Psychrophilic methane fermentation of excess sludge by enrichment culture

This study was carried out to determine the possibility of the psychrophilic methane fermentation of the excess sludge produced from fish-processing wastewater treatment. Three different kinds of seed sludge, named M seed sludge, K seed sludge and KL seed sludge were used for batch methane fermentation in Erlenmeyer flasks. M seed sludge was mesophilic, while the other two were prepared in our laboratory by soil collection to obtain a psychrophilic biological community. The effects of temperature on the total volume of biogas generated and on the rate of biogas generation were examined. The activity of K seen sludge with respect to gas yield at 15°C was found to be 1.6-fold higher than that of M seed sludge. Enrichment techniques were then used to accumulate psychrophilic bacteria in KL seed sludge, and the activity at 15°C increased by about two times due to these techniques. Comparison of the three kinds of seed sludge indicates that methane fermentation at low temperature may be feasible, and therefore further study to isolate the psychrophilic bacteria in anaerobic digestion is needed.     

Effect of temperature on the performance of an anaerobic tubular reactor treating fruit and vegetable waste

This study compares the performance of anaerobic digestion of fruit and vegetable waste (FVW) in the thermophilic (55 °C) process with those under psychrophilic (20 °C) and mesophilic (35 °C) conditions in a tubular anaerobic digesters on a laboratory scale. The hydraulic retention time (HRT) ranged from 10 to 20 days, and raw fruit and vegetable waste was supplied in a semi-continuous mode at various concentrations of total solids (TS) (4, 6, 8 and 10% on dry weight). Biogas production from the experimental thermophilic digester was higher on average than from psychrophilic and mesophilic digesters by 144 and 41%, respectively. The net energy production in the thermophilic digester was 195.7 and 49.07 kJ per day higher than that for the psychrophilic and mesophilic digesters, respectively. The relation between the daily production of biogas and the temperature indicates that for the same produced quantity of biogas, the size of the thermophilic digester can be reduced with regard to that of the psychrophilic and the mesophilic digesters.     

An effective cold inducible expression system developed in Pseudoalteromonas haloplanktis TAC125

A regulative two-component system previously identified in Pseudoalteromonas haloplanktis TAC125 was used to construct a cold inducible expression system that is under the control of l-malate. Performances of the inducible system were tested for both psychrophilic and mesophilic protein production using two "difficult" proteins as control. The results obtained demonstrated that both psychrophilic beta-galactosidase and yeast alpha-glucosidase are produced in a fully soluble and catalytically competent form. Optimal conditions for protein production, including growth temperature, growth medium and l-malate concentration were also investigated. Under optimized conditions yields of 620 and 27 mg/l were obtained for beta-galactosidase and alpha-glucosidase, respectively.     

RUBISCO ADAPTATION TO LOW TEMPERATURES: A COMPARATIVE STUDY IN PSYCHROPHILIC AND MESOPHILIC UNICELLULAR ALGAE

Some properties of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) from two psychrophilic Chloromonas species have been investigated in relation to their adaptation to cold environments. Contrary to the situation usually encountered with psychrophilic enzymes, the carboxylase activity of both purified "cold" RUBISCO enzymes was lower at low temperatures than that found with the enzyme of the mesophilic alga Chlamydomonas reinhardtii Dangeard. Moreover, the apparent optimal temperature for RUBISCO carboxylase activity was similar for psychrophilic and mesophilic enzymes. Psychrophilic RUBISCOs, however, showed a greater thermosensitivity than the C. reinhardtii enzyme. Genes encoding small and large subunits of RUBISCO from one psychrophilic isolate were sequenced. Comparison of the deduced amino acid sequences to those of higher plants and green algae revealed the substitution of a very highly conserved residue (cysteine₂₄₇ → serine in the large subunit) that could be responsible, at least in part, for the increased thermosensitivity of the "cold" enzyme. Interestingly, the relative amount of RUBISCO subunits found in the psychrophilic isolates was about twice as high as the amount observed in C. reinhardtii and five other mesophilic algae. The high production of a key enzyme to counterbalance its poor catalytic efficiency at low temperature could constitute a novel type of adaptive mechanism to cold environments.     

Stepwise adaptations to low temperature as revealed by multiple mutants of psychrophilic α-amylase from Antarctic Bacterium

The mutants Mut5 and Mut5CC from a psychrophilic α-amylase bear representative stabilizing interactions found in the heat-stable porcine pancreatic α-amylase but lacking in the cold-active enzyme from an Antarctic bacterium. From an evolutionary perspective, these mutants can be regarded as structural intermediates between the psychrophilic and the mesophilic enzymes. We found that these engineered interactions improve all the investigated parameters related to protein stability as follows: compactness; kinetically driven stability; thermodynamic stability; resistance toward chemical denaturation, and the kinetics of unfolding/refolding. Concomitantly to this improved stability, both mutants have lost the kinetic optimization to low temperature activity displayed by the parent psychrophilic enzyme. These results provide strong experimental support to the hypothesis assuming that the disappearance of stabilizing interactions in psychrophilic enzymes increases the amplitude of concerted motions required by catalysis and the dynamics of active site residues at low temperature, leading to a higher activity.     

A low temperature microbial biosensor using immobilised psychrophilic bacteria

A psychrophilic bacteria, Deinococcus radiodurans, was used to construct a biosensor to be used in a flow injection system. The transducer used was an O₂ electrode. The response of this cell-based electrode was studied towards a number of sugars. The temperature dependence of the electrode response correlated well with the behavior of the cells. Thus, the optimum temperature for measurement of glucose (0.55 mM) was about +5°C. Since the organism used is psychrophilic, a response time at this low temperature is similar to what is achieved with mesophilic organisms at room temperature. This is the first biosensor constructed using a psychrophilic microorganism.     

A meta-analysis of the activity,stability, and mutational characteristics of temperature-adapted enzymes

Understanding the characteristics that define temperature-adapted enzymes has been a major goal of extremophile enzymology in recent decades. In the present study, we explore these characteristics by comparing psychrophilic, mesophilic, and thermophilic enzymes. Through a meta-analysis of existing data, we show that psychrophilic enzymes exhibit a significantly larger gap (T_g) between their optimum and melting temperatures compared with mesophilic and thermophilic enzymes. These results suggest that T_g may be a useful indicator as to whether an enzyme is psychrophilic or not and that models of psychrophilic enzyme catalysis need to account for this gap. Additionally, by using predictive protein stability software, HoTMuSiC and PoPMuSiC, we show that the deleterious nature of amino acid substitutions to protein stability increases from psychrophiles to thermophiles. How this ultimately affects the mutational tolerance and evolutionary rate of temperature adapted organisms is currently unknown.     

Comparative void-volume analysis of psychrophilic and mesophilic enzymes: Structural bioinformatics of psychrophilic enzymes reveals sources of core flexibility

Background

Psychrophiles, cold-adapted organisms, have adapted to live at low temperatures by using a variety of mechanisms. Their enzymes are active at cold temperatures by being structurally more flexible than mesophilic enzymes. Even though, there are some indications of the possible structural mechanisms by which psychrophilic enzymes are catalytic active at cold temperatures, there is not a generalized structural property common to all psychrophilic enzymes.

Results

We examine twenty homologous enzyme pairs from psychrophiles and mesophiles to investigate flexibility as a key characteristic for cold adaptation. B-factors in protein X-ray structures are one way to measure flexibility. Comparing psychrophilic to mesophilic protein B-factors reveals that psychrophilic enzymes are more flexible in 5-turn and strand secondary structures. Enzyme cavities, identified using CASTp at various probe sizes, indicate that psychrophilic enzymes have larger average cavity sizes at probe radii of 1.4-1.5 Å, sufficient for water molecules. Furthermore, amino acid side chains lining these cavities show an increased frequency of acidic groups in psychrophilic enzymes.

Conclusions

These findings suggest that embedded water molecules may play a significant role in cavity flexibility, and therefore, overall protein flexibility. Thus, our results point to the important role enzyme flexibility plays in adaptation to cold environments.

     

On the potential of mass spectrometry-based metabolite profiling approaches to the study of biochemical adaptation in psychrophilic yeast

To move beyond targeted approaches to the biochemical characterization of psychrophilic yeast and provide a more holistic understanding of the chemistry of physiological adaptation of psychrophiles at the molecular level, ultraperformance liquid chromatography combined with simultaneous acquisition of low- and high-collision energy mass spectra (UPLC/MS^e) was employed for a preliminary comparative analysis of cell extracts of psychrophilic Antarctic yeasts Cryptococcus vishniacii CBS 10616 and Dioszegia cryoxerica CBS 10919 versus the mesophile Saccharomyces cerevisiae ‘cry havoc’. A detailed workflow for providing high-confidence preliminary identifications of psychrophilic yeast-specific molecular features is presented. Preliminary identifications of psychrophile-specific features in C. vishniacii and D. cryoxerica determined with the described method include the glycerophospholipids lysophosphatidylcholine 18:2, lysophosphatidylcholine 18:3, lysophosphatidylethanolamine 18:3, and lysophosphatidylethanolamine 18:2. In addition, levels of guanosine diphosphate appear significantly elevated in cell extracts of the psychrophilic yeasts as compared to Saccharomyces cerevisiae. Finally, five psychrophilic yeast-specific peptides have been discovered. All of these are demonstrated to be glycine- and/or proline-rich, a known structural characteristic of many naturally occurring bioactive peptides. The potential of this untargeted metabolite profiling approach as a tool for knowledge discovery and hypothesis generation in the study of biodiversity and microbial adaptation is highlighted.     

Structural adaptation to low temperatures--analysis of the subunit interface of oligomeric psychrophilic enzymes

Enzymes from psychrophiles show higher catalytic efficiency in the 0-20 degrees C temperature range and often lower thermostability in comparison with meso/thermophilic homologs. Physical and chemical characterization of these enzymes is currently underway in order to understand the molecular basis of cold adaptation. Psychrophilic enzymes are often characterized by higher flexibility, which allows for better interaction with substrates, and by a lower activation energy requirement in comparison with meso/thermophilic counterparts. In their tertiary structure, psychrophilic enzymes present fewer stabilizing interactions, longer and more hydrophilic loops, higher glycine content, and lower proline and arginine content. In this study, a comparative analysis of the structural characteristics of the interfaces between oligomeric psychrophilic enzyme subunits was carried out. Crystallographic structures of oligomeric psychrophilic enzymes, and their meso/thermophilic homologs belonging to five different protein families, were retrieved from the Protein Data Bank. The following structural parameters were calculated: overall and core interface area, characterization of polar/apolar contributions to the interface, hydrophobic contact area, quantity of ion pairs and hydrogen bonds between monomers, internal area and total volume of non-solvent-exposed cavities at the interface, and average packing of interface residues. These properties were compared to those of meso/thermophilic enzymes. The results were analyzed using Student's t-test. The most significant differences between psychrophilic and mesophilic proteins were found in the number of ion pairs and hydrogen bonds, and in the apolarity of their subunit interface. Interestingly, the number of ion pairs at the interface shows an opposite adaptation to those occurring at the monomer core and surface.     

Simulation of low temperature anaerobic digestion of dairy and swine manure

The data reported by L.M. Safley, P.W. Westerman [Bioresource Technology 47 (2) (1994) 165-171] from the laboratory digestion of dairy and swine manure at psychrophilic temperatures (i.e., 10-23 degrees C) have been used to determine the response of the latest comprehensive dynamic mathematical model of methanogenesis [D.T. Hill, S.A. Cobb, Transactions of the ASAE 39 (2) (1996) 565-573] in this low temperature range. Extensive performance data from digesters using animal waste in this temperature range have been lacking, thus allowing limited validation of the comprehensive model. The results of the comprehensive model simulations were compared with the actual data reported by Safley and Westerman (loc. cit.) and with their empirical regression models. Results indicate that the comprehensive model is as accurate as Safley and Westerman's models for three of the four cases reported, but shows a great dissimilarity for the fourth case.     

Electricity generation in single-chamber microbial fuel cells using a carbon source sampled from anaerobic reactors utilizing grass silage

Production of electricity from samples obtained during anaerobic digestion of grass silage was examined using single-chamber air-cathode mediator-less microbial fuel cells (MFCs). The samples were obtained from anaerobic reactors at start-up conditions after 3 and 10 days of operation under psychrophilic (15 °C) and mesophilic (37 °C) temperatures. Electricity was directly produced from all samples at a concentration of 1500 mg COD L⁻¹. Power density obtained from the samples, as a sole carbon source, ranged from 56 ± 3 W m⁻³ to 31 ± 1 W m⁻³ for the mesophilic and psychrophilic samples, respectively. Coulombic efficiencies ranged from 18 ± 1% to 12 ± 1% for the same samples. The relationship between the maximum voltage output and initial COD concentration appeared to follow saturation kinetics at the external resistance of 217 Ω. Chemical oxygen demand (COD) removal was over 90% and total phenolics removal was in the range of 30-75% for all samples tested, with a standard amount of 60 mg L⁻¹ total phenolics removed for every sample. Our results indicate that generating electricity from solution samples of anaerobic reactors utilizing grass silage is possible, opening the possibility for combination of anaerobic digestion with MFC technology for energy generation.     

Kinetics and energetics of ligand binding determined by microcalorimetry: insights into active site mobility in a psychrophilic alpha-amylase

A new microcalorimetric method for recording the kinetic parameters k(cat), K(m) and K(i) of alpha-amylases using polysaccharides and oligosaccharides as substrates is described. This method is based on the heat released by glycosidic bond hydrolysis. The method has been developed to study the active site properties of the cold-active alpha-amylase produced by an Antarctic psychrophilic bacterium in comparison with its closest structural homolog from pig pancreas. It is shown that the psychrophilic alpha-amylase is more active on large macromolecular substrates and that the higher rate constants k(cat) are gained at the expense of a lower affinity for the substrate. The active site is able to accommodate larger inhibitory complexes, resulting in a mixed-type inhibition of starch hydrolysis by maltose. A method for recording the binding enthalpies by isothermal titration calorimetry in a low-affinity system has been developed, allowing analysis of the energetics of weak ligand binding using the allosteric activator chloride. It is shown that the low affinity of the psychrophilic alpha-amylase for chloride is entropically driven. The high enthalpic and entropic contributions of activator binding suggest large structural fluctuations between the free and the bound states of the cold-active enzyme. The kinetic and thermodynamic data for the psychrophilic alpha-amylase indicate that the strictly conserved side-chains involved in substrate binding and catalysis possess an improved mobility, responsible for activity in the cold, and resulting from the disappearance of stabilizing interactions far from the active site.     

Xerotolerant mycobiota from high altitude Anapurna soil, Nepal

Xerophilic and xerotolerant microfungi were isolated from soil samples collected in Anapurna Mountains, Nepal, at altitudes from 3000 to 5400 m. The total numbers and proportions of xerotolerant and psychrophilic strains in comparison with mesophilic mycobiota were determined by using different enumeration, selective media and four isolation methods. The most extreme xerophilic fungi were taxonomically identified as belonging to the genera Eurotium and Aspergillus. The low water activity of the soil due to dry climate and frequent binding of water in ice crystals favors a high proportion of xerotolerant fungal species. The correlation between xerotolerant and psychrophilic fungi was observed.     

Improvement in thermostability and psychrophilicity of psychrophilic alanine racemase by site-directed mutagenesis

A psychrophilic alanine racemase from Bacillus psychrosaccharolyticus has a higher catalytic activity than a thermophilic alanine racemase from Bacillus stearothermophilus even at 60 °C in the presence of pyridoxal 5′-phosphate (PLP), although the thermostability of the former enzyme is lower than that of the latter one [FEMS Microbial. Lett. 192 (2000) 169]. In order to improve the thermostability of the psychrophilic enzyme, two hydrophilic amino acid residues (Glu150 and Arg151) at a surface loop surrounding the active site of the enzyme were substituted with the corresponding residues (Val and Ala) in the B. stearothermophilus alanine racemase. The mutant enzyme (ER150,151VA) showed a higher thermostability, and a markedly lower K_m value for PLP, than the wild type one. In addition, the catalytic activities at low temperatures and kinetic parameters of the two enzymes indicated that the mutant enzyme was more psychrophilic than the wild type one. Thus, the psychrophilic alanine racemase was improved in both psychrophilicity and thermostability by the site-directed mutagenesis. The mutant enzyme may be useful for the production of stereospecifically deuterated NADH and various -amino acids.     

Growth, lipid accumulation, and fatty acid composition in obligate psychrophilic, facultative psychrophilic, and mesophilic yeasts

Obligate psychrophilic, facultative psychrophilic, and mesophilic yeasts were cultured in a carbon-rich medium at different temperatures to investigate whether growth parameters, lipid accumulation, and fatty acid (FA) composition were adaptive and/or acclimatory responses. Acclimation of facultative psychrophiles and mesophiles to a lower temperature decreased their specific growth rate, but did not affect their biomass yield (Y_X/S). Obligate and facultative psychrophiles exhibited the highest Y_X/S. Acclimation to lower temperature decreased the lipid yield (Y_L/X) in mesophilic yeasts, but did not affect Y_L/X in facultative psychrophilic ones. Similar Y_L/X were found in both groups of psychrophiles, suggesting that lipid accumulation is not a distinctive characteristic of adaptation to permanently cold environments. The unsaturation of FAs was one major adaptive feature of the yeasts colonizing permanently cold ecosystems. Remarkable amounts of α-linolenic acid were found in obligate psychrophiles at the expense of linoleic acid, whereas it was scarce or absent in all the other strains. Increased unsaturation of FAs was also a general acclimatory response of facultative psychrophiles to a lower temperature. These results improve the knowledge of the responses enabling psychrophilic yeasts to cope with the cold and may be of support for potential biotechnological exploitation of these strains.     

Antibiotic Complex SP-351 Produced by a Psychrophile,Streptomyces sp. No. 351

In the course of a screening study for antibiotics using psychrophilic microorganisms a water-insoluble antibiotic complex, SP–351, was found in the culture filtrate of a psychrophilic actinomycete, strain No. 351. This active principle was isolated and characterized as a cyclicpolylactone antibiotic. The SP–351-producing strain was classified as a facultative psychrophile and identified as Streptomyces phaeochromogenes.

The main components of the antibiotic complex SP–351 changed with the composition of the culture medium but not with culture temperatures. Component A was exclusively produced in a medium composed of roasted soybean powder and glycerol; components B and C in a medium composed of soybean, glycerol and potassium nitrate; and components A and D in a synthetic medium containing a hydrocarbon, alcohol or ester as the sole carbon source. Maximum production of SP–351 from n-paraffin and methyl acetate was 10 and 15 mcg/ml, respectively.

SP–351 showed strong antibacterial activity against Gram-positive bacteria and acid-fast bacteria at 0.1~0.3 mcg/ml concentrations.     

Psychrophilic yeasts from worldwide glacial habitats: diversity, adaptation strategies and biotechnological potential

Glacial habitats (cryosphere) include some of the largest unexplored and extreme biospheres on Earth. These habitats harbor a wide diversity of psychrophilic prokaryotic and eukaryotic microorganisms. These highly specialized microorganisms have developed adaptation strategies to overcome the direct and indirect life-endangering influence of low temperatures. For many years Antarctica has been the geographic area preferred by microbiologists for studying the diversity of psychrophilic microorganisms (including yeasts). However, there have been an increasing number of studies on psychrophilic yeasts sharing the non-Antarctic cryosphere. The present paper provides an overview of the distribution and adaptation strategies of psychrophilic yeasts worldwide. Attention is also focused on their biotechnological potential, especially on their exploitation as a source of cold-active enzymes and for bioremediation purposes.     

Manipulations of AMP metabolic genes increase growth rate and cold tolerance in Escherichia coli: implications for psychrophilic evolution

Diverse organisms have adapted to thrive at low temperatures (i.e., <20 °C, termed psychrophiles), colonizing the majority of earth's biosphere. In contrast with mesophiles (20-40 °C thermal range), all observed psychrophiles increase intracellular adenosine 5'-triphosphate concentrations as temperatures decline; this phenomenon has been described as an important compensatory mechanism to deal with decreases in thermal energy and molecular motion. We considered purine metabolic pathways in class gammaproteobacteria (n = 115) to investigate metabolic and evolutionary bases of this process. A survey of the KEGG database indicated that psychrophilic purine metabolic pathways tend to be enriched with de novo adenosine 5'-monophosphate (AMP) synthetic enzymes, whereas mesophiles tend to be enriched with AMP degradative enzymes. Function of the observed psychrophilic pathway structure was tested by engineering the mesophilic gammaproteobacterium Escherichia coli to reflect psychrophilic purine metabolism, specifically by expressing adenylosuccinate synthetase (purA) from the psychrophilic gammaproteobacterium, Psychrobacter cryohalolentis, in an AMP nucleosidase (amn)-deficient background. Modified E. coli was capable of growing up to ～70% faster at low temperatures and became up to ～10-fold more cold tolerant relative to wild type. These findings highlight potentially important transitional steps in psychrophilic evolution.     

Paradoxical thermostable enzymes from psychrophile: molecular characterization and potentiality for biotechnological application

NAD(P)⁺-dependent aldehyde dehydrogenase (EC 1.2.1.5) and aspartase (EC 4.3.1.1) in the cells of an atypical psychrophile from Antarctic seawater, Cytophaga sp. KUC-1, were paradoxically thermostable, although they derived from a psychrophile. Both enzymes showed the highest activity at about 55 °C, and also active even under cold conditions. The enzymes contained more Ile residues than the enzymes from mesophiles. The Ile/Ile + Val + Leu ratio of the Cytophaga thermostable enzymes was much higher than that of the enzymes from mesophiles. As compared with the enzymes from other microorganisms, the Cytophaga thermostable enzymes have the structural differences in the C-terminal region of the enzymes. Therefore, the C-terminal region might be important for the paradoxical thermostability of the enzymes. The psychrophilic microorganism produces not only psychrophilic enzyme, but thermostable enzyme with psychrophilicity. Therefore, the psychrophilic microorganism is one of the candidates for isolation of novel biocatalysts, which have potential for various industrial applications.