Production,by filamentous,nitrogen-fixing cyanobacteria,of a bacteriocin and of other antibiotics that kill related strains

Colonies of sixty-five filamentous cyanobacteria were screened for the production of temperate phages and/or antibiotics on solid medium. None of them was observed to release phages. However, seven N₂-fixing strains were found to produce antibiotics very active against other cyanobacteria. The antibiotic produced by Nostoc sp. 78-11 A-E represents a bacteriocin of low molecular weight. Nostoc sp. ATCC 29132 appears to secrete, together with an antibiotic, a protein that inhibits its action.     

Development of genetic markers in cyanobacteria and stability of genetically marked strains in soil

Reporter marker GUS (-glucuronidase gene from Escherichia coli) and luc operon from the American firefly were introduced into cyanobacteria and the stability of these markers in soil was examined. To transfer the integrational vector into cyanobacteria, the genomic DNA library of Synechocystis sp. or Anabaena cylindrica maintained in pBR 322, pCY 100 and pCY 101 were transformed with HB 101 containing pRL 528 and selected for Cm^r and Amp^r. These clones of HB 101 containing pRL 528 and the vectors carrying different cyanobacterial chromosomal DNA fragments were used for triparental mating with HB 101 [pRK 2013/pRK 2073] and cyanobacteria. The frequency of transconjugants for integrational vectors was between 2.1 × 10^–5 and 4.0 × 10^–4. The transfer frequency of RSF 1010 based vectors (pDSK 519 and pCY 106) was 1.0–4.5 × 10^–4 in Synechocystis sp. whereas A. cylindrica failed to maintain these vectors. Low frequency transfer (2.0–2.3 × 10^–6) of RK 2 based vectors pVK 100 and pCY 104 was observed in A. cylindrica but these were unable to replicate in Synechocystis sp. The vectors in general were stable at least by 74.9% for 60 days of incubation in BG-11 medium. The markers were less stable in A. cylindrica (74.9–84.2%) compared to Synechocystis sp. (80.1–88.8%) at 60 days of incubation. Integrational vectors were almost 85% stable in both the strains. The RK 2 derivative of pCY 104 was less stable in A. cylindrica (74.9–77.3%) than the RSF 1010-based vector pCY 106 in Synechocystis sp. (80.1–81.0%). A maximum of 64.7% of the markers were lost in soil. The chromosomal markers through integrational vectors were found to be highly stable and 68.2–72.7% of these markers were retained in cyanobacteria at 60 days of incubation. Plasmid markers were less stable, with a loss of 64.7–48.7% at the end of the experiment. In A. cylindrica 58–65% of the RK 2 vector was lost whereas in Synechocystis sp. 49–61% of RSF 1010 was lost at 60 days of incubation.     

Regulation of nitrogenase levels in Anabaena sp. ATCC 33047 and other filamentous cyanobacteria

Incubation in the dark of photoautotrophically grown N₂-fixing heterocystous cyanobacteria leads to a loss of nitrogenase activity. Original levels of nitrogenase activity are rapidly regained upon re-illumination of the filaments, in a process dependent on de novo protein synthesis. Ammonia, acting indirectly through some of its metabolic derivatives, inhibits the light-promoted development of nitrogenase activity in filaments of Anabaena sp. ATCC 33047 and several other cyanobacteria containing mature heterocysts. The ammonia-mediated control system is also operative in N₂-fixing filaments in the absence of any added source of combined nitrogen, with the ammonia resulting from N₂-fixation already partially inhibiting full expression of nitrogenase. High nitrogenase levels, about two-fold higher than those in normal N₂-fixing Anabaena sp. ATCC 33047, are found in cell suspensions which have been treated with the glutamine synthetase inhibitor l-methionine-d,l-sulfoximine or subjected to nitrogen starvation. Filaments treated in either way are insensitive to the ammonia-promoted inhibition of nitrogenase development, although this insensitivity is only transitory for the nitrogen-starved filaments, which become ammonia-sensitive once they regain their normal nitrogen status.Abbreviations Chl chlorophyll - EDTA ethylenediaminetetraacetic acid - MSX l-methionine-d,l-sulfoximine     

Effect of <Emphasis Type="Italic">Nostoc</Emphasis> sp. on soil characteristics,plant growth and nutrient uptake

The arid desert area is expanding by 6 million ha year⁻¹. Nostoc, a terrestrial cyanobacterium, is known as a pioneer organism which can photosynthesize, fix atmospheric nitrogen, and secrete polysaccharides. In order to explore the potential of Nostoc as a countermeasure to soil desertification, we have investigated the effects of Nostoc on the chemical and physical properties of soil and on plant growth in outdoor and laboratory experiments. The effect of Nostoc on soil properties, when applied on the surface of soil in plastic containers, was determined after cultivation for 90 days outdoors. Its effect on plant growth and nutrient uptake was determined by growing Nostoc with Komatsuna (Brassica rapa var. peruviridis) on a soil-less culture in a plastic petri dish incubated inside a growth chamber for 14 days. Two colonies of Nostoc with different shapes were also examined for their tolerance to dryness and salinity. Nostoc application resulted in an increase in the organic carbon and nitrogen content of the surface soil and enhanced plant growth and plant iron uptake, but soil temperature and moisture were maintained. Spherical-shaped Nostoc showed a higher tolerance to dryness and salinity than irregular-shaped Nostoc, and the former was able to rectify problematic soil better than the latter. The results indicate that the application of Nostoc to soil has a potential for increasing soil organic matter and reclaiming degraded soil ecosystems. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Rhizobium meliloti distribution in the soil following alfalfa inoculation

Summary Alfalfa seeds, inoculated with an antibiotic-resistantRhizobium meliloti strain, were planted in three replicated field plots at Clayton, N.C. Core samples were taken three times in the next year at 0, 10, and 20 cm from the edge of each plot. Soil subsamples were taken from within each core sample at 0, 6, 12, and 18 cm depths. The numbers of the inoculum Rhizobium strain in each soil subsample were determined by inoculation of alfalfa plants with diluted soil samples. In general the distribution of rhizobia showed some movement outward and downward in the soil. Lower counts were obtained at the surface during summer. The Rhizobium persistence pattern in the soil differed in the three plots which is consistent with the variability in Rhizobium numbers often observed in established alfalfa stands. Cooperative investigation of the United States Department of Agriculture, Science and Education Administration, Agricultural Research and the North Carolina Agricultural Research Service, Raleigh, North Carolina. Paper No. 6818 of the Journal Series of the North Carolina Agricultural Research Service at Raleigh.     

Carbohydrate formation in rewetted terrestrial cyanobacteria

Summary In the terrestrial cyanobacterium Nostoc commune Vauch. formation of carbohydrate polymers was measured upon rewetting the mats in a light-dark regime. To discriminate between carbohydrates of different physiological function, total carbohydrate was determined as anthrone-reactive material (ARM) and storage carbohydrate (glycogen) assayed by an enzymic test. In the dry thalli glycogen was found to represent less than one tenth of the ARM. After rewetting an increase of total carbohydrate was observed in illuminated samples. Only glycogen, however, showed a regular pattern of synthesis and degradation during a 12:12 h light-dark cycle. This indicates that most carbohydrates detected by anthrone belong to the metabolically inert sheath material.When illuminated colonies were kept submerged after rewetting glycogen was hydrolyzed indicative of being used in the rapid recovery of cellular functions as observed in rewetted colonies. Apparently, photosynthesis allowed for net glycogen synthesis only, provided the mats were sufficiently aerated. These findings give evidence that the (carbohydrate) sheath plays an important role in water retention in an organism bound to a terrestrial habitat.     

Frankia inoculation,soil biota,and host tissue amendment influence Casuarina nodulation capacity of a tropical soil

The effects of soil biota, Frankia inoculation and tissue amendment on nodulation capacity of a soil was investigated in a factorial study using bulked soil from beneath a Casuarina cunninghamiana tree and bioassays with C. cunninghamiana seedlings as capture plants. Nodulation capacities were determined from soils incubated in sterile jars at 21 °C for 1, 7, and 28 days, after receiving all combinations of the following treatments: ± steam pasteurization, ± inoculation with Frankia isolate CjI82001, and ± amendment with different concentrations of Casuarina cladode extracts. Soil respiration within sealed containers was determined periodically during the incubation period as a measure of overall microbial activity. Soil respiration, and thus overall microbial activity, was positively correlated with increasing concentrations of Casuarina cladode extracts. The nodulation capacity of soils inoculated with Frankia strain Cj82001 decreased over time, while those of unpasteurized soils without inoculation either increased or remained unaffected. The mean nodulation capacity of unpasteurized soil inoculated with Frankia CjI82001 was two to three times greater than the sum of values for unpasteurized and inoculated pasteurized soils. Our results suggest a positive synergism between soil biota as a whole and Frankia inoculum with respect to host infection.     

Short-term ammonium inhibition of nitrate utilization by Anacystis nidulans and other cyanobacteria

Ammonium at low concentrations caused a rapid and effective inhibition of nitrate utilization in the light by the cyanobacterium Anacystis nidulans without affecting the cellular level of nitrate reductase activity. The inhibition was reversible, and the ability of the cells to utilize nitrate was restored immediately after ammonium had been exhausted. The inhibitory effect was dependent on consumption by the cells of the added ammonium which was rapidly incorporated into amino acids. In the presence of L-methionine-d,l-sulfoximine (MSX) or azaserine, inhibitors of the glutamine synthetase-glutamate synthase pathway, ammonium did not exhibit any inhibitory effect on nitrate utilization. Ammonium assimilation, rather than ammonium itself, seems to regulate nitrate utilization in A. nidulans. Short-term inhibition by ammonium of nitrate utilization and its prevention by MSX were also demonstrated in the filamentous cyanobacteria Anabaena and Nostoc.Abbreviations MSX L-Methionine-d-l-sulfoximine     

Phylogeny of symbiotic cyanobacteria within the genus <Emphasis Type="Italic">Nostoc</Emphasis> based on 16S rDNA sequence analyses

A phylogenetic analysis of selected symbiotic Nostoc strain sequences and available database 16S rDNA sequences of both symbiotic and free-living cyanobacteria was carried out using maximum likelihood and Bayesian inference techniques. Most of the symbiotic strains fell into well separated clades. One clade consisted of a mixture of symbiotic and free-living isolates. This clade includes Nostoc sp. strain PCC 73102, the reference strain proposed for Nostoc punctiforme. A separate symbiotic clade with isolates exclusively from Gunnera species was also obtained, suggesting that not all symbiotic Nostoc species can be assigned to N. punctiforme. Moreover, isolates from Azolla filiculoides and one from Gunnera dentata were well nested within a clade comprising most of the Anabaena sequences. This result supports the affiliation of the Azolla isolates with the genus Anabaena and shows that strains within this genus can form symbioses with additional hosts. Furthermore, these symbiotic strains produced hormogonia, thereby verifying that hormogonia formation is not absent in Anabaena and cannot be used as a criterion to distinguish it from Nostoc.The GenBank accession numbers for the cyanobacterial 16S rRNA gene sequences determined in this study are AY742447-AY742454.     

Effects of manganese on chlorophyll fluorescence in epiphytic cyano- and chlorolichens

Incubation with 10 mM MnCl₂ for 1 h decreased the effective quantum yield of photochemical energy conversion in photosystem 2 in the bipartite chlorolichen Hypogymnia physodes as well as in the bipartite cyanolichens Leptogium saturninum and Nephroma helveticum, but not in the tripartite lichen Lobaria pulmonaria. Among the bipartite species, Mn sensitivity increased in the order H. physodes < N. helveticum < L. saturninum. This equals the sequence heteromerous chlorolichen < heteromerous cyanolichen < homoiomerous (gelatinous) cyanolichen. MnCl₂ reduced non-photochemical quenching of chlorophyll fluorescence in the bipartite cyanolichens and in H. physodes; in the latter, however, this decrease was limited to light intensities above the adapted growth light intensity. Photochemical quenching was increased in H. physodes, but reduced in the bipartite cyanolichens. The results indicate that the bipartite cyanolichens L. saturninum and N. helveticum are even more sensitive to high Mn concentrations than the chlorolichen H. physodes, the low Mn tolerance of which has been already demonstrated. This agrees with results of field studies from western North America, where conifer bark under cyanolichens (including L. saturninum and N. helveticum) was found to contain less Mn than bark which only supported chlorolichens. The high sensitivity of the bipartite cyanolichens probably results from high sensitivity of the Nostoc photobiont. The high Mn tolerance of L. pulmonaria is probably not due to its being a tripartite lichen, but might be caused by high tolerance of the green-algal primary photobiont Dictyochloropsis, which is, however, not experimentally proven. The high Mn tolerance of the highly SO₂-sensitive L. pulmonaria shows that different mechanisms are responsible for Mn and SO₂ toxicity in lichens.     

Akinetes of the cyanobacteriumNostoc PCC 7524: morphological changes during synchronous germination

Following dilution into fresh medium in the light, akinetes ofNostoc PCC 7524 germinated synchronously. Synchrony was maintained at a high level during the first 24 h, at which time the young filaments were composed either of three cells (with N₂ as nitrogen source) or four cells (with NO ₃ ^- or NH ₄ ⁺ ), and at a slightly lower level during the next 24 h of growth. The pattern of cell division was similar in media containing the different nitrogen sources although the timing of the major events varied. In the presence of N₂ or NO ₃ ^- , heterocysts differentiated synchronously; the first developed invariably from a terminal cell of the young filament at approximately 19 h, the second from the other terminal cell after further vegetative cell division. Heterocyst differentiation did not occur in the presence of NH ₄ ⁺ . In the absence of nitrogen (gas phase argon: CO₂) akinete germination initially followed the same pattern as that observed in N₂, this early stage probably occurring at the expense of intracellular reserve materials.During germination, a new laminated layer, similar in structure and position to that found in the heterocyst envelope, appeared in the akinete envelope. This layer was not present in the germinating akinetes of a mutant which was incapable of forming heterocysts.     

Effects of water stress on cryptoendolithic cyanobacteria from hot desert rocks

Four strains of Chroococcidiopsis and one Chroococcus, all isolated from extreme arid desert rocks, and one marine Chroococcus, were subjected to water stress using both matric and osmotic control methods. For all Chroococcidiopsis strains, photosynthetic rates decreased with decreasing water potential. After 24h preincubation the decrease was linear but after 72h there was a sharp drop below-3400 kPa (a _w0.976). In contrast, the two Chroococcus strains showed optimum photosynthesis between-3000 and-4000 KPa. It appears, therefore, that Chroococcidiopsis in deserts may have a different survival strategy in response to aridity than Chroococcus (rare in deserts).Absolute rates of ¹⁴CO₂ uptake were higher in matric than in osmotic control systems. It is suggested that, in a matric experimental system, the water status is more representative of the natural conditions in arid environments.The consistent differences between different strains in their response to water stress suggest that this character in Cyanobacteria may be of taxonomic significance.     

Isolation and characterization of a temperate cyanophage for a tropical Anabaena strain

In this paper we describe the isolation and characterization of a temperate cyanophage N(S)1 of the genus cyanopodovirus which produces turbid plaques on the host Anabaena 77S15 isolated from tropical soil. Its properties have been compared to those of other well-characterized cyanophages. In addition, two strains of Anabaena 77S15 lysogenic for N(S)1 were isolated. N(S)1 seems to be integrated into the chromosome of the two lysogens, and a 2 kb plasmid present at a low copy number in the non-lysogenic strain is amplified significantly.     

固氮树种马占相思对巨尾桉人工林土壤团聚体粒径分布及稳定性的影响

土壤团聚体稳定性是评价土壤结构和土壤肥力的重要指标。为探究固氮树种马占相思对巨尾桉人工林土壤团聚体粒径分布及稳定性的影响,该文以17年生的巨尾桉纯林(PP)与巨尾桉/马占相思(固氮树种)混交林(MP)为研究对象,采用干筛法和湿筛法分别测定0～10 cm和10～20 cm土层团聚体粒径分布及平均重量直径(MWD)、几何平均直径(GMD)、分形维数(Dm)、水稳定性团聚体含量(WSA)、团聚体破坏率(PAD)和团聚体稳定性指数(ASI)等稳定性指标。结果表明:(1)与PP相比,MP的土壤理化性质有不同程度的提升,其中以土壤pH、有机碳(SOC)及全氮(TN)最为显著。(2)MP的土壤团聚体粒径分布优于PP,差异主要体现在>2.00 mm和<0.25 mm粒径中,均以大团聚体(>0.25 mm)为主; 相较于PP,MP的土壤团聚体机械稳定性仅在0～10 cm土层显著提高,但其团聚体水稳定性在0～10 cm和10～20 cm土层均显著提高。(3)Mantel分析结果显示团聚体稳定性与TN相关性最强,通过RDA分析进一步说明TN是驱动其团聚体稳定性变异的最关键因子。综上认为,固氮树种马占相思对巨尾桉人工林土壤团聚体稳定性具有明显改善作用,该研究结果为南亚热带桉树人工林水土保持、养分管理及可持续经营等提供了科学的理论依据。     

Bioactivity in free-living and symbiotic cyanobacteria of the genus Nostoc

Fifty cyanobacterial strains from different habitats(symbioses, soil, fresh and marine waters) belongingto the genus Nostoc were cultured and tested forbioactivity. Thirty-seven strains were isolated in ourlaboratory, the remaining were supplied by officialculture collections. All the organisms were grownunder controlled laboratory conditions. The biomasseswere lyophilised and extracted with ethanol:water toobtain a hydrophilic extract and then withdichloromethane:isopropanol to obtain a lipophilicextract. Both crude extracts were tested forantifungal (against Penicillium expansum andRhizoctonia solani) and antibacterial activity(against Agrobacterium vitis, Escherichiacoli and Staphylococcus epidermidis), and fortoxicity against Artemia salina nauplii.Twenty-four strains showed activity against at leastone of the target organisms. Bioactivity was equallydistributed between lipophilic and hydrophilicextracts, and was mostly directed against fungi (15strains) and Artemia nauplii (12 strains);antibacterial activity was less frequent (8 strains).The presence of bioactivity was independent of thestrain origin.     

Organization of the nif genes in cyanobacteria in symbiotic association with Azolla and Anthoceros

The sizes of endonuclease digestion fragments of DNA from cyanobacteria in symbiotic association with Azolla caroliniana or Anthoceros punctatus, or in free-living culture, were compared by Southern hybridization using cloned nitrogenase (nif) genes from Anabaena sp. PCC 7120 as probes. The restriction fragment pattern produced by cyanobacteria isolated from A. caroliniana by culture through symbiotic association with Anthoceros differed from that of the major symbiotic cyanobacterium freshly separated from A. caroliniana. The results indicate that minor cyanobacterial symbionts occur in association with Azolla and that the dominant symbiont was not cultured in the free-living state. Both the absence of hybridization to an xisA gene probe and the mapping of restriction fragments indicated a contiguous nifHDK organization in all cells of the symbiont in association with Azolla. On the other hand, in the cultured isolate from Azolla and in Nostoc sp. 7801, the nifD and nifK genes are nominally separated by an interval of unknown length, compatible with the interruption of the nifHDK operon by a DNA element as observed in Anabaena sp. PCC 7120. In the above cultured strains, restriction fragments consistent with a contiguous nifHDK operon were also present at varying hybridization intensities, especially in Nostoc sp. 7801 grown in association with Anthoceros, presumably due to gene rearrangement in a fraction of the cells.Non-standard abbreviations bp base pairs - kb kilobase pairs - kd kilodaltons     

Effect of yeast inoculation rate on the metabolism of contaminating lactobacilli during fermentation of corn mash

Two separate 4 (bacterial concentrations)×6 (yeast concentrations) full factorial experiments were conducted in an attempt to identify a novel approach to minimize the effects caused by bacterial contamination during industrial production of ethanol from corn. Lactobacillus plantarum and Lactobacillus paracasei, commonly occurring bacterial contaminants in ethanol plants, were used in separate fermentation experiments conducted in duplicate using an industrial strain of Saccharomyces cerevisiae, Allyeast Superstart. Bacterial concentrations were 0, 1×10⁶, 1×10⁷ and 1×10⁸ cells/ml mash. Yeast concentrations were 0, 1×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, and 4×10⁷ cells/ml mash. An increased yeast inoculation rate of 3×10⁷ cells/ml resulted in a greater than 80% decrease (P<0.001) and a greater than 55% decrease (P<0.001) in lactic acid production by L. plantarum and L. paracasei, respectively, when mash was infected with 1×10⁸ lactobacilli/ml. No differences (P>0.25) were observed in the final ethanol concentration produced by yeast at any of the inoculation rates studied, in the absence of lactobacilli. However, when the mash was infected with 1×10⁷ or 1×10⁸ lactobacilli/ml, a reduction of 0.7–0.9% v/v (P<0.005) and a reduction of 0.4–0.6% v/v (P<0.005) in the final ethanol produced was observed in mashes inoculated with 1×10⁶ and 1×10⁷ yeast cells/ml, respectively. At higher yeast inoculation rates of 3×10⁷ or 4×10⁷ cells/ml, no differences (P>0.35) were observed in the final ethanol produced even when the mash was infected with 1×10⁸ lactobacilli/ml. The increase in ethanol corresponded to the reduction in lactic acid production by lactobacilli. This suggests that using an inoculation rate of 3×10⁷ yeast cells/ml reduces the growth and metabolism of contaminating lactic bacteria significantly, which results in reduced lactic acid production and a concomitant increase in ethanol production by yeast.     

Successful inoculation of mature pine with Tricholoma matsutake

Our finding demonstrates, for the first time, that the roots of mature pine trees can be successfully inoculated with a symbiotic ectomycorrhizal fungus, the valuable matsutake mushroom. Long root segments (ca. 5–10 mm in diameter, ca. 50 cm in length) of 50-year-old Pinus densiflora trees were excavated, washed, auxin-treated (2–5 mg indole butyric acid, IBA, per root) and incubated in moist Spagnum moss. Twelve months later, short roots were regenerated, of which approximately 90% were free of mycorrhizae. Mycorrhiza-free short roots were inoculated with mycelial pieces of Tricholoma matsutake and incubated further in a sterilized substrate. Four-and-a-half months later, roots putatively colonized by Matsutake were sampled near the inoculation points. A T. matsutake-specific ITS-rDNA fragment was amplified by nested PCR from approximately 80% of the root samples analyzed, whereas approximately 66% of the root samples processed for staining with Chlorazol black E displayed characteristic T. matsutake Hartig net structures. These results confirm the symbiotic infection of mature P. densiflora roots by matsutake.     

Effect of soil liming and vesicular-arbuscular-mycorrhizal inoculation on the growth and micronutrient content of the teff plant

In a greenhouse experiment involving an acid soil teff [Eragrostis tef (Zucc.) Trotter] plants failed to grow unless the soil was limed or inoculated with either of two vesicular-arbuscular-mycorrhizal (VAM) fungi,Glomus mosseae orGlomus macrocarpum. Plant growth increased by liming and to a lesser extent by VAM fungal inoculation. Liming also enhanced root colonization by VAM fungi. Shoot micronutrient content generally increased as a result of inoculation, and decreased by increased lime applications.     

Acetate uptake by the unicellular cyanobacteria Synechococcus and Aphanocapsa

Acetate uptake by strains of Synechococcus and Aphanocapsa in short experiments required light, and was strongly inhibited by m-dichlorocarbonyl cyanide phenylhydrazone and dichlorophenyl dimethyl urea. Acetate carbon was distributed in amino acids and in the acyl portion of lipids in the same way as during growth experiments when CO₂ was available, but the reduced incorporation in the absence of CO₂ was primarily into the lipid fraction. An apparent K _m for uptake by Synechococcus and for Aphanocapsa 6308 of 20 and 180 M at pH 7.4 was obtained; corresponding V _max values were 6 and 11 nmol x min^-1 x mg protein^-1. Uptake with Synechococcus was affected by pH, with affinity decreased and maximal rate increase with rising pH. Acetate uptake was not affected by propionate or butyrate when both were added at the same time, but a light and concentration dependent inhibition developed if suspensions were preincubated with propionate. Acetate carbon moved rapidly into acid insoluble material, but after 10–15 s 75% or more of the recovered intracellular counts were in acetyl CoA. Counts in this compound were reduced by preincubation with propionate.Kinetic measurements of acetyl CoA synthetase in fractionated cell extracts gave values for K _m of about 50 M for acetate, 5 mM for propionate, 100 M for CoA and 0.38 mM for ATP. The internal pool of free CoA was measured to be about 20 M, and was reduced by preincubation with propionate. This suggests that the activity of CoA-mediated reactions may be regulated by the availability of this cofactor.Abbreviations Used CCCP m-Dichlorocarbonyl cyanide phenyl hydrazone - DCMU dichlorophenyl dimethyl urea - TCA trichloroacetic acid - Tris trishydroxymethyl amino methane - HEPES N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid