Alternative agar substitutes for culturing unculturable microorganisms.

Gelling agents are necessary for the preparation of solid or semisolid media. For more than a hundred years, agar has been the primary gelling agent. However, a substantial body of evidence has accumulated suggesting that agar-based media inhibit the growth of many microbial species through the generation of reactive oxygen species (ROS), toxic organic contaminants, or competitive exclusion effects. In this review we have compiled the largest amount of data to date on the use of various gelling agents in microbial isolation and cultivation, with the particular emphasis on rare microbe isolation cases. Our analysis suggested that microbial-derived compounds (especially gellan gum), as gelling agents, are superior to agar in their ability to isolate and maintain either new or known microbial species. We analyzed the reasons behind this success and concluded that there are phylum-level differences in microbial responses to the changes in conditions from natural to the laboratory conditions (with respect to gelling agent usage). Consequently, we hypothesize that at least partial success of microbial-derived gelling agents lies in the recreation of the natural microenvironment conditions (which we address as the "familiarity of conditions" hypothesis). Finally, we present a list of recommendations and suggestions for further microbial ecology studies.

Scaling Laws in Polysaccharide Rheology: Comparative Analysis of Water and Ionic Liquid Systems.

This study investigates the rheological behavior of two plant-based polysaccharides, with different degrees of hydrophilicity, agar (highly hydrophilic) and guar gum (hydrophilic), in water and 1-ethyl-3-methylimidazolium acetate (EMImAc). The rheological response of these polymers is highly dependent on the solvent's ability to disrupt intermolecular associations. In water, agar forms hydrogels, while guar gum behaves as a viscoelastic liquid with slow modes. The plateau modulus (GN0) scales with polymer concentration (c) as GN0 ∼ c3, consistent with other natural polymers. In EMImAc, both polysaccharides form viscoelastic liquids, exhibiting GN0 ∼ c2.3, as expected for semiflexible polymer solutions. However, the terminal relaxation time, τD, and the specific viscosity, ηsp, scale as τD ∼ c5.3 and ηsp ∼ c7.6, indicative of intermolecular chain-chain associations. Despite the solvent or polysaccharide, the fractional viscosity overshoot and the shear strain at the maximum stress show a terminal Weissenberg number dependence similar to other synthetic polymers.

Characterization of gelling agents in callus inducing media: Physical properties and their effect on callus growth.

In plant tissue culture, callus formation serves as a crucial mechanism for regenerating entire plants, enabling the differentiation of diverse tissues. Researchers have extensively studied the influence of media composition, particularly plant growth regulators, on callus behavior. However, the impact of the physical properties of the media, a well-established factor in mammalian cell studies, has received limited attention in the context of plant tissue culture. Previous research has highlighted the significance of gelling agents in affecting callus growth and differentiation, with Agar, Phytagel, and Gelrite being the most used options. Despite their widespread use, a comprehensive comparison of their physical properties and their subsequent effects on callus behavior remains lacking. Our study provides insights into optimizing plant tissue culture media by analyzing the physical properties of gelling agents and their impact on callus induction and differentiation. We compared the phenotypes of calli grown on media composed of these different gelling agents and correlated them to the physical properties of these media. We tested water retention, examined pore size using cryo-SEM, measured the media mechanical properties, and studied diffusion characteristics. We found that the mechanical properties of the media are the only quality correlated with callus phenotype.

A straightforward procedure to build a non-toxic relaxometry phantom with desired T1 and T2 times at 3T.

OBJECTIVE: To prepare and analyze soy-lecithin-agar gels for non-toxic relaxometry phantoms with tissue-like relaxation times at 3T. METHODS: Phantoms mimicking the relaxation times of various tissues (gray and white matter, kidney cortex and medulla, spleen, muscle, liver) were built and tested with a clinical 3T whole-body MR scanner. Simple equations were derived to calculate the appropriate concentrations of soy lecithin and agar in aqueous solutions to achieve the desired relaxation times. Phantoms were tested for correspondence between measurements and calculated T1 and T2 values, reproducibility, spatial homogeneity, and temporal stability. T1 and T2 mapping techniques and a 3D T1-weighted sequence with high spatial resolution were applied. RESULTS: Except for the liver relaxation phantom, all phantoms were successfully and reproducibly produced. Good agreement was found between the targeted and measured relaxation times. The percentage deviations from the targeted relaxation times were less than 3% for T1 and less than 6.5% for T2. In addition, the phantoms were homogeneous and had little to no air bubbles. However, the phantoms were unstable over time: after a storage period of 4 weeks, mold growth and also changes in relaxation times were detected in almost all phantoms. CONCLUSION: Soy-lecithin-agar gels are a non-toxic material for the construction of relaxometry phantoms with tissue-like relaxation times. They are easy to prepare, inexpensive and allow independent adjustment of T1 and T2. However, there is still work to be done to improve the long-term stability of the phantoms.

Red Seaweed (Rhodophyta) Phycocolloids: A Road from the Species to the Industry Application.

Seaweed polysaccharides are versatile both in their functions in seaweed physiology and in their practical applications in society. However, their content and quality vary greatly. This review discusses the main factors that influence the yield and quality of polysaccharides, specifically carrageenans and agars (sulfated galactans) found in red algae species (Rhodophyta). In addition, its historical, current, and emerging applications are also discussed. Carrageenan has been influenced mainly by photosynthetically active radiation (PAR) and nitrogen, while its relationship with temperature has not yet been replicated by recent studies. Agar's seasonal trend has also been found to be more ambiguous than stated before, with light, temperature, nutrients, and pH being influencing factors. In this review, it is also shown that, depending on the compound type, seaweed polysaccharides are influenced by very different key factors, which can be crucial in seaweed aquaculture to promote a high yield and quality of polysaccharides. Additionally, factors like the extraction method and storage of polysaccharides also influence the yield and quality of these compounds. This review also highlights the drawbacks and inadequacy inherent from the conventional (or current) extraction technology approaches.

Statistical optimization of process variables for agarase production using Microbacterium sp. SS5 strain from non-marine sources.

Agar oligosaccharides are thought to be valuable biomolecules with high bioactivity potential, along with a wide range of applications and advantages. The current study aimed to optimize the culture parameters required to produce agarase enzyme and agar oligosaccharides from industrial waste agar. Microbacterium spp. strain SS5 was isolated from a non-marine source and could synthesize oligo derivatives for use in a variety of industries ranging from food to pharmaceuticals. In addition, the strain and culture conditions were optimized to maximize extracellular agarase production. The bacterium grew best at pH 5.0 - 9.0, with an optimal pH of 7.5 - 8.0; temperatures ranging from 25 to 45 °C, with an optimal of 35 °C; and carbon and nitrogen concentrations of 0.5% each. Plackett-Burman experimental design and response surface methods were used to optimize various process parameters for agarase production by Microbacterium spp. strain SS5. Using the Plackett-Burman experimental design, eleven process factors were screened, and agar, beef extract, CaCl2, and beginning pH were found as the most significant independent variables affecting agarase production with confidence levels above 90%. To determine the optimal concentrations of the identified process factors on agarase production, the Box- Behnken design was used. Agarase production by Microbacterium spp. strain SS5 after optimization was 0.272 U/mL, which was determined to be greater than the result obtained from the basal medium (0.132 U/mL) before screening using Plackett-Burman and BBD with a fold increase of 2.06.

Seaweed polysaccharides: Emerging extraction technologies, chemical modifications and bioactive properties.

Nowadays, consumers are increasingly aware of the relationship between diet and health, showing a greater preference of products from natural origin. In the last decade, seaweeds have outlined as one of the natural sources with more potential to obtain bioactive carbohydrates. Numerous seaweed polysaccharides have aroused the interest of the scientific community, due to their biological activities and their high potential on biomedical, functional food and technological applications. To obtain polysaccharides from seaweeds, it is necessary to find methodologies that improve both yield and quality and that they are profitable. Nowadays, environmentally friendly extraction technologies are a viable alternative to conventional methods for obtaining these products, providing several advantages like reduced number of solvents, energy and time. On the other hand, chemical modification of their structure is a useful approach to improve their solubility and biological properties, and thus enhance the extent of their potential applications since some uses of polysaccharides are still limited. The present review aimed to compile current information about the most relevant seaweed polysaccharides, available extraction and modification methods, as well as a summary of their biological activities, to evaluate knowledge gaps and future trends for the industrial applications of these compounds.Key teaching pointsStructure and biological functions of main seaweed polysaccharides.Emerging extraction methods for sulfate polysaccharides.Chemical modification of seaweeds polysaccharides.Potential industrial applications of seaweed polysaccharides.Biological activities, knowledge gaps and future trends of seaweed polysaccharides.

Headspace control and antimicrobials: Inhibition strategies to prevent the growth of Alicyclobacillus acidoterrestris in orange juice.

Alicyclobacillus acidoterrestris can cause spoilage in orange juice that leads to consumer rejection. Six different orange juices were physiochemically characterized (pH, total soluble solids, titratable acidity, total polyphenols and vitamin C). A bottle for each sampling point per juice was filled (headspace: 40% volume) and inoculated with 102 -103 CFU per ml of A. acidoterrestris ATCC® 49025™ (heat shocked before inoculation: 75°C, 20 min). Samples were stored for 21 days at 45 ± 1°C and plate counted periodically on acidified YSG agar (pH 3·7) incubated at 45 ± 1°C for 3 days. The effect of headspace (6% versus 40% volume) on A. acidoterrestris growth was also evaluated. The effect of nisin (0·006, 0·003, 0·0015, and 0·00075%), sodium benzoate (0·1%), potassium sorbate (0·1%) and a mix of benzoate and sorbate (0·05% each) on A. acidoterrestris was additionally addressed. Alicyclobacillus acidoterrestris reached up to 107 CFU per ml in five of the six juices in less than 1 week. Headspace significantly impacted (P < 0·05) A. acidoterrestris maximum population, which reached the critical value of 5 log CFU per ml at 40% headspace. All preservatives, regardless of concentration, showed a bacteriostatic effect during 22 days of storage with no significant differences amongst treatments (P > 0·05).

Comparison of BCYEα+AB agar and MWY agar for detection and enumeration of Legionella spp. in hospital water samples.

BACKGROUND: This study illustrates for the first time the performance (sensitivity and selectivity) of the selective medium BCYEα +AB suggested by the new edition of ISO 11731 for legionella isolation and enumeration. We compared the efficacy of the selective BCYEα +AB medium with that of the highly selective MWY medium. RESULTS: Legionella spp. was detected in 48.2 and 47.1% of the samples by BCYEα +AB and MWY agar, respectively. For optimal detection of Legionella spp., most protocols recommend using selective media to reduce the number of non-Legionella bacteria. Agreement between the two media was 86.7%. CONCLUSIONS: According to the results, both media have a very similar performance and they both have advantages and disadvantages over each other. In AB medium there is the risk of being less selective so more interfering microbiota may grow but in MWY medium there is the risk of being too selective. The low selectivity of the AB medium could be resolved if other treatments are applied after filtration, e.g. acid and/or heat treatment, but it must be taken into account that these treatments still reduce the number of viable Legionella. In conclusion, we recommend using MWY as a selective medium for the detection of Legionella spp. as it is easier discern suspected colonies and facilitate the final Legionella spp.

On the interference from agar in chemical exchange saturation transfer MRI parameter optimization in model solutions.

Chemical exchange saturation transfer (CEST) MRI is currently set to become part of clinical routine as it enables indirect detection of low concentrated molecules and proteins. Recently, intermediate to fast exchanging functional groups of glucose and its derivatives, glutamate and dextran, have gained attention as promising CEST contrast agents. To increase the specificity of CEST MRI for certain functional groups, the presaturation module is commonly optimized. At an early stage, this is performed in well-defined model solutions, in which, for instance, the relaxation times are adjusted to mimic in vivo conditions. This often involves agar, assuming the substance would not yield significant CEST effects by itself, which the current study proves to be an invalid assumption. Model solutions at different pH values and concentrations of agar were investigated at different temperatures at a 9.4 T human whole body MR scanner. High power presaturation of around 4 µT, optimal for investigating intermediate to fast exchanging groups, was applied. Postprocessing included spatiotemporal corrections for B0 and spatial corrections for B1+ . CEST effects of up to 3 % of the bulk water signal were observed. From pH, concentration and temperature dependency, it was concluded that the observed behavior reflects a CEST effect of agar. It was also shown how to remove this undesirable contribution from CEST MRI data. It was concluded that if agar is involved in the CEST MRI parameter optimization process, its contribution to the observed effects has to be taken into account. CEST agent concentration must be sufficiently high to be able to neglect the contribution of agar, or a control sample at matched pH is necessary for correction. Experiments on pure agarose showed reduced CEST effects compared with agar but did not provide a neutral baseline either.

Evaluation of CHROMagar™-Serratia agar, a new chromogenic medium for the detection and isolation of Serratia marcescens.

A comparative analysis of the performance of the new selective chromogenic CHROMagar™-Serratia culture medium for detection and isolation of Serratia marcescens was undertaken. A total of 134 clinical isolates (95 S. marcescens with and without carbapenemase production and 39 non-S. marcescens isolates) and 96 epidemiological samples (46 rectal swabs and 50 from environmental surfaces) were studied. Diagnostic values when compared with CHROMagar™-Orientation medium were 96.8% sensitivity, 100% specificity, 100% positive predictive value and 88.5% negative predictive value. In conclusion, CHROMagar™-Serratia shows an excellent ability for differentiation of S. marcescens among clinical isolates and in environmental samples.

CHROMagarTM Candida Plus: A novel chromogenic agar that permits the rapid identification of Candida auris.

Candida auris is a serious nosocomial health risk, with widespread outbreaks in hospitals worldwide. Successful management of such outbreaks has depended upon intensive screening of patients to identify those that are colonized and the subsequent isolation or cohorting of affected patients to prevent onward transmission. Here we describe the evaluation of a novel chromogenic agar, CHROMagarTM Candida Plus, for the specific identification of Candida auris isolates from patient samples. Candida auris colonies on CHROMagarTM Candida Plus are pale cream with a distinctive blue halo that diffuses into the surrounding agar. Of over 50 different species of Candida and related genera that were cultured in parallel, only the vanishingly rare species Candida diddensiae gave a similar appearance. Moreover, both the rate of growth and number of colonies of C. auris recovered from swabs of pure and mixed Candida species were substantially increased on CHROMagarTM Candida Plus agar when compared with growth on the traditional mycological isolation medium, Sabouraud dextrose agar. Taken together, the present data suggest that CHROMagarTM Candida Plus agar is an excellent alternative to current conventional mycological media for the screening of patients who are potentially colonized/infected with Candida auris, can be reliably used to identify this emerging fungal pathogen, and should be tested in a clinical setting. LAY ABSTRACT: Candida auris is a novel pathogenic yeast that has been associated with large hospital outbreaks across several continents. Affected patients become colonized, predominantly on the skin, with large quantities of C. auris which they then shed into the hospital environment. Identification of C. auris is challenging using routine laboratory methods, and time consuming when patients are colonized with a mixture of different Candida species. Here we demonstrate that a novel chromogenic agar, CHROMagarTM Candida Plus, permits the rapid differentiation of C. auris from a wide range of other yeast species and is potentially ideally suited to screening of patients that are suspected of being colonized or infected with this medically important yeast.

Determination of available breaking stress of agar and gellan gum plate culture methods and the duration of bacterial culture under strong acidic conditions.

AIMS: Several acidophilic bacteria have not been cultured, primarily owing to the lack of suitable culture methods under strong acidic conditions. This study aimed to quantitatively evaluate the strengths of the agar plates (AP) and gellan gum plates (GP), and optimal culture periods under strong acidic conditions. METHODS AND RESULTS: To define the lower limit of plate strength for bacterial isolation culture, the diameter of Escherichia coli K12 colonies and the breaking stress of plates at different concentrations of gelling agents, medium composition and pH conditions were determined. The lower limit of available strength of AP and GP was 19·6 and 14·8 kPa, respectively. Medium composition slightly affected AP breaking stress, although GP with a high cationic concentration medium could not be prepared. CONCLUSIONS: Assessment of the strength limits of AP and GP revealed that AP is not suitable for prolonged bacterial culture (≥72 h). Furthermore, GP was completely ineffective for bacterial culture under highly acidic conditions (≤pH 1·0). SIGNIFICANCE AND IMPACT OF THE STUDY: Our quantitative evaluation method based on breaking stress is a potentially valuable tool to understand the state and the suitable limit of plate culture methods in more detail under various conditions.

Performance of polymyxin B agar-based tests among carbapenem-resistant Enterobacterales.

Therapeutic options for infections caused by Carbapenem-resistant Enterobacterales (CRE) are restricted and include polymyxins-centred schemes. Evaluation of in vitro susceptibility is difficult and time consuming. Agar-based methodologies are an alternative to broth microdilution (BMD) and we aimed to evaluate the accuracy of those methods among Enterobacterales. A total of 137 non-duplicated CRE were subjected to polymyxin B BMD, agar screening test (Mueller Hinton plates containing 3 µg ml-1 of polymyxin B) and agar dilution (antibiotic serially diluted 0·25-64 µg ml-1 ). CRE of 42·3% were resistant to polymyxin B (MICs range: 0·25->64 µg ml-1 ) and 16·8% presented borderline MICs. Sensitivity, specificity, PPV and NPV were 86·2, 98·7, 98 and 90·7% for screening test and 86·2, 97·5, 96·1 and 90·6% for agar dilution. ME was 0·73 and 1·5% for screening and agar dilution respectively; VME was 5·8% for both techniques. In general, agar-based methods had a good performance. As far as we know, this is the first study to propose an agar screening test using polymyxin B instead of colistin.

Simple stain-free screening method for pectinolytic microorganisms under alkalophilic conditions.

OBJECTIVES: To develop a simple pectin-degrading microorganism screening method. RESULTS: We developed a method utilizing the phenomenon whereby cooling an alkaline agar medium containing pectin causes the agar to become cloudy. This highly simplified method involves culturing the microorganisms on pectin-containing agar medium until colony formation is observed, and subsequent overnight cooling of the agar medium to 4 °C. Using this simple procedure, we successfully identified pectin-degrading microorganisms by observing colonies with halos on the clouded agar medium. We used alkaline pectinase and Bacillus halodurans, which is known to secrete alkaline pectinase, to establish the screening method. We demonstrated the screening of pectin-degrading microorganisms using the developed method and successfully isolated pectin-degrading microorganisms (Paenibacillus sp., Bacillus clausii, and Bacillus halodurans) from a soil sample. CONCLUSIONS: The developed method is useful for identifying pectin-degrading microorganisms.

Pretreatment Techniques and Green Extraction Technologies for Agar from Gracilaria lemaneiformis.

Optimizing the alkali treatment process alone without tracking the changes of algae and agar quality with each pretreatment process will not achieve the optimal agar yield and final quality. In this study, we monitored the changes of the morphology and weight of algae with each treatment process, and comprehensively analyzed the effects of each pretreatment process on the quality of agar by combining the changes of the physicochemical properties of agar. In conventional alkali-extraction technology, alkali treatment (7%, w/v) alone significantly reduced the weight of algae (52%), but hindered the dissolution of algae, resulting in a lower yield (4%). Acidification could solve the problem of algal hardening after alkali treatment to improve the yield (12%). In enzymatic extraction technology, agar with high purity cannot be obtained by enzyme treatment alone, but low gel strength (405 g/cm2) and high sulfate content (3.4%) can be obtained by subsequent acidification and bleaching. In enzyme-assisted extraction technology, enzyme damage to the surface fiber of algae promoted the penetration of low-concentration alkali (3%, w/v), which ensured a high desulfurization efficiency and a low gel degradation rate, thus improving yield (24.7%) and gel strength (706 g/cm2), which has the potential to replace the traditional alkali-extraction technology.

Gracilaria gracilis (Gracilariales, Rhodophyta) from Dakhla (Southern Moroccan Atlantic Coast) as Source of Agar: Content, Chemical Characteristics, and Gelling Properties.

Agar is a sulfated polysaccharide extracted from certain marine red algae, and its gel properties depend on the seaweed source and extraction conditions. In the present study, the seaweed Gracilaria gracilis (Gracilariales, Rhodophyta) from Dakhla (Moroccan Atlantic Coast) was investigated for its agar content, structure, and gel properties. The agar yields of G. gracilis were 20.5% and 15.6% from alkaline pretreatment and native extraction, respectively. Agar with alkaline pretreatment showed a better gelling property supported by higher gel strength (377 g·cm-2), gelling (35.4 °C), and melting (82.1 °C) temperatures with a notable increase in 3,6-anhydro-galactose (11.85%) and decrease in sulphate (0.32%) contents. The sulfate falling subsequent to alkaline pretreatment was verified through FT-IR spectroscopy. The 13C NMR spectroscopy showed that alkaline-pretreated agar has a typical unsubstituted agar pattern. However, native agar had a partially methylated agarose structure. Overall, this study suggested the possibility of the exploitation of G. gracilis to produce a fine-quality agar. Yet, further investigation may need to determine the seasonal variability of this biopolymer according to the life cycle of G. gracilis.

Valorization of the Red Algae Gelidium sesquipedale by Extracting a Broad Spectrum of Minor Compounds Using Green Approaches.

Until now, the red algae Gelidium sesquipedale has been primarily exploited for agar production, leaving an undervalued biomass. In this work, the use of eco-friendly approaches employing ultrasound-assisted extraction (UAE) and green solvents was investigated to valorize the algal minor compounds. The green methods used herein showed an attractive alternative to efficiently extract a broad spectrum of bioactive compounds in short extraction times (15 to 30 min vs. 8 h of the conventional method). Using the best UAE conditions, red seaweed extracts were characterized in terms of total phenolics (189.3 ± 11.7 mg GAE/100 g dw), flavonoids (310.7 ± 9.7 mg QE/100 g dw), mycosporine-like amino acids (MAAs) (Σ MAAs = 1271 mg/100 g dw), and phycobiliproteins (72.4 ± 0.5 mg/100 g dw). Additionally, produced algal extracts exhibited interesting antioxidant and anti-enzymatic activities for potential applications in medical and/or cosmetic products. Thus, this study provides the basis to reach a superior valorization of algal biomass by using alternative methods to extract biologically active compounds following eco-friendly approaches. Moreover, the strategies developed not only open new possibilities for the commercial use of Gelidium sesquipedale, but also for the valorization of different algae species since the techniques established can be easily adapted.

Convenient Agarose Preparation with Hydrogen Peroxide and Desulfation Process Analysis.

Agarose is a natural seaweed polysaccharide and widely used in the medicine, food, and biological fields because of its high gel strength, non-toxicity, and electrical neutrality. The sulfate group is one of the main charged groups that affect the performance of agarose. In the present study, a simple, eco-friendly, and efficient method was explored for agarose preparation. After desulfation with hydrogen peroxide (H2O2), the sulfate content of agar reached 0.21%. Together with gel strength, electroendosmosis, gelling and melting temperature, the indicators of desulfated agar met the standards of commercially available agarose. Notably, the desulfated agar can be used as an agarose gel electrophoresis medium to separate DNA molecules, and the separation effect is as good as that of commercially available agarose. Further, the H2O2 desulfation process was analyzed. The addition of a hydroxyl radical (HO•) scavenger remarkably decreased the H2O2 desulfation rate, indicating that HO• has a certain role in agar desulfation. Sulfate content detection indicated that sulfur was removed from agar molecules in the form of sulfate ions (SO42-) and metal sulfate. The band absence at 850 cm-1 indicated that the sulfate groups at C-4 of D-galactose in sulfated galactan were eliminated.

Improvement of cadaverine production in whole cell system with baker's yeast for cofactor regeneration.

Cadaverine, 1,5-diaminopentane, is one of the most promising chemicals for biobased-polyamide production and it has been successfully produced up to molar concentration. Pyridoxal 5'-phosphate (PLP) is a critical cofactor for inducible lysine decarboxylase (CadA) and is required up to micromolar concentration level. Previously the regeneration of PLP in cadaverine bioconversion has been studied and salvage pathway pyridoxal kinase (PdxY) was successfully introduced; however, this system also required a continuous supply of adenosine 5'-triphosphate (ATP) for PLP regeneration from pyridoxal (PL) which add in cost. Herein, to improve the process further a method of ATP regeneration was established by applying baker's yeast with jhAY strain harboring CadA and PdxY, and demonstrated that providing a moderate amount of adenosine 5'-triphosphate (ATP) with the simple addition of baker's yeast could increase cadaverine production dramatically. After optimization of reaction conditions, such as PL, adenosine 5'-diphosphate, MgCl2, and phosphate buffer, we able to achieve high production (1740 mM, 87% yield) from 2 M L-lysine. Moreover, this approach could give averaged 80.4% of cadaverine yield after three times reactions with baker's yeast and jhAY strain. It is expected that baker's yeast could be applied to other reactions requiring an ATP regeneration system.