk plate experiment showed that G. stearothermophilus H6 could produce extracellular protease that functioned at 60 °C, and the genome predictions included 18 secreted proteases with signal peptides. By analyzing the sequence of the strain genome, a protease gene gs-sp1 was successfully screened. The gene sequence was analyzed and heterologously expressed, and the protease was successfully expressed in Escherichia coli. These results could provide a theoretical basis for the development and application of industrial strains.

Geobacillus stearothermophilus and Bacillus atrophaeus spores exhibit linear inactivation kinetic performance when treated with an industrial scale vaporized hydrogen peroxide (VH2O2) sterilization process.

AIMS: This study aims to determine the inactivation kinetics of Geobacillus stearothermophilus and Bacillus atrophaeus biological indicators, treated with vaporized hydrogen peroxide (VH2O2) at an industrial scale. There is an assumption that sterilization processes generate linear kinetic plots of treated biological indicators that are used for informing probability-based decision-making by the MedTech industry for effective sterilization treatments; however, this has not been reported for sterilization using VH2O2. METHODS AND RESULTS: Survivor curves were generated, and sterilization performances were separately determined using G. stearothermophilus and B. atrophaeus biological indicators following the development of appropriate process challenge devices (PCDs). Regression analysis revealed that the inactivation kinetics for VH2O2-treated microorganisms exhibited log linear profiles. The use of scanning electron microscope (SEM) revealed no significant topographical changes in the outer surface of these VH2O2-treated spores. CONCLUSIONS: Both biological indicators exhibited log linear inactivation kinetics when treated with an industrial scale vaporized hydrogen peroxide (VH2O2) sterilization process. Therefore, this novel finding corroborates and proves the appropriateness of using VH2O2 as a sterilization method in accordance with applicable ISO standards.