ARTP mutation and genome shuffling of ABE fermentation symbiotic system for improvement of butanol production.

Butanol is an ideal renewable biofuel which possesses superior fuel properties. Previously, butanol-producing symbiotic system TSH06 was isolated in our lab, with microoxygen tolerance ability. To boost butanol yield for large-scale industrial production, TSH06 was used as parental strain and subjected to atmospheric and room temperature plasma (ARTP) and four rounds of genome shuffling (GS). ARTP mutant and GS strain were co-cultured with facultative anaerobic Bacillus cereus TSH2 to form a symbiotic system with microoxygen tolerance, which was then subjected to fermentation. Relative messenger RNA (mRNA) level of key enzyme gene was measured by real-time PCR. The highest butanol titer of TS4-30 reached 15.63 g/L, which was 34% higher than TSH06 (12.19 g/L). Compared with parental strain, mRNA of acid-forming gene in TS4-30 decreased in acidogenesis phase, while solvent-forming gene increased in solventogenesis phase. This gene expression pattern was consistent with high butanol yield and low acid level in TS4-30. In summary, symbiotic system TS4-30 was obtained with butanol titer improvement and microoxygen tolerance.

Interactions between Bacillus cereus CGMCC 1.895 and Clostridium beijerinckii NCIMB 8052 in coculture for butanol production under nonanaerobic conditions.

Low oxygen tolerance and substrate restriction continues to hamper the process of biobutanol industrialization. In this work, butanol fermentation with cocultures of Bacillus cereus China General Microbiological Culture Collection Center (CGMCC) 1.895 and Clostridium beijerinckii NCIMB 8052 under nonanaerobic conditions was investigated, and the interactions between these two strains were examined. The addition of B. cereus CGMCC 1.895 resulted in higher oxygen tolerance and a wider range of substrate utilization, compared with the pure culture of C. beijerinckii NCIMB 8052. Butanol concentration reached 10.49 g/L with an optimized inoculation size of 90% under nonanaerobic conditions, and this concentration was close to that of pure C. beijerinckii NCIMB 8052 culture under anaerobic conditions. Dynamic relative abundance analysis demonstrated that the ratio of C. beijerinckii NCIMB 8052 accounted for nearly 99% of the cocultured cells. Furthermore, the substrate utilization range was expanded, allowing the use of corn mash for butanol production. The final concentration of butanol and total solvents was 6.78 and 10.52 g/L, respectively. Coculture also was performed successfully in a 5-L fermenter and 8.75 g/L butanol was obtained. Dynamic dissolved oxygen analysis demonstrated that B. cereus consumed the dissolved oxygen in the broth and resulted in the anaerobic condition for C. beijerinckii.

Phenotypic H-Antigen Typing by Mass Spectrometry Combined with Genetic Typing of H Antigens, O Antigens, and Toxins by Whole-Genome Sequencing Enhances Identification of Escherichia coli Isolates.

Mass spectrometry-based phenotypic H-antigen typing (MS-H) combined with whole-genome-sequencing-based genetic identification of H antigens, O antigens, and toxins (WGS-HOT) was used to type 60 clinical Escherichia coli isolates, 43 of which were previously identified as nonmotile, H type undetermined, or O rough by serotyping or having shown discordant MS-H and serotyping results. Whole-genome sequencing confirmed that MS-H was able to provide more accurate data regarding H antigen expression than serotyping. Further, enhanced and more confident O antigen identification resulted from gene cluster based typing in combination with conventional typing based on the gene pair comprising wzx and wzy and that comprising wzm and wzt The O antigen was identified in 94.6% of the isolates when the two genetic O typing approaches (gene pair and gene cluster) were used in conjunction, in comparison to 78.6% when the gene pair database was used alone. In addition, 98.2% of the isolates showed the existence of genes for various toxins and/or virulence factors, among which verotoxins (Shiga toxin 1 and/or Shiga toxin 2) were 100% concordant with conventional PCR based testing results. With more applications of mass spectrometry and whole-genome sequencing in clinical microbiology laboratories, this combined phenotypic and genetic typing platform (MS-H plus WGS-HOT) should be ideal for pathogenic E. coli typing.

Mass Spectrometry-Based Escherichia coli H Antigen/Flagella Typing: Validation and Comparison with Traditional Serotyping.

BACKGROUND: Escherichia coli H antigen typing with antisera, a useful method for flagella clinical identification and classification, is a time-consuming process because of the need to induce flagella growth and the occurrence of undetermined strains. We developed an alternative rapid and analytically sensitive mass spectrometry (MS) method, termed MS-based H antigen typing (MS-H), and applied it at the protein sequence level for H antigen typing. We also performed a comparison with traditional serotyping on reference strains and clinical isolates. METHODS: On the basis of international guidelines, the analytical selectivity and sensitivity, imprecision, correlation, repeatability, and reproducibility of the MS-H platform was evaluated using reference strains. Comparison of MS-H typing and serotyping was performed using 302 clinical isolates from 5 Canadian provinces, and discrepant results between the 2 platforms were resolved through whole genome sequencing. RESULTS: Repeated tests on reference strain EDL933 demonstrated a lower limit of the measuring interval at the subsingle colony (16.97 µg or 1.465 × 10(7) cells) level and close correlation (r(2) > 0.99) between cell culture biomass and sequence coverage. The CV was <10.0% among multiple repeats with 4 reference strains. Intra- and interlaboratory tests demonstrated that the MS-H method was robust and reproducible under various sample preparation and instrumentation conditions. Using discrepancy analysis via whole genome sequencing, performed on isolates with discrepant results, MS-H accurately identified 12.3% more isolates than conventional serotyping. CONCLUSIONS: MS-H typing of E. coli is useful for fast and accurate flagella typing and could be very useful during E. coli outbreaks.

Butanol production under microaerobic conditions with a symbiotic system of Clostridium acetobutylicum and Bacillus cereus.

BACKGROUND: One major problem of ABE (acetone, butanol and ethanol) fermentation is high oxygen sensitivity of Clostridium acetobutylicum. Currently, no single strain has been isolated or genetically engineered to produce butanol effectively under aerobic conditions. In our previous work, a symbiotic system TSH06 has been developed successfully by our group, and two strains, C. acetobutylicum TSH1 and Bacillus cereus TSH2, were isolated from TSH06. RESULTS: Compared with single culture, TSH06 showed promotion on cell growth and solvent accumulation under microaerobic conditions. To simulate TSH06, a new symbiotic system was successfully re-constructed by adding living cells of B. cereus TSH2 into C. acetobutylicum TSH1 cultures. During the fermentation process, the function of B. cereus TSH2 was found to deplete oxygen and provide anaerobic environment for C. acetobutylicum TSH1. Furthermore, inoculation ratio of C. acetobutylicum TSH1 and B. cereus TSH2 affected butanol production. In a batch fermentation with optimized inoculation ratio of 5 % C. acetobutylicum TSH1 and 0.5 % B. cereus TSH2, 11.0 g/L butanol and 18.1 g/L ABE were produced under microaerobic static condition. In contrast to the single culture of C. acetobutylicum TSH1, the symbiotic system became more aerotolerant and was able to produce 11.2 g/L butanol in a 5 L bioreactor even with continuous 0.15 L/min air sparging. In addition, qPCR assay demonstrated that the abundance of B. cereus TSH2 increased quickly at first and then decreased sharply to lower than 1 %, whereas C. acetobutylicum TSH1 accounted for more than 99 % of the whole population in solventogenic phase. CONCLUSIONS: The characterization of a novel symbiotic system on butanol fermentation was studied. The new symbiotic system re-constructed by co-culture of C. acetobutylicum TSH1 and B. cereus TSH2 showed excellent performance on butanol production under microaerobic conditions. B. cereus TSH2 was a good partner for C. acetobutylicum TSH1 by providing an anaerobic environment. During fermentation process, the high ratio of Clostridium and low ratio of Bacillus composition indicated that this symbiotic system was an effective and easily controlled cultivation model for ABE fermentation under microaerobic conditions.

Rapid, Sensitive, and Specific Escherichia coli H Antigen Typing by Matrix-Assisted Laser Desorption Ionization-Time of Flight-Based Peptide Mass Fingerprinting.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has gained popularity in recent years for rapid bacterial identification, mostly at the genus or species level. In this study, a rapid method to identify the Escherichia coli flagellar antigen (H antigen) at the subspecies level was developed using a MALDI-TOF MS platform with high specificity and sensitivity. Flagella were trapped on a filter membrane, and on-filter trypsin digestion was performed. The tryptic digests of each flagellin then were collected and analyzed by MALDI-TOF MS through peptide mass fingerprinting. Sixty-one reference strains containing all 53 H types and 85 clinical strains were tested and compared to serotyping designations. Whole-genome sequencing was used to resolve conflicting results between the two methods. It was found that DHB (2,5-dihydroxybenzoic acid) worked better than CHCA (α-cyano-4-hydroxycinnamic acid) as the matrix for MALDI-TOF MS, with higher confidence during protein identification. After method optimization, reference strains representing all 53 E. coli H types were identified correctly by MALDI-TOF MS. A custom E. coli flagellar/H antigen database was crucial for clearly identifying the E. coli H antigens. Of 85 clinical isolates tested by MALDI-TOF MS-H, 75 identified MS-H types (88.2%) matched results obtained from traditional serotyping. Among 10 isolates where the results of MALDI-TOF MS-H and serotyping did not agree, 60% of H types characterized by whole-genome sequencing agreed with those identified by MALDI-TOF MS-H, compared to only 20% by serotyping. This MALDI-TOF MS-H platform can be used for rapid and cost-effective E. coli H antigen identification, especially during E. coli outbreaks.

Isolation and characterisation of non-anaerobic butanol-producing symbiotic system TSH06.

Butanol-producing microorganisms are all obligate anaerobes. In this study, a unique symbiotic system TSH06 was isolated to be capable of producing butanol under non-anaerobic condition. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S ribosomal RNA (rRNA) revealed that two strains coexist in TSH06. The two strains were identical to Clostridium acetobutylicum and Bacillus cereus, respectively. They were isolated individually and named as C. acetobutylicum TSH1 and B. cereus TSH2. C. acetobutylicum TSH1 is a butanol-producing, obligate anaerobic strain. Facultative anaerobic B. cereus TSH2 did not possess the ability of butanol production; however, it offered C. acetobutylicum TSH1 the viability under non-anaerobic condition. Moreover, B. cereus TSH2 enhanced butanol yield and speed of fermentation. TSH06 produced 12.97 g/L butanol and 15.39 g/L total solvent under non-anaerobic condition, which is 25 and 24 %, respectively, higher than those of C. acetobutylicum TSH1. In addition, TSH06 produced butanol faster under non-anaerobic condition than under anaerobic condition. Butanol accounted for more than 80 % of total solvent, which is higher than the known report. TSH06 was stable during passage. In all, TSH06 is a promising candidate for industrialisation of biobutanol with high yield, high butanol proportion, easy-handling and time-saving system. These results demonstrated the potential advantage of symbiosis. This study also provides a promising strategy for butanol fermentation.

Sequence-level and dual-phase identification of Salmonella flagellum antigens by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Forty-three reference strains involving the 24 most common serovars of Salmonella enterica were examined by using a mass spectrometry-based H antigen typing platform (MS-H). The results indicate that MS-H can be used as a sensitive, rapid, and straightforward approach for the typing of Salmonella flagella at the molecular level without antiserum and phase inversion.

Mass spectrometry-based Shiga toxin identification: A clinical validation.

After we published our preliminary study on the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and curated E. coli toxin databases on the identification of E. coli Shiga toxins (Stxs) in the Journal of Proteomics in year 2018, we were encouraged to further refine the method and test clinical isolates. In this study, different concentrations of mitomycin C (MMC) and ciprofloxacin (CF), two common antibiotic/chemotherapy agents capable of stimulating Stx production, were first tested and compared on three reference strains and eight clinical isolates to observe the toxin induction and subsequent identification. Notably, no differences were observed between the two agents other than the concentrations applied. Seventeen more clinical isolates were then tested using fixed MMC and CF concentrations and sample amount. This study confirms that the majority of stx2-positive E. coli strains can be stimulated to produce sufficient toxin for confident identification. This does not occur with stx1-positive E. coli isolates, however, despite the fact that both Stxs can be identified for several isolates without MMC or CF stimulation. BIOLOGICAL SIGNIFICANCE: Stxs, especially Stx2, are very important causes of severe food-borne disease, even death. This study confirms that receptor analogue-based affinity enrichment of Stxs, after MMC or CF treatment of E. coli, is useful for fast and accurate Stx2 identification through LC-MS/MS.

Mass spectrometry-based Shiga toxin identification: An optimized approach.

Toxin expression is a key factor in Shiga toxin (Stx)-producing E. coli, a common pathogen involved in foodborne disease outbreaks. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) based approach has been used in this study to identify commonly reported E. coli toxins, with a focus on Shiga toxins (Stxs). Different sample preparation methods using variable culture conditions and concentrations of mitomycin C (MMC), a common antibiotic/chemotherapy agent capable of stimulating Stx production, were first tested on reference strains EDL933 and 90-2380 by LC-MS/MS detection of tryptic digests of receptor-analogue affinity binding enriched Stx preparations from culture supernatants and lysates. A curated E. coli protein toxin database was also used for faster and more straightforward toxin identification. With eight more genetically confirmed E. coli strains examined to verify the method, this preliminary study indicates that receptor-analogue based affinity enrichment on cell lysate or supernatant is a sensitive and accurate method for Stx identification. BIOLOGICAL SIGNIFICANCE: The existence of Stx is very important for identifying Stx-producing E. coli and implementing a clinical treatment regime. This study demonstrates for the first time that using a curated E. coli toxin database, together with receptor-analogue-based affinity enrichment of Stxs after MMC treatment of E. coli, is an easy and appropriate approach for fast and accurate Stx identification through LC-MS/MS.

Traditional approaches versus mass spectrometry in bacterial identification and typing.

Biochemical methods such as metabolite testing and serotyping are traditionally used in clinical microbiology laboratories to identify and categorize microorganisms. Due to the large variety of bacteria, identifying representative metabolites is tedious, while raising high-quality antisera or antibodies unique to specific biomarkers used in serotyping is very challenging, sometimes even impossible. Although serotyping is a certified approach for differentiating bacteria such as E. coli and Salmonella at the subspecies level, the method is tedious, laborious, and not practical during an infectious disease outbreak. Mass spectrometry (MS) platforms, especially matrix assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS), have recently become popular in the field of bacterial identification due to their fast speed and low cost. In the past few years, we have used liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approaches to solve various problems hindering serotyping and have overcome some insufficiencies of the MALDI-TOF-MS platform. The current article aims to review the characteristics, advantages, and disadvantages of MS-based platforms over traditional approaches in bacterial identification and categorization.

[Lipid synthesis by an acidic acid tolerant Rhodotorula glutinis].

Acetic acid, as a main by-product generated in the pretreatment process of lignocellulose hydrolysis, significantly affects cell growth and lipid synthesis of oleaginous microorganisms. Therefore, we studied the tolerance of Rhodotorula glutinis to acetic acid and its lipid synthesis from substrate containing acetic acid. In the mixed sugar medium containing 6 g/L glucose and 44 g/L xylose, and supplemented with acetic acid, the cell growth was not:inhibited when the acetic acid concentration was below 10 g/L. Compared with the control, the biomass, lipid concentration and lipid content of R. glutinis increased 21.5%, 171% and 122% respectively when acetic acid concentration was 10 g/L. Furthermore, R. glutinis could accumulate lipid with acetate as the sole carbon source. Lipid concentration and lipid yield reached 3.20 g/L and 13% respectively with the initial acetic acid concentration of 25 g/L. The lipid composition was analyzed by gas chromatograph. The main composition of lipid produced with acetic acid was palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid, including 40.9% saturated fatty acids and 59.1% unsaturated fatty acids. The lipid composition was similar to that of plant oil, indicating that lipid from oleaginous yeast R. glutinis had potential as the feedstock of biodiesel production. These results demonstrated that a certain concentration of acetic acid need not to be removed in the detoxification process when using lignocelluloses hydrolysate to produce microbial lipid by R. glutinis.

Recent development of mass spectrometry and proteomics applications in identification and typing of bacteria.

Identification and typing of bacteria occupy a large fraction of time and work in clinical microbiology laboratories. With the certification of some MS platforms in recent years, more applications and tests of MS-based diagnosis methods for bacteria identification and typing have been created, not only on well-accepted MALDI-TOF-MS-based fingerprint matches, but also on solving the insufficiencies of MALDI-TOF-MS-based platforms and advancing the technology to areas such as targeted MS identification and typing of bacteria, bacterial toxin identification, antibiotics susceptibility/resistance tests, and MS-based diagnostic method development on unique bacteria such as Clostridium and Mycobacteria. This review summarizes the recent development in MS platforms and applications in bacteria identification and typing of common pathogenic bacteria.

Bacterial DNA decontamination for reverse transcription polymerase chain reaction (RT-PCR).

For RT-PCR, removal of contaminating genomic DNA in RNA samples using manganese sulfate was more effective than magnesium. DNA contamination was removed in 3 microg of nucleic acid using 10 U of RNase-free DNase I in 10-microl reaction volumes. The digestion procedure was compatible with commercial RNA extraction kits and was suitable for RT-PCR assay.

Fit-for-purpose curated database application in mass spectrometry-based targeted protein identification and validation.

BACKGROUND: Mass spectrometry (MS) is a very sensitive and specific method for protein identification, biomarker discovery, and biomarker validation. Protein identification is commonly carried out by comparing MS data with public databases. However, with the development of high throughput and accurate genomic sequencing technology, public databases are being overwhelmed with new entries from different species every day. The application of these databases can also be problematic due to factors such as size, specificity, and unharmonized annotation of the molecules of interest. Current databases representing liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based searches focus on enzyme digestion patterns and sequence information and consequently, important functional information can be missed within the search output. Protein variants displaying similar sequence homology can interfere with database identification when only certain homologues are examined. In addition, recombinant DNA technology can result in products that may not be accurately annotated in public databases. Curated databases, which focus on the molecule of interest with clearer functional annotation and sequence information, are necessary for accurate protein identification and validation. Here, four cases of curated database application have been explored and summarized. FINDINGS: The four presented curated databases were constructed with clear goals regarding application and have proven very useful for targeted protein identification and biomarker application in different fields. They include a sheeppox virus database created for accurate identification of proteins with strong antigenicity, a custom database containing clearly annotated protein variants such as tau transcript variant 2 for accurate biomarker identification, a sheep-hamster chimeric prion protein (PrP) database constructed for assay development of prion diseases, and a custom Escherichia coli (E. coli) flagella (H antigen) database produced for MS-H, a new H-typing technique. Clearly annotating the proteins of interest was essential for highly accurate, specific, and sensitive sequence identification, and searching against public databases resulted in inaccurate identification of the sequence of interest, while combining the curated database with a public database reduced both the confidence and sequence coverage of the protein search. CONCLUSION: Curated protein sequence databases incorporating clear annotations are very useful for accurate protein identification and fit-for-purpose application through MS-based biomarker validation.

Quality evaluation of LC-MS/MS-based E. coli H antigen typing (MS-H) through label-free quantitative data analysis in a clinical sample setup.

PURPOSE: The need for rapid and accurate H typing is evident during Escherichia coli outbreak situations. This study explores the transition of MS-H, a method originally developed for rapid H antigen typing of E. coli using LC-MS/MS of flagella digest of reference strains and some clinical strains, to E. coli isolates in clinical scenario through quantitative analysis and method validation. EXPERIMENTAL DESIGN: Motile and nonmotile strains were examined in batches to simulate clinical sample scenario. Various LC-MS/MS batch run procedures and MS-H typing rules were compared and summarized through quantitative analysis of MS-H data output for a standard method development. RESULTS: Label-free quantitative data analysis of MS-H typing was proven very useful for examining the quality of MS-H result and the effects of some sample carryovers from motile E. coli isolates. Based on this, a refined procedure and protein identification rule specific for clinical MS-H typing was established and validated. CONCLUSIONS AND CLINICAL RELEVANCE: With LC-MS/MS batch run procedure and database search parameter unique for E. coli MS-H typing, the standard procedure maintained high accuracy and specificity in clinical situations, and its potential to be used in a clinical setting was clearly established.

MS-H: a novel proteomic approach to isolate and type the E. coli H antigen using membrane filtration and liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Serotyping is the long-standing gold standard method to determine E. coli H antigens; however, this method requires a panel of H-antigen specific antibodies and often culture-based induction of the H-antigen flagellar motility. In this study, a rapid and accurate method to isolate and identify the Escherichia coli (E. coli) H flagellar antigen was developed using membrane filtration and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Flagella were isolated from pure culture, digested with trypsin, and then subjected to LC-MS/MS using one of two systems (Agilent-nano-LC-QSTAR XL or Proxeon-nano-LC-LTQ-Orbitrap XL). The resulting peptide sequence data were searched against a custom E. coli flagella/H antigen database. This approach was evaluated using flagella isolated from reference E. coli strains representing all 53 known H antigen types and 41 clinical E. coli strains. The resulting LC-MS/MS classifications of H antigen types (MS-H) were concordant with the known H serogroup for all 53 reference types, and of 41 clinical isolates tested, 38 (92.7%) were concordant with the known H serogroup. MS-H clearly also identified two clinical isolates (4.9%) that were untypeable by serotyping. Notably, successful detection and classification of flagellar antigens with MS-H did not generally require induction of motility, establishing this proteomic approach as more rapid and cost-effective than traditional methods, while providing equitable specificity for typing E. coli H antigens.

Catalytic conversion of xylose to furfural over the solid acid SO4 ²â»-/ZrO2-Al2O3/SBA-15 catalysts.

Al-promoted SO4 ²â»-/ZrO2SBA-15 catalysts were prepared and characterized by XRD, BET, ICP and NH3-TPD techniques. The influence of introducing aluminum on the structure and surface properties of the catalyst and the catalytic activity for dehydration of xylose to furfural has been investigated. The introduction of the Al stabilizes the tetragonal phase of the ZrO2 and thus increases the number and intensity of acid sites. Based on the characterization of the deactivated catalyst, the accumulation of byproducts is the main reason for the deactivation of the catalyst. Regeneration with H2O2 can completely recover the catalytic activity of the deactivated catalyst.

Statistical optimization of recycled-paper enzymatic hydrolysis for simultaneous saccharification and fermentation via central composite design.

A central composite design of the response surface methodology (RSM) was employed to study the effects of temperature, enzyme concentration, and stirring rate on recycled-paper enzymatic hydrolysis. Among the three variables, temperature and enzyme concentration significantly affected the conversion efficiency of substrate, whereas stirring rate was not effective. A quadratic polynomial equation was obtained for enzymatic hydrolysis by multiple regression analysis using RSM. The results of validation experiments were coincident with the predicted model. The optimum conditions for enzymatic hydrolysis were temperature, enzyme concentration, and stirring rate of 43.1 degrees C, 20 FPU g(-1) substrate, and 145 rpm, respectively. In the subsequent simultaneous saccharification and fermentation (SSF) experiment under the optimum conditions, the highest 28.7 g ethanol l(-1) was reached in the fed-batch SSF when 5% (w/v) substrate concentration was used initially, and another 5% added after 12 h fermentation. This ethanol output corresponded to 77.7% of the theoretical yield based on the glucose content in the raw material.