scholarly journals Characterization and adaptation of Caldicellulosiruptor strains to higher sugar concentrations, targeting enhanced hydrogen production from lignocellulosic hydrolysates

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Eoin Byrne ◽  
Johanna Björkmalm ◽  
James P. Bostick ◽  
Krishnan Sreenivas ◽  
Karin Willquist ◽  
...  
Keyword(s):  
Mixed Culture ◽  
Mixed Population ◽  
Sugar Concentration ◽  
Monod Kinetics ◽  
Adaptive Laboratory Evolution ◽  
Kinetic Growth ◽  
Wide Range ◽  
Elevated Glucose ◽  
Lignocellulose Hydrolysates ◽  
Sugar Conversion

Abstract Background The members of the genus Caldicellulosiruptor have the potential for future integration into a biorefinery system due to their capacity to generate hydrogen close to the theoretical limit of 4 mol H2/mol hexose, use a wide range of sugars and can grow on numerous lignocellulose hydrolysates. However, members of this genus are unable to survive in high sugar concentrations, limiting their ability to grow on more concentrated hydrolysates, thus impeding their industrial applicability. In this study five members of this genus, C.owensensis, C. kronotskyensis, C.bescii, C.acetigenus and C.kristjanssonii, were developed to tolerate higher sugar concentrations through an adaptive laboratory evolution (ALE) process. The developed mixed population C.owensensis CO80 was further studied and accompanied by the development of a kinetic model based on Monod kinetics to quantitatively compare it with the parental strain. Results Mixed populations of Caldicellulosiruptor tolerant to higher glucose concentrations were obtained with C.owensensis adapted to grow up to 80 g/L glucose; other strains in particular C. kristjanssonii demonstrated a greater restriction to adaptation. The C.owensensis CO80 mixed population was further studied and demonstrated the ability to grow in glucose concentrations up to 80 g/L glucose, but with reduced volumetric hydrogen productivities ($$Q_{{{\text{H}}_{2} }}$$ Q H 2 ) and incomplete sugar conversion at elevated glucose concentrations. In addition, the carbon yield decreased with elevated concentrations of glucose. The ability of the mixed population C.owensensis CO80 to grow in high glucose concentrations was further described with a kinetic growth model, which revealed that the critical sugar concentration of the cells increased fourfold when cultivated at higher concentrations. When co-cultured with the adapted C.saccharolyticus G5 mixed culture at a hydraulic retention time (HRT) of 20 h, C.owensensis constituted only 0.09–1.58% of the population in suspension. Conclusions The adaptation of members of the Caldicellulosiruptor genus to higher sugar concentrations established that the ability to develop improved strains via ALE is species dependent, with C.owensensis adapted to grow on 80 g/L, whereas C.kristjanssonii could only be adapted to 30 g/L glucose. Although C.owensensis CO80 was adapted to a higher sugar concentration, this mixed population demonstrated reduced $$Q_{{{\text{H}}_{2} }}$$ Q H 2 with elevated glucose concentrations. This would indicate that while ALE permits adaptation to elevated sugar concentrations, this approach does not result in improved fermentation performances at these higher sugar concentrations. Moreover, the observation that planktonic mixed culture of CO80 was outcompeted by an adapted C.saccharolyticus, when co-cultivated in continuous mode, indicates that the robustness of CO80 mixed culture should be improved for industrial application.

scholarly journals Characterization and Development of Osmotolerant Caldicellulosiruptor Strains Targeting Enhanced Hydrogen Production from Lignocellulosic Hydrolysates

2020 ◽  
Author(s):  
Eoin Byrne ◽  
Johanna Björkmalm ◽  
James Bostick ◽  
Krishnan Sreeni ◽  
Karin Willquist ◽  
...  
Keyword(s):  
Hydraulic Retention Time ◽  
Monod Kinetics ◽  
Theoretical Limit ◽  
Carbon Yield ◽  
Adaptive Laboratory Evolution ◽  
Kinetic Growth ◽  
Wide Range ◽  
Elevated Glucose ◽  
Lignocellulose Hydrolysates ◽  
Sugar Conversion

Abstract Background The members of the genus Caldicellulosiruptor have the potential for future integration into a biorefinery system due to their capacity to generate hydrogen close to the theoretical limit of 4 mol H 2 /mol hexose, use a wide range of sugars and can grow on numerous lignocellulose hydrolysates. However, members of this genus are unable to survive in high osmolarity conditions, limiting their ability to grow on more concentrated hydrolysates, thus impeding their industrial applicability. In this study five members of this genus, C. owensensis , C. kronotskyensis , C. bescii, C. acetigenus and C. kristjanssonii , were developed to tolerate higher osmolarities through an adaptive laboratory evolution (ALE) process. The developed strain C. owensensis CO80 was further studied accompanied by the development of a kinetic model based on Monod kinetics. Results Osmotolerant strains of Caldicellulosiruptor were obtained with C. owensensis adapted to grow up to 80 g/l glucose; other strains in particular C. kristjanssonii demonstrated a greater restriction to adaptation. C. owensensis CO80 was further studied and demonstrated the ability to grow in glucose concentrations up to 80 g/l glucose but with reduced volumetric hydrogen productivities (Q H2 ) and incomplete sugar conversion at elevated glucose concentrations. In addition, the carbon yield decreased with elevated concentrations of glucose. The ability of C. owensensis CO80 to grow in high glucose concentrations was further described with a kinetic growth model, which revealed that the critical osmolarity of the cells increased fourfold when cultivated at higher osmolarity. When co-cultured with the osmotolerant strain C. saccharolyticus G5 at a hydraulic retention time (HRT) of 20h, C. owensensis constituted only 0.09-1.58% of the population in suspension.Conclusions The adaptation of members of the Caldicellulosiruptor genus to higher osmolarity established that the ability to develop improved strains via ALE is species dependent, with C. owensensis adapted to grow on 80 g/l, whereas C. kristjanssonii could only be adapted to 30 g/l glucose. Although, C. owensensis CO80 was adapted to a higher osmolarity medium, the strain demonstrated reduced Q H2 with elevated glucose concentrations. This would indicate that while ALE permits adaptation to elevated osmolarities, this approach does not result in improved fermentation performances at these higher osmolarities. Moreover, the observation that planktonic culture of CO80 was outcompeted by an osmotolerant strain of C. saccharolyticus, when co-cultivated in continuous mode, indicates that the robustness of strain CO80 should be improved for industrial application .

scholarly journals PRODUCTION AND STABILITY OF MYCO-FLOCCULANTS FROM LENTINUS SQUARROSULUS RWF5 AND SIMPLICILLIUM OBCLAVATUM RWF6 FOR REDUCTION OF WATER TURBIDITY

2018 ◽  
Vol 19 (1) ◽  
pp. 48-58
Author(s):  
Nessa X Jebun ◽  
Abdullah Al Mamun ◽  
Md. Zahangir Alam ◽  
Raha Ahmad Raus
Keyword(s):  
Mixed Culture ◽  
Removal Rate ◽  
Production Optimization ◽  
Screening Tests ◽  
Malt Extract ◽  
Water And Wastewater Treatment ◽  
Turbidity Removal ◽  
Single Culture ◽  
Inoculum Dose ◽  
Wide Range

The production and stability of two novel myco-flocculants produced by river water fungus (RWF) were investigated. Screening tests were conducted to find suitable nutrients, pH, nutrient concentration, inoculum dose, and stability for two myco-flocculants L. squarrosulus (RWF5) and S. obclavatum (RWF6). The strains showed good flocculating activity in reducing turbidity of kaolin suspension while malt extract was used as nutrient source. Supernatants of RWF5 and RWF6 were able to reduce turbidity from 900±10 NTU to 46 NTU (95%) and 195 NTU (78%), respectively. In order to enhance the production, optimization of cultivation conditions were studied using a one-factor-at-a-time (OFAT) method. L. squarrosulus (RWF5) reduced 96% of turbidity at optimum conditions, comprising of 0.1% (w/v) malt extract, 3% (v/v) inoculum dose, and initial pH 7.0 for 6 days. The results of the compatible mixed culture showed good flocculation activity at 88% compared to a single culture of S. obclavatum at 78%. On the other hand, L. squarrosulus showed better turbidity reduction in the single culture rather than the mixed culture. The stability of L. squarrosulus and S. obclavatum supernatants showed excellent turbidity reduction over a wide pH range of 4-8 with the maximal flocculation rate of 96% and 90%, respectively, at pH 7.0. They also exhibited high turbidity removal ability in a temperature range of 4 oC – 55 oC for 24h with a maximum turbidity removal rate of 96% (RW5) and 87% (RW6) at 25 oC. Time stability of the L. squarrosulus supernatant showed good turbidity removal potential at above 90% at room temperature (28± 2 oC) and 85% at low temperature (4 oC) for 12 days. The high flocculating rate of the myco-flocculants and their good stability under wide range of temperature indicated their potentiality as biodegradable flocculants for water and wastewater treatment industry. ABSTRAK: Keberhasilan dan kestabilan dua myco-gumpalan baharu oleh kulat air sungai (RWF) telah dikaji. Ujian penapisan telah dijalankan untuk mencari nutrien sesuai, pH, kepekatan nutrien, dos inokulum dan kestabilan dua myco-gumpalan L. squarrosulus (RWF5) dan S. obclavatum (RWF6). Rantaian menunjukkan aktiviti gumpalan yang baik dalam mengurangkan kekeruhan air sungai dengan penggantungan koalin di mana ekstrak malt telah digunakan sebagai sumber nutrien. Larutan supernatan RWF5 dan RWF6, masing-masing mampu mengurangkan kekeruhan dari 900±10 NTU kepada 46 NTU (95%) dan 195 NTU (78%). Bagi meningkatkan pengeluaran, keadaan optimum bagi menggalakkan hasil telah diselidiki menggunakan kaedah Satu Faktor pada Tiap Masa (OFAT). Pada takat optimum, L. squarrosulus (RWF5) mengurangkan 96% kekeruhan, ianya terdiri daripada ekstrak malt 0.1% (w/v), dos inokulum 3% (v/v) dan pH awal 7.0 selama 6 hari. Keputusan kultur campuran yang sesuai menunjukkan aktiviti penggumpalan yang baik pada 88% berbanding kultur sendirian S. obclavatum pada 78%. Pada waktu sama, L. squarrosulus menunjukkan pengurangan kekeruhan yang lebih baik dalam kultur sendirian berbanding kultur campuran. Kestabilan larutan supernatan L. squarrosulus dan S. obclavatum menunjukkan pengurangan kekeruhan yang sangat baik pada pH yang luas iaitu 4-8 dengan kadar maksimum kekeruhan pada 96% dan 90%, pada pH 7.0 masing-masing. Keduanya menunjukkan kebolehan penyingkiran kekeruhan yang tinggi pada skala suhu 4 oC – 55 oC selama 24 jam dengan kadar nyah kekeruhan maksimum pada 96% (RW5) dan 87% (RW6) pada suhu 25 oC. Kestabilan masa larutan supernatan L. Squarrosulus menunjukkan potensi penyingkiran kekeruhan terbaik atas 90% pada suhu bilik (28± 2 oC) dan 85% pada suhu rendah (4 oC) selama 12 hari. Kadar gumpalan yang tinggi oleh myro-gumpalan dan kestabilan yang baik pada julat suhu yang luas menunjukkan potensinya sebagai agen biodegradasi gumpalan kepada air dan industri rawatan loji air.

scholarly journals Selection of Resistant Streptococcus pneumoniae during Penicillin Treatment In Vitro and in Three Animal Models

2003 ◽  
Vol 47 (8) ◽  
pp. 2499-2506 ◽  
Author(s):  
Jenny Dahl Knudsen ◽  
Inga Odenholt ◽  
Helga Erlendsdottir ◽  
Magnus Gottfredsson ◽  
Otto Cars ◽  
...  
Keyword(s):  
Animal Models ◽  
Mixed Culture ◽  
Observation Time ◽  
Treatment Regimens ◽  
Rabbit Tissue ◽  
Drug Concentrations ◽  
Wide Range ◽  
Different Strains ◽  
Selection Of

ABSTRACT Pharmacokinetic (PK) and pharmacodynamic (PD) properties for the selection of resistant pneumococci were studied by using three strains of the same serotype (6B) for mixed-culture infection in time-kill experiments in vitro and in three different animal models, the mouse peritonitis, the mouse thigh, and the rabbit tissue cage models. Treatment regimens with penicillin were designed to give a wide range of T>MICs, the amounts of time for which the drug concentrations in serum were above the MIC. The mixed culture of the three pneumococcal strains, 107 CFU of strain A (MIC of penicillin, 0.016 μg/ml; erythromycin resistant)/ml, 106 CFU of strain B (MIC of penicillin, 0.25 μg/ml)/ml, and 105 CFU of strain C (MIC of penicillin, 4 μg/ml)/ml, was used in the two mouse models, and a mixture of 105 CFU of strain A/ml, 104 CFU of strain B/ml, and 103 CFU of strain C/ml was used in the rabbit tissue cage model. During the different treatment regimens, the differences in numbers of CFU between treated and control animals were calculated to measure the efficacies of the regimens. Selective media with erythromycin or different penicillin concentrations were used to quantify the strains separately. The efficacies of penicillin in vitro were similar when individual strains or mixed cultures were studied. The eradication of the bacteria, independent of the susceptibility of the strain or strains or the presence of the strains in a mixture or on their own, followed the well-known PK and PD rules for treatment with β-lactams: a maximum efficacy was seen when the T>MIC was >40 to 50% of the observation time and the ratio of the maximum concentration of the drug in serum to the MIC was >10. It was possible in all three models to select for the less-susceptible strains by using insufficient treatments. In the rabbit tissue cage model, a regrowth of pneumococci was observed; in the mouse thigh model, the ratio between the different strains changed in favor of the less-susceptible strains; and in the mouse peritonitis model, the susceptible strain disappeared and was overgrown by the less-susceptible strains. These findings with the experimental infection models confirm the importance of eradicating all the bacteria taking part in the infectious process in order to avoid selection of resistant clones.

Simulation and Application of Stock Composition Estimators

1983 ◽  
Vol 40 (12) ◽  
pp. 2113-2118 ◽  
Author(s):  
Rodney C. Cook
Keyword(s):  
Confidence Intervals ◽  
Classification Scheme ◽  
Mixed Population ◽  
Stock Identification ◽  
Dramatic Reduction ◽  
Simulation Studies ◽  
Practical Application ◽  
Wide Range ◽  
Mixed Populations

Stock composition estimates for mixed populations may be obtained with a classification scheme and the results of classifying individuals from this mixed population and from the component subpopulations. Several estimators have been proposed. Simulation studies were conducted over a wide range of practical possibilities. These studies showed that the simplest estimator, together with a constraint, was more than adequate for practical application. Implementation of the constraint provides for a dramatic reduction in effort for scientists involved in stock identification work. Statistically valid, conservative confidence intervals were obtained with existing variance formulae.

scholarly journals Population systems of Eurasian water frogs (Pelophylax) in the south of Ukraine

2020 ◽  
Vol 28 (2) ◽  
pp. 154-162
Author(s):  
N. M. Suriadna ◽  
G. I. Mykytynets ◽  
M. Pupiņš ◽  
V. Y. Gasso
Keyword(s):  
Taxonomic Diversity ◽  
Mixed Population ◽  
Large Rivers ◽  
Mixed System ◽  
The South ◽  
Isolated Populations ◽  
Sex Structure ◽  
Water Frogs ◽  
Wide Range ◽  
Freshwater Bodies

Ecological and evolutionary consequences of population-genetic processes that occur because of natural cross-species hybridization can show mechanisms of overcoming the reproductive barrier and obtaining the species status by a hybrid taxon. This is clearly seen in the population systems of Eurasian water frogs – Pelophylax esculentus complex. The P. esculentus (E) hybrid usually discards one of the parental genomes of P. lessonae (L) or P. ridibundus (R) and reproduces semi-clonally. The genetic structure and direction of gene flows precisely depend on the type and distribution of mixed or pure population systems of water frogs. Three population systems in the south of Ukraine were identified and confirmed as RR, RE and REL. The populations of P. ridibundus are most common (76.2%). A mixed population systems of P. ridibundus and P. esculentus (20.0%) are concentrated in the floodplains of large rivers where triploids were found and the unisexual hybrids (1.0♂ : 0.1♀) were proved. Parent species populations having different ploidy of P. esculentus such as 3n and for the first time 4n were found. A mixed system of three taxa (REL) is rare (3.8%) and locally concentrated in the lower Danube and Dnieper with the smallest proportion of P. lessonae. We did not find populations of P. lessonae (LL), P. esculentus (EE, very rare system of hybrids only), and two mixed populations of parental species RL and semi-clonal LE in the south of Ukraine, but they are known for northern areas. The high number of P. ridibundus tends to decrease; the scarce P. esculentus and the extremely rare P. lessonae require special conservation measures. P. ridibundus (RR) occupies a wide range of diverse natural, permanent, temporary, coastal, continental, and artificial freshwater bodies, including synanthropic ecosystems. Mixed population systems inhabit willow and poplar forests in the floodplains of large rivers. In the south of Ukraine rare and isolated populations of the water frogs occurring outside the main range can be relict. Biotopic preferences, ratio and number of constituent taxa are crucial for an adequate assessment of biological (taxonomic) diversity and development of an appropriate strategy for the population systems’ conservation. Such characteristics as unisexuality of hybrids, their spreading patterns, specific sex structure and ploidy in different population systems of the P. esculentus complex contribute to the understanding of the hybridogenetic dynamics; produce new tendencies of becoming independent hybridogenous taxa and emergence of new evolutionary relationships.

scholarly journals Mutant Variants of the Substrate-Binding Protein DppA fromEscherichia coliEnhance Growth on Nonstandard γ-Glutamyl Amide-Containing Peptides

2018 ◽  
Vol 84 (13) ◽  
Author(s):  
Tilmann Kuenzl ◽  
Xiaochun Li-Blatter ◽  
Puneet Srivastava ◽  
Piet Herdewijn ◽  
Timothy Sharpe ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Protein ◽  
Substrate Binding ◽  
Building Blocks ◽  
Side Chains ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Wide Range ◽  
Amino Acid Side Chains ◽  
Substrate Binding Protein

ABSTRACTThe import of nonnatural molecules is a recurring problem in fundamental and applied aspects of microbiology. The dipeptide permease (Dpp) ofEscherichia coliis an ABC-type multicomponent transporter system located in the cytoplasmic membrane, which is capable of transporting a wide range of di- and tripeptides with structurally and chemically diverse amino acid side chains into the cell. Given this low degree of specificity, Dpp was previously used as an entry gate to deliver natural and nonnatural cargo molecules into the cell by attaching them to amino acid side chains of peptides, in particular, the γ-carboxyl group of glutamate residues. However, the binding affinity of the substrate-binding protein dipeptide permease A (DppA), which is responsible for the initial binding of peptides in the periplasmic space, is significantly higher for peptides consisting of standard amino acids than for peptides containing side-chain modifications. Here, we used adaptive laboratory evolution to identify strains that utilize dipeptides containing γ-substituted glutamate residues more efficiently and linked this phenotype to different mutations in DppA.In vitrocharacterization of these mutants by thermal denaturation midpoint shift assays and isothermal titration calorimetry revealed significantly higher binding affinities of these variants toward peptides containing γ-glutamyl amides, presumably resulting in improved uptake and therefore faster growth in media supplemented with these nonstandard peptides.IMPORTANCEFundamental and synthetic biology frequently suffer from insufficient delivery of unnatural building blocks or substrates for metabolic pathways into bacterial cells. The use of peptide-based transport vectors represents an established strategy to enable the uptake of such molecules as a cargo. We expand the scope of peptide-based uptake and characterize in detail the obtained DppA mutant variants. Furthermore, we highlight the potential of adaptive laboratory evolution to identify beneficial insertion mutations that are unlikely to be identified with existing directed evolution strategies.

Liquid Chromatographic Determination of Sugars in Licorice Extracts: Collaborative Study

1984 ◽  
Vol 67 (4) ◽  
pp. 764-767
Author(s):  
Pramathesh S Vora ◽  
Raymond M Tuorto ◽  
◽  
F Atchley ◽  
W Conway ◽  
...  
Keyword(s):  
Liquid Chromatography ◽  
Collaborative Study ◽  
Chromatographic Determination ◽  
Sugar Concentration ◽  
Wide Range ◽  
Liquid Chromatographic Determination ◽  
The Individual ◽  
Liquid Chromatographic

Abstract A collaborative study to determine fructose, glucose, sucrose, and maltose in a variety of licorice extracts has been performed using liquid chromatography. Six collaborators participated in the study, and each collaborator was sent 5 samples which were analyzed in blind replicates. A practice sample was used with a specific range of 8-10% fructose, 10-12% glucose, and 5-7% sucrose. The individual sugar concentration of the 5 samples ranged from 2.0 to 10%. AH samples were quantitated by comparing results with a pure sugar standard containing fructose, glucose, sucrose, and maltose and analyzed under the same conditions. No significant problems were encountered running the practice or other samples despite the wide range of instruments and columns used. Results showed less deviation within laboratories compared with results obtained among laboratories. The method has been adopted official first action.

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