Extreme Culture Conditions Research Articles

Effect of Cultivation Parameters on Fermentation and Hydrogen Production in the Phylum Thermotogae.

The phylum Thermotogae is composed of a single class (Thermotogae), 4 orders (Thermotogales, Kosmotogales, Petrotogales, Mesoaciditogales), 5 families (Thermatogaceae, Fervidobacteriaceae, Kosmotogaceae, Petrotogaceae, Mesoaciditogaceae), and 13 genera. They have been isolated from extremely hot environments whose characteristics are reflected in the metabolic and phenotypic properties of the Thermotogae species. The metabolic versatility of Thermotogae members leads to a pool of high value-added products with application potentials in many industry fields. The low risk of contamination associated with their extreme culture conditions has made most species of the phylum attractive candidates in biotechnological processes. Almost all members of the phylum, especially those in the order Thermotogales, can produce bio-hydrogen from a variety of simple and complex sugars with yields close to the theoretical Thauer limit of 4 mol H2/mol consumed glucose. Acetate, lactate, and L-alanine are the major organic end products. Thermotagae fermentation processes are influenced by various factors, such as hydrogen partial pressure, agitation, gas sparging, culture/headspace ratio, inoculum, pH, temperature, nitrogen sources, sulfur sources, inorganic compounds, metal ions, etc. Optimization of these parameters will help to fully unleash the biotechnological potentials of Thermotogae and promote their applications in industry. This article gives an overview of how these operational parameters could impact Thermotogae fermentation in terms of sugar consumption, hydrogen yields, and organic acids production.

Effect of Mild Salinity Stress on the Growth, Fatty Acid and Carotenoid Compositions, and Biological Activities of the Thermal Freshwater Microalgae Scenedesmus sp.

Carotenoids have strong antioxidant activity as well as therapeutic value. Their production has been induced in algae under stressful culture conditions. However, the extreme culture conditions lead to the Programmed Cell Death (PCD) of algae, which affects their growth and productivity. This study was performed to evaluate the effect of salinity on the physiological and biochemical traits of Scenedesmus sp., thermal freshwater microalgae from Northern Tunisia. It was cultured under different NaCl concentrations ranging from 0 to 60 g/L. Results showed a good growth and high contents of total chlorophyll and carotenoids in Scenedesmus sp. cultured at 10 g/L of NaCl (salt-stressed 10 (Ss10)). The pigment composition of the Ss10 extract was acquired using HPLC–MS, and showed that the carotenoid fraction is particularly rich in xanthophylls. Moreover, the antioxidant (DPPH and FRAP) and enzymatic inhibition (tyrosinase and elastase) activities of the Ss10 extract were higher compared to those of the control culture. In addition, the cytotoxicity test on B16 cells showed that the Ss10 extract was non-toxic for all tested concentrations below 100 µg/mL. It also showed a rich unsaturated fatty acid (FA) composition. Therefore, these findings suggest that Scenedesmus sp. strain cultivated under mild stress salinity could be a source of biomolecules that have potential applications in the nutraceutical and cosmeceutical industries.

Algal wastewater treatment: Photoautotrophic vs. mixotrophic processes

Photoautotrophic algal systems have been investigated as potentially greener and more sustainable alternatives to the traditional bacteria-based wastewater treatment (WWT) systems. This paper presents mixotrophic algal systems as better suited for WWT. Since the literature is void of mixotrophic WWT systems, a brief review of the laboratory results from the literature comparing the different types of algal metabolisms is presented first. Details of a mixotrophic WWT system driven by an extremophilic alga, Galdieria sulphuraria (pH = 1 to 4; temperature = 25 to 56 °C) are presented next. Results from pilot scale version of this mixotrophic system (700 L) are summarized to show that it can reduce biochemical oxygen demand (BOD) and nutrients in primary-settled wastewater in a single step to yield discharge-ready effluent in <3 days of fed-batch processing. Average volumetric removal rates of BOD5 (16.5 ± 3.6 mg L−1 d−1) and ammoniacal nitrogen (6.09 ± 0.92 mg L−1 d−1) in this mixotrophic system were found to be not different from those reported for the photoautotrophic high rate algal ponds (significance level of 0.05); but, that of phosphates (1.40 ± 0.57 mg L−1 d−1) is greater than that reported for the photoautotrophic high rate algal ponds (significance level of 0.05). Additionally, the extreme culture conditions in this particular mixotrophic system enabled concurrent reductions of E. coli (>5 log) and other pathogenic bacteria.

Survival/Adaptation of Bone Marrow-Derived Mesenchymal Stem Cells After Long-Term Starvation Through Selective Processes.

After in vivo transplantation, mesenchymal stem cells (MSC) face an ischemic microenvironment, characterized by nutrient deprivation and reduced oxygen tension, which reduces their viability and thus their therapeutic potential. Therefore, MSC response to models of in vitro ischemia is of relevance for improving their survival and therapeutic efficacy. The aim of this study was to understand the survival/adaptive response mechanism that MSC use to respond to extreme culture conditions. Specifically, the effect of a long-term starvation on human bone marrow (hBM)-derived MSC cultured in a chemically defined medium (fetal bovine serum-free [SF] and human SF), either in hypoxic or normoxic conditions. We observed that hBM-MSC that were isolated and cultured in SF medium and subjected to a complete starvation for up to 75 days transiently changed their behavior and phenotype. However, at the end of that period, hBM-MSC retained their characteristics as determined by their morphology, DNA damage resistance, proliferation kinetic, and differentiation potential. This survival mode involved a quiescent state, confirmed by increased expression of cell cycle regulators p16, p27, and p57 and decreased expression of proliferating cell nuclear antigen (PCNA), Ki-67, mTOR, and Nanog. In addition, Jak/STAT (STAT6) antiapoptotic activity selected which cells conserved stemness and that supported metabolic, bioenergetic, and scavenging requirements. We also demonstrated that hBM-MSC exploited an autophagic process which induced lipid β-oxidation as an alternative energy source. Priming MSC by concomitant starvation and culture in hypoxic conditions to induce their quiescence would be of benefit to increase MSC survival when transplanted in vivo. Stem Cells 2019;37:813-827.

Microbial enzymes with special characteristics for biotechnological applications.

This article overviews the enzymes produced by microorganisms, which have been extensively studied worldwide for their isolation, purification and characterization of their specific properties. Researchers have isolated specific microorganisms from extreme sources under extreme culture conditions, with the objective that such isolated microbes would possess the capability to bio-synthesize special enzymes. Various Bio-industries require enzymes possessing special characteristics for their applications in processing of substrates and raw materials. The microbial enzymes act as bio-catalysts to perform reactions in bio-processes in an economical and environmentally-friendly way as opposed to the use of chemical catalysts. The special characteristics of enzymes are exploited for their commercial interest and industrial applications, which include: thermotolerance, thermophilic nature, tolerance to a varied range of pH, stability of enzyme activity over a range of temperature and pH, and other harsh reaction conditions. Such enzymes have proven their utility in bio-industries such as food, leather, textiles, animal feed, and in bio-conversions and bio-remediations.

Effect of reducing the top concentration used in the in vitro chromosomal aberration test in CHL cells on the evaluation of industrial chemical genotoxicity

A current concern with in vitro mammalian cell genotoxicity testing is the high frequency of false or misleading positive results caused in part by the past use of excessively high test concentrations. A dataset of 249 industrial chemicals used in Japan and tested for genotoxicity was analyzed. Of these, 116 (46.6%) were positive in the in vitro chromosomal aberration (CA) test, including 6 that were positive only at test concentrations >10 mM. There were 59 CA-positive chemicals at test concentrations ≤1 mM. At >1 mM, 51 chemicals were CA-positive, including 13 Ames-positive chemicals, which were therefore not “missed” by the test battery. Thus, 38 potentially positive chemicals would not have been detected in the test battery if the top test concentration was limited to 1 mM in CA test. Analysis of the relevance of CA results on the 38 missed chemicals was conducted based on a weight of evidence approach, including evaluations of effects of extreme culture conditions (low pH, high toxicity, or precipitation), in silico structural alert analysis, in vivo genotoxicity and carcinogenicity test data (where available), mode of action, or information from closely related chemicals. After an exhaustive review, there were four chemicals with some concern for human health risk assessment, nine with minimal concern, and the remaining 25 with negligible concern. We apply different top concentrations to the 38 missed chemicals to identify the most accurate approach for predicting the genotoxicity of industrial chemicals. Of these 2 mM or 1 mg/mL, whichever is higher, was the most effective in detecting these chemicals, i.e., relatively higher (8/13) or lower (17/25) detection among 13 chemicals with some or minimal concern, or 25 with negligible concern, respectively. Lower top concentration limits, 1 mM or 0.5 mg/mL, whichever is higher, are not as effective (2/13) for detecting these chemicals with concern. Therefore, we conclude 2 mM or 1 mg/mL, whichever is higher, would be an appropriate top concentration limit for testing industrial chemicals for chromosome damage.

A battery of genotoxicity studies with an allergy vaccine adjuvanted with monophosphoryl lipid A (MPL®) for the treatment of grass pollen allergy

The allergy vaccine Pollinex® Quattro Grass, developed for the prevention/relief of allergic symptoms caused by grass pollen in adults and children, contains extracts of 12 grass pollens and rye cereal (all chemically modified by glutaraldehyde), adsorbed onto L-tyrosine with addition of the immunostimulatory adjuvant monophosphoryl lipid A (MPL®). This paper represents the first publication on genetic toxicology data for such a vaccine. An Ames test using five strains of Salmonella typhimurium at concentrations up to 2000 standardized units (SU) per plate in the absence (-) and presence (+) of metabolic activation (rat liver S9) showed negative results, as did treatment - S9 in the mouse lymphoma assay (MLA). However, a reproducible positive response was noted in the MLA + S9, which could not be attributed to extreme culture conditions, but may have been an artefact of the exogenous metabolizing system. Up to 60% false positive results are reported for the MLA with noncarcinogens. Hence, in vivo genotoxicity studies were conducted. These further assessments comprised a rat bone marrow micronucleus test following subcutaneous (s.c.) doses of 10 000, 20 000 or 40 000 SU kg⁻¹ per day for 2 days, and a comet assay in liver and kidney cells from rats treated with Pollinex® Quattro Grass at 20 000 or 40 000 SU kg⁻¹ per day s.c. for 2 days. Plasma analysis showed evidence of systemic antibodies to Pollinex® Quattro Grass immunogens, but the exposure levels could not be quantified. No evidence of genotoxicity was observed in either of the in vivo studies. Overall, the genotoxicity test results supported safe clinical use of Pollinex® Quattro Grass.

Culturability and Secondary Metabolite Diversity of Extreme Microbes: Expanding Contribution of Deep Sea and Deep-Sea Vent Microbes to Natural Product Discovery

Microbes from extreme environments do not necessarily require extreme culture conditions. Perhaps the most extreme environments known, deep-sea hydrothermal vent sites, support an incredible array of archaea, bacteria, and fungi, many of which have now been cultured. Microbes cultured from extreme environments have not disappointed in the natural products arena; diverse bioactive secondary metabolites have been isolated from cultured extreme-tolerant microbes, extremophiles, and deep-sea microbes. The contribution of vent microbes to our arsenal of natural products will likely grow, given the culturability of vent microbes; their metabolic, physiologic, and phylogenetic diversity; numerous reports of bioactive natural products from microbes inhabiting high acid, high temperature, or high pressure environments; and the recent isolation of new chroman derivatives and siderophores from deep-sea hydrothermal vent bacteria.

UV induced mutations in Acidianus brierleyi growing in a continuous stirred tank reactor generated a strain with improved bioleaching capabilities

Use of microorganisms for bioleaching processes is commonly limited by low tolerance of extreme culture conditions such as temperature, pH, and pulp density. Adaptation to extreme conditions is the main approach for strain improvement. Traditional methods for breeding strains with tolerance of extreme condition are difficult to apply without genetic tools because of many of such strains are not readily purely cultivated in liquid or agar medium. Here, we developed a method using ultraviolet (UV) irradiation for mutation in a continuous stirred tank reactor (CSTR) for screening to improve the bioleaching performance of a poorly characterized strain of Acidianus brierleyi ( A. brierleyi). An improved strain developed with our method exhibited a good tolerance to Cu 2+ and a high bioleaching rate of chalcopyrite, which was four-fold better than that of a strain selected by traditional adaptation method. We also designed a countercurrent bioleaching process in stirred tanks in series, and copper recovery in 7–8 days at a 15% chalcopyrite pulp density was more than 90% in a two stage countercurrent reactor.

Apoptosis may contribute to false-positive results in the in vitro micronucleus test performed in extreme osmolality, ionic strength and pH conditions

Conditions in which clastogens produce positive responses have been increasingly challenged, and several situations have been described in which clastogenic responses would be considered not to be relevant. For example, extreme culture conditions lead to high variations of pH, osmolality or ionic strength. Apoptosis is induced in extreme culture conditions and contributes to false-positive results in the in vitro micronucleus test performed with CTLL-2 cells. These cells can enter apoptosis when exposed to apoptosis stimuli or after IL-2 deprivation, whereas the CTLL-2 Bcl2 cell line is protected from apoptosis due to the over-expression of the apoptosis inhibitor Bcl2 in bcl2- transfected CTLL-2 cells. The two cell lines were treated in extreme culture conditions of either pH or osmolality or were submitted to high ionic strength. The apoptosis level was measured in parallel with the in vitro micronucleus test using the annexin V-FITC method. Data obtained in the two cell lines suggested that apoptosis caused by extreme culture condition induces the formation of micronucleated cells, which leads to false-positive results in the in vitro micronucleus test.

Apoptosis may contribute to false-positive results in the in vitro micronucleus test performed in extreme osmolality, ionic strength and pH conditions

Conditions in which clastogens produce positive responses have been increasingly challenged, and several situations have been described in which clastogenic responses would be considered not to be relevant. For example, extreme culture conditions lead to high variations of pH, osmolality or ionic strength. Apoptosis is induced in extreme culture conditions and contributes to false-positive results in the in vitro micronucleus test performed with CTLL-2 cells. These cells can enter apoptosis when exposed to apoptosis stimuli or after IL-2 deprivation, whereas the CTLL-2 Bcl2 cell line is protected from apoptosis due to the over-expression of the apoptosis inhibitor Bcl2 in bcl2-transfected CTLL-2 cells. The two cell lines were treated in extreme culture conditions of either pH or osmolality or were submitted to high ionic strength. The apoptosis level was measured in parallel with the in vitro micronucleus test using the annexin V-FITC method. Data obtained in the two cell lines suggested that apoptosis caused by extreme culture condition induces the formation of micronucleated cells, which leads to false-positive results in the in vitro micronucleus test.

Biochemistry and biotechnological applications of Gluconobacter strains.

The genus Gluconobacter belongs to the group of acetic acid bacteria, which are characterized by their ability to incompletely oxidize a wide range of carbohydrates and alcohols. The corresponding products (aldehydes, ketones and organic acids) are excreted almost completely into the medium. In most cases, the reactions are catalyzed by dehydrogenases connected to the respiratory chain. Since the reactive centers of the enzymes are oriented towards the periplasmic space, transport of substrates and products into, and out of, the cell is not necessary. Thus, rapid accumulation of incompletely oxidized products in the medium is facilitated. These organisms are able to grow in highly concentrated sugar solutions and at low pH-values. High oxidation rates correlate with low biomass production, which makes Gluconobacter strains interesting organisms for industrial applications. Modern fermentation processes, such as the production of L-sorbose (vitamin C synthesis) and 6-amino- L-sorbose (synthesis of the antidiabetic drug miglitol) are carried out with members of this genus. Other important products are dihydroxyacetone, gluconate and ketogluconates. The bacteria belonging to the genus Gluconobacter exhibit extraordinary uniqueness not only in their biochemistry but also in their growth behavior and response to extreme culture conditions. This uniqueness makes them ideal organisms for microbial process development.

Chromosomal aberration tests in vitro: problems with protocol design and interpretation of results.

In vitro chromosomal aberration (CA) tests have come to play a central role in testing for mutagenic/carcinogenic potential of chemicals in most countries. Guidelines on the conduct of such assays have therefore been published by a variety of sources, and, if anything, recommended protocols have become even more extensive as time has progressed and revisions have been made. Yet there is very little comparative data from within or between laboratories to form a basis for these recommendations. Some claims that certain cell types were more sensitive than others for CA testing has led to comparisons between Chinese hamster ovary (CHO), Chinese hamster lung cells and human lymphocytes, and some examination of critical factors such as exposure periods and sampling times has been undertaken, but much more needs to be done. Clones of CHO and V79 cells from different sources show karyotypic variability are not equally stable, further confounding any comparisons of sensitivity. There would therefore seem to be a need to acknowledge genetic diversity in these cell lines, particularly when making comparisons of sensitivity. As in vitro CA tests employ more and more comprehensive protocols, an understanding or artefacts becomes more critical. Extreme culture conditions (pH shift, high osmolality, high ionic strength) have given rise to significant CA, but are unlikely to occur in vivo. It is possible that chemicals which only produce CA detectable at high levels of cytotoxicity are also not in vivo hazards, although experimental data is urgently needed to confirm or deny this. High toxicity clastogens (HTC) would not have the same biological importance as those inducing CA at low levels of toxicity [(low toxicity clastogens (LTC)]. However, current study design is generally inadequate to discriminate LTC from HTC, and agreement on the level of toxicity that divides them will be a contentious issue. Proposals are made for improved study design, in particular the spacing of doses to permit categorization of chemicals as HTC or LTC. A concept of comparing minimal positive (clastogenic) dose with an arbitrary level of toxicity (e.g. 40-50% inhibitory dose) is introduced to permit this categorization. As an indication of likely in vivo hazard, categorizing a chemical as either HTC or LTC can help with decision making in industry and risk assessment by industrialists and regulators.

Genotoxicity under extreme culture conditions: A report from ICPEMC task group 9

Genotoxicity under extreme culture conditions: A report from ICPEMC task group 9

Genotoxicity under extreme culture conditions

Genotoxicity under extreme culture conditions

Temperature and salinity limits for growth and survival of some planktonic foraminifers in laboratory cultures

The biological response to extreme temperatures and salinities is investigated in the laboratory for seven species of planktonic foraminifera: Globigerinoides sacculi/er (Brady), Globigerinoides ruber (d'Orbigny), Globigerinoides conglobatus (Brady), Globigerine/la siphonifera (d'Orbigny), Orbulina universa d'Orbigny, Neogloboquadrina dutertrei (d'Orbigny) and Globorotalia menardii (d'Orbigny). When one of the vital processes, food acceptance, growth or reproduction is inhibited by a culture variable, the absolute survival limit is reached. The measured in vitro temperature ranges compare well with the global temperature distribution patterns of these species, suggesting that this parameter plays a major role in their biogeographical distribution. The salinity ranges that are tolerated in laboratory cultures exceed the range encountered in modern oceans. Thus salinity does not limit the distribution of the species investigated herein. In general, larger mean final shell sizes are attained and the total shell length increase is larger at optimum temperatures and salinities than at extreme culture conditions, but the differences were not always statistically significant. Marginal temperature and salinity conditions do not induce contained growth in expatriated specimens. Under extreme culture conditions, the relative frequency of the different shell morphologies is altered relative to normal conditions. Abnormal phenotypes are more frequent under normal conditions and the normal morphology is found more often under extreme conditions. As opposed to previous reports, the frequency of kummerform chambers generally decreases toward extreme temperature and salinity culture conditions, indicating that kummerform phenotypes are not indicative of environmental stress. The incidence of sac-like chambers in G. sacculi/er and the formation of spherical chambers in adult 0. universa decrease toward extreme temperature and salinity culture conditions, demonstrating that maturation is suppressed in stress situations. SEM investigations show that changes in shell porosity are correlated with treatment variables in culture. The highest porosities are attained at higher temperatures and lower salinities. Generally, an increase in total porosity is achieved by an increase of the pore area accompanied by a reduction of the pore density. The in vitro experiments explain the changes that occurred in the Pleistocene foraminiferal assemblages from the Red Sea around 18 thousand years ago and earlier. During glacial periods, salinity approximated or even exceeded the upper thresholds that were tolerated under laboratory conditions. Under these circumstances, species disappeared from the water column. The order of disappearance as recorded in the sediments may be explained with the upper salinity limits found in this study. Also, the recurrent shifts of dominance between G. sacculi/er and G. ruber are well documented for this fossil assemblage. The present experiments support the conclusion that salinity is the driving mechanism behind this phenomenon. Observations in modern oceans suggest that the fertility of the water mass is probably also an important factor behind the shifts of dominance between G. sacculi/er and G. ruber.

Genotoxicity in vitro under extreme culture conditions: An ICPEMC investigation

Genotoxicity in vitro under extreme culture conditions: An ICPEMC investigation

Genotoxicity in vitro under extreme culture conditions: An ICPEMC investigation

Genotoxicity in vitro under extreme culture conditions: An ICPEMC investigation

Genotoxicity in vitro under extreme culture conditions: an ICPEMC investigation - call for information

Genotoxicity in vitro under extreme culture conditions: an ICPEMC investigation - call for information

The influence of pH and medium osmolarity on a monoxenic culture of Entamoeba moshkovskii

The behaviour of Entamoeba moshkovskii was tested in hypotonic culture media (beef serum and distilled water) of varying osmotic pressure and pH, using phosphate buffers. 600 m.osm∗ was the highest concentration assayed; the pH was varied between 5.9 and 7.2. It has been established that the amoeba has the ability to produce similar yields at all concentrations of the medium from the most dilute to 600 m.osm. At the upper and lower limits of concentration a pH of between 6.2 and 6.8 was found optimum for maximum yield. Yields, especially in predominately hypo- and hypertonic media, were found to be also dependent on the size of the initial inoculum. Under extreme culture conditions reduced phagocytosis was observed and the decline phase of the culture was independent of the starch consumption. Under more normal conditions decline took place when the starch had been consumed.