Nursing biocrusts: isolation,cultivation, and fitness test of indigenous cyanobacteria

Biological soil crusts (biocrust) are microbial communities that develop at the soil surface of drylands and play an important role in erosion control and fertility. Soil surface disturbance from a broad range of natural and human processes (e.g. fire, livestock grazing, off‐road traffic) cause significant losses in biocrust cover and associated ecosystems services. Hence, biocrust restoration is emerging as an important intervention strategy to rehabilitate degraded dryland soils. In a multistep process, we designed protocols for the establishment of “microbial nurseries” to produce photosynthetic cyanobacterial inoculum for biocrust seeding at scale. We first report on the strategy for isolation, directly from the target site, of a large culture collection of cyanobacteria that included multiple representatives of the five most common biocrust taxa. After genetic pedigreeing of these isolates, we could select those that best matched field populations genetically for scale‐up cultivation. We then developed protocols for effective cyanobacterial biomass production to obtain sufficient inoculum. This was followed by conditioning treatments (hardening off) to preacclimate this inoculum to the stressful conditions expected in the field. Finally, we show that the inoculum obtained was fit to thrive in its original soil under natural outdoor conditions if sufficient water was available. We repeated this process successfully for four sites, two in the hot Chihuanuan desert and two in the cooler Great Basin Desert, and on two textural types of soils in each. The cyanobacterial biocrust nursery approach represents a versatile, viable, and safe tool for the rehabilitation of dryland soils.     

Comparison of different methods for the isolation and purification of total community DNA from soil

The efficiency and reproducibility of DNA extraction from soil was tested for variations in lytic and purification treatments and their effect on yield and purity of DNA. The extraction yield was improved by increasing the concentration of EDTA or monovalent ions in isolation buffers, by the introduction of mechanical lysis treatments, and by the use of ethanol precipitation in place of PEG precipitation. Purity was improved using buffers with decreasing concentration of EDTA or by reducing the ionic strength of the buffer, and by all mechanical treatments. No lytic treatment was efficient on its own, the highest purity was achieved using Crombach buffer and a combination of bead-beating with lysozyme and SDS lysis followed by potassium acetate and PEG precipitation, phenol/chloroform purification, isopropanol precipitation, and spermine-HCl precipitation. Sonication sheared the DNA more than bead-beating. Lysozyme and SDS lysis without any mechanical treatments allowed isolation of larger fragments (40-90 kb). Denaturing gradient gel electrophoresis analysis of DNA isolated using a range of lytic treatments revealed alterations in band patterns which might reflect differences in the efficiency of lytic treatments.     

Effective methods for isolation and purification of extracellular vesicles from plants

Plant extracellular vesicles (EVs) play critical roles in the cross-kingdom trafficking of molecules from hosts to interacting microbes, most notably in plant defense responses. However, the isolation of pure, intact EVs from plants remains challenging. A variety of methods have been utilized to isolate plant EVs from apoplastic washing fluid (AWF). Here, we compare published plant EV isolation methods, and provide our recommended method for the isolation and purification of plant EVs. This method includes a detailed protocol for clean AWF collection from Arabidopsis thaliana leaves, followed by EV isolation via differential centrifugation. To further separate and purify specific subclasses of EVs from heterogeneous vesicle populations, density gradient ultracentrifugation and immunoaffinity capture are then utilized. We found that immunoaffinity capture is the most precise method for specific EV subclass isolation when suitable specific EV biomarkers and their corresponding antibodies are available. Overall, this study provides a guide for the selection and optimization of EV isolation methods for desired downstream applications.     

Photobioreactor cultivation strategies for microalgae and cyanobacteria

The current burden on fossil‐derived chemicals and fuels combined with the rapidly increasing global population has led to a crucial need to develop renewable and sustainable sources of chemicals and biofuels. Photoautotrophic microorganisms, including cyanobacteria and microalgae, have garnered a great deal of attention for their capability to produce these chemicals from carbon dioxide, mineralized water, and solar energy. While there have been substantial amounts of research directed at scaling‐up production from these microorganisms, several factors have proven difficult to overcome, including high costs associated with cultivation, photobioreactor construction, and artificial lighting. Decreasing these costs will substantially increase the economic feasibility of these production processes. Thus, the purpose of this review is to describe various photobioreactor designs, and then provide an overview on lighting systems, mixing, gas transfer, and the hydrodynamics of bubbles. These factors must be considered when the goal of a production process is economic feasibility. Targets for improving microalgae and cyanobacteria cultivation media, including water reduction strategies will also be described. As fossil fuel reserves continue to be depleted and the world population continues to increase, it is imperative that renewable chemical and biofuel production processes be developed toward becoming economically feasible. Thus, it is essential that future research is directed toward improving these processes. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:811–827, 2018     

Dialysis cultivation of cyanobacteria

Various aspects and the significance of the dialysis cultivation method for physiology, ecology, and biotechnology are considered. In this method, the cell culture is separated by a semipermeable membrane with a medium volume that is greater by a factor of 5–10. Dialysis cultivation is a promising method for isolating symbiotic components, selecting bionts for mixed cultures, for exometabolite production, environmental monitoring, etc. Dialysis cultures are characterized by high rates of photosynthesis and growth and by a considerable increase in the duration of the stationary stage. The small volume of the dialysis bag contains high concentrations of physiologically active cells that can be used for the production of biomass and organic substances.     

Rapid small-scale plasmid isolation by several methods from filamentous cyanobacteria

Fifteen strains of cyanobacteria, mainly Nostoc and Anabaena species, were screened for plasmids using five rapid procedures. Two of these methods, based on alkaline extraction and phenol extraction of cleared lysates respectively, were successful with a total of ten species, the latter method proving more sensitive. Plasmids ranging from less than 2.6 to at least 30 mD were isolated; most of the strains examined possessed one or two plasmids, while five lacked detectable plasmid DNA.     

Principal methods for isolation and identification of soil microbial communities

Soil microbial populations play crucial role in soil properties and influence below-ground ecosystem processes. Microbial composition and functioning changes the soil quality through decomposition of organic matter, recycling of nutrients, and biological control of parasites of plants. Moreover, the discovery that soil microbes may translate into benefits for biotechnology, management of agricultural, forest, and natural ecosystems, biodegradation of pollutants, and waste treatment systems maximized the need of scientists for the isolation and their characterization. Operations such as the production of antibiotics and enzymic activities from microorganisms of soil constitute objectives of industry in her effort to cope with the increase of population of earth and disturbance of environment and may ameliorate the effects of global climate change. In the past decades, new biochemical and molecular techniques have been developed in our effort to identify and classify soil bacteria. The goal of measuring the soil microbial diversity is difficult because of the limited knowledge about bacteria species and classification through families and orders. Molecular techniques extend our knowledge about microbial diversity and help the taxonomy of species. Measuring and monitoring soil microbial communities can lead us to better understanding of their composition and function in many ecosystem processes.     

Vertical thin-layer photoreactor for controlled cultivation of cyanobacteria

Nostoc sp. was cultivated in an air-lift reactor with continuous recirculation of the head gas phase that aerated and agitated the cyanobacterial suspension at regulated flow rates. The supply of inorganic carbon for growth was coupled with pH control, in the range of 7.7 to 8.1, by intermittent sparging of CO₂-head gas mixtures. The formation of irregular bubbles with swirling motion at the photostage of the reactor promoted efficient CO₂ transference in dense populations of Nostoc sp. (1.1 g/l) when bubbling at flow rates of 10 l/min. Biomass productivity was almost six-fold higher in the photoreactor (16.4 mg/l.h) than in a conventional system (2.8 mg/l.h). The exponential growth phase of cultures in the photoreactor amounted to 60% of the total growth period.The authors are with the Laboratorio de Alimentos, Area Microbiologia, Facultad de Quimica Bioquimica y Farmacia, Universidad Nacional de San Luis, Chacabuco y Pedernera, 5700 San Luis, Argentina     

Methods for the isolation, sterilization and cultivation of Lemnaceae

SUMMARY. A method is described for isolating and sterilizing fronds of Lemnà gibba, L. minor, L. trisulca, L. polyrrhiza and Wolffia arrhiza , employing enrichment followed by sodium hypochlorite treatment. The method enables about 4% of the total number of treated fronds to be used as initiators of clonal cultures in which bacteria responding to organic enrichment and algae are excluded. Appropriate growth media, culture vessels and culture conditions are detailed.     

动物源活性蛋白多肽的分离纯化方法研究进展

动物类中药的有效成分以蛋白多肽为主,因此活性蛋白多肽具有重要的医疗保健价值。文章分析了沉淀法、色谱法、膜分离法以及电泳法的基本原理和主要适用范围,综述了这些方法在动物源活性蛋白多肽的分离纯化中的应用,为动物源蛋白多肽的分离纯化与进一步研究提供参考,以期开发出高效、经济和环保的蛋白多肽分离纯化新技术。     

The horizontal movement of seeds in arable soil by different soil cultivation methods

1. To assess the impact of soil cultivation on the horizontal movement of seeds in arable soil, plastic beads and barley or triticale seeds were used as seed models. Different coloured beads were introduced in the field immediately before each of five cultivations: ploughing, two tine cultivations, harrowing and seed drilling. Beads were recovered from 20-cm soil cores divided into four 5-cm deep soil horizons.
2. After a typical cultivation sequence of five operations, beads were found up to 15 m from their source, although most beads were found within 2 m. Most beads were recovered from the surface 5 cm of the soil profile, except for those introduced onto the surface or at 20 cm depth before ploughing, which were concentrated below 10 cm.
3. Regression analysis was used to determine the pattern of bead movement by seed drilling. A novel analysis using Fast Fourier Transforms established the probability distribution functions of the remaining cultivation operations for horizontal movement. Using the final seed distributions, the effects of each cultivation were sequentially deconvoluted and the probability distribution functions smoothed. The proportions of beads moved were also calculated.
4. Ploughing and seed drilling moved seed the least distance compared with other cultivations. The mean distances moved were 0·36 m and 0·26 m, respectively. Tine cultivations moved beads 0·71 m and 1·21 m, while harrowing moved seed a mean distance of 1·58 m. Cultivation sequences based on ploughing are likely to limit seed movement in soil.
5. The Fourier deconvolution approach has potential for predicting future seed distributions and thus the spatial behaviour of weed patches within fields.     

Miniprep DNA isolation from unicellular and filamentous cyanobacteria

A rapid miniprep method for isolation of DNA from 12 strains of cyanobacteria belonging to groups I, III, IV and V is described. The protocol is a modification of the methods of Boyle and Lew [Boyle, J.S., Lew, A.M., 1995. An inexpensive alternative to glassmilk for DNA purification. Trends Genet. 11, 8] and the cetyltrimethyl ammonium bromide (CTAB) extraction method of Sahgai-Maroof et al. [Sahgai-Maroof, M.A., Soliman, K.M., Jorgensen, R.A., Allard, R.W., 1984. Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location and population dynamics. Proc. Natl. Acad. Sci. USA 81, 8014-80181. The new method is especially useful for obtaining cyanobacterial DNA from unicellular, filamentous and filamentous branched species. The method does not require phenol extraction and the product can be used directly for PCR amplification and restriction digestion.     

A rapid procedure for the isolation of phycobilisomes from cyanobacteria

This paper describes a method for the rapid isolation of phycobilisomes using a cationic detergent, CTAB (cetyltrimethylammonium bromide). The method has distinct advantages over those currently in use in that (i) release of intact phycobilisomes from cells in the presence of CTAB occurs in 40 s (as compared to 40-60 min of incubation required with Triton X-100), thereby reducing the chances of proteolysis of the component phycobiliproteins; and (ii) these phycobilisome preparations have reduced chlorophyll contamination in the initial stages. In addition this method also helps retain the structural and functional properties, as evidenced by spectroscopy and sodium dodecyl sulfate-polyacrylamide gel analysis.     

Nutrient medium for the isolation and cultivation of pneumococcus

A culture medium for the isolation and cultivation of pneumococci, produced in a solid or liquid form and based on raw material unsuitable for use as foodstuff (human placenta), has been developed. The amino acid composition of the medium has been studied. The medium has been found to contain 19 amino acids, to be free from ballast serum proteins and blood, and to ensure the good growth of pneumococci isolated from pathological material, the formation of the normal capsule, as well as active biological properties. The medium has proved to create elective and selective conditions enhancing the effectiveness of investigations and simplifying the isolation of pneumococci in the microbiological examination of patients.     

土壤放线菌分离方法的初步研究

本文对土壤放线菌的一种新分离技术进行了研究。结果表明,用加有氟哌酸、青霉素、制霉菌素的培养基,可以选择性地从土壤中分离放线菌,０．０５％ＳＤＳ（十二烷基碘酸钠）在４０℃自理土壤样品,可以促进放线菌的孢子萌发而获得更多的放线菌株,进一步提高放线菌的分离效果。