Repeated batch antibiotic fermentation by streptomycetes immobilized on cotton cloth

Summary Streptomyces cattleya and Streptomyces clavuligerus were found to form immobilized populations on cotton cloth. The resulting bacterial films were used as resident inocula in batch fermentations of thienamycin and cephalosporin C. During 20 cycles of 3-day batch fermentation, these antibiotics were produced at the constant yields.     

Environmental microbiome engineering for the mitigation of climate change

Environmental microbiome engineering is emerging as a potential avenue for climate change mitigation. In this process, microbial inocula are introduced to natural microbial communities to tune activities that regulate the long-term stabilization of carbon in ecosystems. In this review, we outline the process of environmental engineering and synthesize key considerations about ecosystem functions to target, means of sourcing microorganisms, strategies for designing microbial inocula, methods to deliver inocula, and the factors that enable inocula to establish within a resident community and modify an ecosystem function target. Recent work, enabled by high-throughput technologies and modeling approaches, indicate that microbial inocula designed from the top-down, particularly through directed evolution, may generally have a higher chance of establishing within existing microbial communities than other historical approaches to microbiome engineering. We address outstanding questions about the determinants of inocula establishment and provide suggestions for further research about the possibilities and challenges of environmental microbiome engineering as a tool to combat climate change.     

Competition Among Rhizobium spp. for Nodulation of Leucaena leucocephala in Two Tropical Soils

The successful nodulation of legumes by a Rhizobium strain is determined by the competitive ability of that strain against the mixture of other native and inoculant rhizobia. Competition among six Leucaena rhizobial strains in single and multistrain inoculants were studied. Field inoculation trials were conducted in an oxisol and a mollisol soil, both of which contained indigenous Leucaena-nodulating rhizobia. Strain-specific fluorescent antibodies were used for the identification of the strains in Leucaena nodules. Mixtures of three recommended inoculum strains for Leucaena spp. (TAL82, TAL582, and TAL1145) were used in peat-based inocula either alone or with one of the three other strains isolated from the sites, B213, B214, and B215. Each of these latter three strains was also used as single-strain inocula to study their competition with the native rhizobia in the two soil systems. In the oxisol soil, strains B213 and B215, when used as single-strain inocula, outcompeted the native rhizobia and formed 92 and 62% of the nodules, respectively. Strain B214 was the least competitive in oxisol soil, where it formed 30% of the nodules, and the best in mollisol soil, where it formed 70% of the nodules. The most successful competitor for nodulation in multistrain inocula was strain TAL1145, which outcompeted native and other inoculum Leucaena rhizobia in both soils. None of the strains in single or multistrain inoculants was capable of completely overcoming the resident rhizobia, which formed 4 to 70% of the total nodules in oxisol soil and 12 to 72% in mollisol soil. No strong relationship was detected between the size of the rhizosphere population of a strain and its successful occupation of nodules.     

Evaluation of commercial arbuscular mycorrhizal inocula in a sand/peat medium

Eight commercial inocula of arbuscular mycorrhizal fungi (AMF) were tested for their ability to colonize plant roots in the sand/peat medium specified by the U.S. Golf Association for use in putting greens. Using the standard assay for potency of inocula (Zea mays grown for 6 weeks in containers), inocula were added at the rate recommended by the manufacturer as well as at five and ten times the recommended rate. To ensure that growth conditions were conducive to AM formation, a soil-based inoculum of native AMF also was assessed for inoculum potential. Only three of the commercial inocula formed mycorrhizas when used at the recommended rate, and the extent of colonization ranged from 0.4 to 8%. Increasing the amount of inoculum resulted in colonization levels of 8.6 to 72.5% at the highest rate (10×). Mean colonization using the native AMF was 60%. One inoculum that did not form mycorrhizas at the recommended rate or at 5× produced 8.6% colonization at 10×. An inoculum that did not produce mycorrhizas at any application rate did contain a fungus tentatively identified as a root pathogen (Olpidium brassicae) that colonized the corn roots. The failure of five of the eight commercial inocula to colonize roots when applied at the recommended rate suggests that preliminary trials should be made before commercial AMF inocula are used in important plantings.     

Spectophotometric method for preparing the inocula of dematiaceous fungi.

The aim of this study was to adapt a spectrophotometric method for preparing the inocula of dematiaceous fungi used for in vitro susceptibility tests. Fifty-two isolates of 17 different species of dematiaceous fungi were used for this purpose. Homogeneous suspensions of conidia and hyphae of these isolates were obtained and adjusted for reading at 530 and 550 nm at 40% and 50% of transmittance. The suspensions were standardised to 1-5 x 10 e6 CFU/ml. Quality controls of the inocula were done by quantitative cultures on agar-Sabouraud plates. The inocula obtained by spectrophotometry showed little variability within all the isolates. This method can be useful for in vitro antifungal evaluation of dematiaceous fungi.     

Enhancement of biodegradability of disposable polyethylene in controlled biological soil

Plastics as polyethylene are widely used in packaging and other agricultural applications. They accumulate in the environment at a rate of 25 million tons per year. Thus, the development and use of degradable plastics was proposed as a solution for plastic waste problem. Because of the ever-increasing use of plastic films, nowadays, biodegradability has become a useful characteristic for plastics. Conversely, the introduction of biodegradable plastics has generated a need for methods to evaluate the biodegradation of these polymers in landfills and solid waste treatment systems such as composting or anaerobic digestion treatment plants. The purpose of this study was to investigate the biodegradation of disposable low-density polyethylene bags containing starch (12%), autoxidizable fatty acid ester and catalytic agents in soil. Structurally this work intended to evaluate the capacity of Phanerochaete chrysosporium (ATCC 34541) to enhance polyethylene film biodegradation in soil microcosms. Soil samples inoculated with P. chrysosporium were mixed with LDPE/starch blend films and biological changes of the films and soil were monitored for 6 months. The biodegradation of polyethylene starch blend film has been determined by the physical, chemical and biological properties of the samples such as pH, biomass, CO₂ formation, percentage elongation, relative viscosity and FTIR spectrum.     

Interactions of Fr genes and mixed-pathogen inocula in the loblolly pine-fusiform rust pathosystem

Open-pollinated loblolly pine seedlings derived from seven maternal parents were inoculated in a greenhouse with 10 different bulked inocula of the fusiform rust fungus and assessed for disease incidence. The maternal parents are heterozygous (Rr) for one or two of nine known pathotype-specific Fr genes (fusiform rust resistance genes). Progeny were genotyped to identify carriers of known R and r alleles inherited from the maternal parents. The R alleles condition resistance to specific genotypes of the fungal pathogen, while r alleles do not condition for resistance. Interactions were tested among different host genotypes and different bulked inocula. Significant differences in virulence against R genotypes were observed in the bulked inocula. Likewise, the inocula were significantly different with regard to their ability to incite disease at the family level and in r genotypes. Across the inocula, disease levels differed significantly among families. Within each family, r genotype seedlings typically exhibited higher disease rates than did R genotype seedlings. The magnitude of difference (odds ratio) between the R versus r genotypes for disease incidence within each family varied from 1 to 32 times. Significant interactions between host and pathogen genotypes were observed in four of the seven families. These greenhouse assessments using bulked inocula sources revealed wide ranges of pathogen virulence levels against the different R alleles. Barring virulence masking by unknown resistance genes, similar virulence assessments should be effective guides for the field deployment of seedlings carrying specific R alleles to regions where inocula samples show low or no corresponding virulence.     

Herpes simplex virus latency in the rabbit trigeminal ganglia: ganglionic superinfection

It has been confirmed and further documented that infection of the rabbit cornea with the E-43 strain of HSV-1 precludes superinfection of the corresponding trigeminal ganglia by another HSV strain, i.e., the challenging virus does not establish latency and can not be recovered from the ganglia. It was shown that after primary infection, a state of resistance is established in the neuronal cells of the ganglia, and although the challenging strain reaches the ganglia, it does not cause discernible acute infection, and does not displace the resident virus in the ganglia. This protection was present 6 months after primary infection, was independent of immune factors such as circulating or secretory antibodies, and was localized to the point of entry of the primary infecting strain and the sensory neurons that innervate that site. The smallest inoculum that provided protection from ganglionic superinfection was that which produced overt disease in the eye, although different degrees of disease resulted from varying inocula above this minimum. Asymptomatic primary infections produced by subminimal inocula of the E-43 strain or by the HSV recombinant strain, F(MP)F, which is avirulent for the rabbit eye, protected against severe disease and death, but the degree of protection against ganglionic superinfection was variable and depended on the time of challenge. These findings suggest that susceptible neurons in the trigeminal ganglion, when "occupied" by an infecting strain, cannot be superinfected by a second strain.     

Variation in Preference for Rhizobium meliloti Within and Between Medicago sativa Cultivars Grown in Soil

Variation in nodulation preferences for Rhizobium strains within and between Medicago sativa cultivars was assessed in the greenhouse with plants grown in Leonard jars and two soils of diverse origin (Lanark and Ottawa), using inocula consisting of effective individual or paired strains of R. meliloti which could be recognized by high-concentration antibiotic resistance. The results indicated considerable variability in host preferences for R. meliloti among plants within cultivars but not between cultivars. The implications of this variation are discussed from the point of view of possible improvement of symbiotic nitrogen fixation. With one exception, the differences in nodulation success between inoculant R. meliloti strains were consistent in Leonard jars and both soils. All introduced strains formed significantly more nodules in Renfrew soil containing few native rhizobia than in Ottawa soil with a large resident R. meliloti population. Plants grown in Lanark soil without inoculation were ineffectively nodulated by native rhizobia and yielded significantly less growth than those receiving inoculation. In contrast, the yield of inoculated plants in Ottawa soil did not significantly differ from those without inoculation due to effective nodulation by native R. meliloti. The data indicated synergistic effects on yield by certain paired strain inocula relative to the same strains inoculated individually in Lanark but not in Ottawa soil or Leonard jars.     

Application of Flow Microcalorimetry to Analytical Problems: the Preparation, Storage and Assay of Frozen Inocula of Saccharomyces cerevisiae

A simple procedure for the freezing of large batches of yeast inocula (up to 150 samples) for storage at liquid nitrogen temperatures with the retention of high viability after thawing is described. The recovered frozen cells were examined using a flow microcalorimetric procedure which enables a rapid determination of viability. The application of such inocula to antibiotic assays and growth studies is discussed.     

Effect of the use of commercial Saccharomyces strains in a newly established winery in Ronda (Málaga, Spain)

An ecological study of the yeasts present in a spontaneous and an inoculated fermentation in red wine was carried out in 2005 vintage in a winery located in the Denomination of Origin "Sierras de Málaga" (Málaga, southern of Spain). The winery operated by the first time with the 2003 vintage and since then, has used commercial yeast inocula to start alcoholic fermentation. Yeast isolates were identified by PCR-RFLP analysis of the 5.8S-ITS region from the ribosomal DNA and by mitochondrial DNA RFLP analysis. Except for non-Saccharomyces yeasts found in the fresh must before fermentation, all the isolates were found to be commercial Saccharomyces cerevisiae strains employed by the winery during the successive vintages; thus, no indigenous Saccharomyces yeasts were isolated during fermentation. The same four restriction patterns were found in non inoculated and inoculated vats, although with different frequencies. The use of commercial yeast starter in a new established winery seems to have prevented the development of a resident indigenous Saccharomyces flora.     

Heligmosomoides polygyrus: inoculum size and glucose, ion, and gas fluxes in the small intestine of mice

Female CDI mice were inoculated with 10, 50, 100, 250, or 500 larvae of Heligmosomoides polygyrus. At Days 7, 9, and 12 after infection, the anterior third of the small intestine was perfused using an in vivo technique. The distribution of worms in the mouse intestine was determined after 7, 9, and 12 days. All worms that were recovered were from the proximal half of the small intestine. When compared to uninfected controls, there was a significant increase (+56%) in glucose absorption of the small intestine at Day 7 after infection with inocula of 50 and 100 larvae; at Day 9, glucose absorption was significantly increased with a 10-larvae inoculum. A decrease in glucose absorption occurred at Days 7 and 9 after infection with a 500-larvae inoculum. Net water absorption was significantly increased (+183%) with the 50- and 100-larvae inocula at Day 7, but was significantly reduced at Day 9 after infection with the 50-, 100-, 250-, and 500-larvae inocula. Both Cl- and Na+ absorption were significantly increased with the 50-, 100-, and 250-larvae inocula at Day 7 after infection; at 9 and 12 days, there was significant net secretion of both ions. In control mice, there was net secretion of K+, while with the 50-, 100-, and 250-larvae inocula on Day 7 there was significant net absorption of K+ ions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sheared-Root Inocula of Vesicular-Arbuscular Mycorrhizal Fungi

For efficient handling, vesicular-arbuscular mycorrhizal fungi should be processed into small and uniform inocula; however, processing can reduce the inoculum density. In this article we describe the preparation and use of sheared-root inocula of Glomus spp. in which inoculum densities were increased during processing. Our objectives were to determine inoculum viability and density after shearing and to ascertain if the sheared inocula could be pelletized or used with a gel carrier. Root samples were harvested from aeroponic cultures, blotted dry, cut into 1-cm lengths, and sheared in a food processor for up to 80 s. After shearing, the inoculum was washed over sieves, and the propagule density in each fraction was determined. Sheared inocula were also encapsulated in carrageenan or used in a gel carrier. Shearing aeroponically produced root inocula reduced particle size. Propagule density increased with decreasing size fraction down to a size of 63 μm, after which propagule density decreased. The weighted-average propagule density of the inoculum was 135,380 propagules g (dry weight) of sheared root material^-1. Sheared roots were encapsulated successfully in carrageenan, and the gel served as an effective carrier. Aeroponic root inoculum was stored dry at 4°C for 23 months without significant reduction in propagule density; however, this material was not appropriate for shearing. Moist roots, useful for shearing, began to lose propagule density after 1 month of storage. Shearing proved to be an excellent method to prepare viable root inocula of small and uniform size, allowing for more efficient and effective use of limited inoculum supplies.     

Culture of Clostridium pasteurianum in Defined Medium and Growth as a Function of Sulfate Concentration

Clostridium pasteurianum strain W-5 was selected as an anaerobe which may be grown from large inocula in defined media with sulfate as its primary sulfur source. Since it is important to keep inocula small in minimizing transfer of sulfur sources, culture conditions were optimized. The medium devised decreased lag period and generation time when compared with other media, but growth could not be induced consistently with 6 x 10(6) cells per ml or less. Addition of trace elements, chelating agents, reducing agents, metabolites, and spent medium from various stages of growth did not stimulate growth from small inocula. Generation time was 85 min on inoculation with 10(7) or more cells per ml taken from young stocks, but the lag period decreased somewhat with larger inocula. On the other hand, generation time and lag period increased with age of the inoculum. The total yield of cells increased when buffer capacity was increased. Growth of C. pasteurianum W-5 was dependent upon sulfate at relatively low sulfate concentrations, and the organism is thus suitable for study of sulfur metabolism. No evidence of a maintenance requirement for sulfate was detected.     

Aerobic and anaerobic degradation and mineralization of 14C-chitin by water column and sediment inocula of the York River estuary, Virginia.

Potential rates of chitin degradation (Cd) and mineralization (Cm) by estuarine water and sediment bacteria were measured as a function of inoculum source, temperature, and oxygen condition. In the water column inoculum, 88 to 93% of the particulate chitin was mineralized to CO2 with no apparent lag between degradation and mineralization. No measurable dissolved pool of radiolabel was found in the water column. For the sediment inocula, 70 to 90% of the chitin was degraded while only 55 to 65% was mineralized to CO2. 14C label recoveries in the dissolved pool were 19 to 21% for sand, 17 to 24% in aerobic mud, and 12 to 21% for the anaerobic mud. This uncoupling between degradation and mineralization occurred in all sediment inocula. More than 98% of the initial 14C-chitin was recovered in the three measured fractions. The highest Cd and Cm values, 30 and 27% day-1, occurred in the water column inoculum at 25 degrees C. The lowest Cd and Cm values were found in the aerobic and anaerobic mud inocula incubated at 15 degrees C. Significant differences in Cd and Cm values among water column and sediment inocula as well as between temperature treatments were evident. An increased incubation temperature resulted in shorter lag times before the onset of chitinoclastic bacterial growth, degradation, and mineralization and resulted in apparent Q10 values of 1.1 for water and 1.3 to 2.1 for sediment inocula. It is clear that chitin degradation and mineralization occur rapidly in the estuary and that water column bacteria may be more important in this process than previously acknowledged.     

Cryopreservation of Holospora undulata, a bacterial parasite of the ciliate Paramecium caudatum

We investigated cryopreservation of horizontal transmission stages of Holospora undulata, a micronucleus-specific bacterial parasite of Paramecium caudatum. Unlike in previous studies on related Holospora species, protocols using glycerol as cryoprotectant failed entirely. In contrast, freezing with dimethyl sulfoxide (Me2SO) conserved infectiousness of nearly all replicate inocula, although infection success was considerably lower than that of fresh inocula. Infection probability was enhanced by increasing the Me2SO concentration from 5 to 10%, and by freezing at -196 degrees C rather than -80 degrees C. Prolonged storage of up to 3 months had no significant effect on the viability of the inocula.     

Development of Fungal Inocula for Bioaugmentation of Contaminated Soils

This report describes novel fungal inocula for bioaugmentation of soils contaminated with hazardous organic compounds. The inocula are in the form of pelleted solid substrates coated with a sodium alginate suspension of fungal spores or mycelial fragments and incubated until overgrown with the mycelium of selected lignin-degrading fungi. The organisms evaluated were Phanerochaete chrysosporium (BKM F-1767, ATCC 42725), P. sordida (HHB-8922-Sp), Irpex lacteus (Mad-517, ATCC 11245), Bjerkandera adusta (FP-135160-Sp, ATCC 62023), and Trametes versicolor (MD-277). The pelleted fungal inocula resisted competition and proliferation from indigenous soil microbes, were lower in moisture content than current fungal inocula, and had sufficient mechanical strength to allow handling and introduction into the soil without a change in the mechanical consistency of the pellets. Inoculated at a rate of 3% in artificially contaminated nonsterile soil, I. lacteus, B. adusta, and T. versicolor removed 86, 82, and 90%, respectively, of the pentachlorophenol in 4 weeks. A mathematical model was developed to explain moisture distribution in a hydrogel-coated pelleted substrate.     

Biodegradability and adsorption on lake sediments of cyanobacterial hepatotoxins and anatoxin-a

Cyanobacterial hepatotoxins and anatoxin-a, a neurotoxin, were shown to be degraded when crude extracts of lysed toxic laboratory strains of cyanobacteria were exposed to natural populations of micro-organisms from lakes. While anatoxin-a decayed equally fast with all the inocula from lake sediment and water, the degradation rate of hepatotoxins was higher with inocula from places at which cyanobacterial water blooms had occurred than with inocula from places with no known mass occurrences of cyanobacteria. Degradation was slowest when an inoculum from a humic lake was used. A part of the loss of the toxins was shown to be due to adsorption on lake sediments.     

Staphylococcus aureus pneumonia in hamsters with elastase-induced emphysema--the virulence enhancing activity of mucin

Staphylococcus aureus pneumonia was studied in hamsters with elastase-induced emphysema and in saline-treated controls. Emphysematous animals cleared endotracheally administered inocula of S. aureus in saline as rapidly as controls. After infection with S. aureus in 1% mucin, emphysematous animals had impaired clearance compared with controls; after infection with S. aureus in 5% mucin, emphysematous animals had decreased survival at 96 hr compared to controls (6/24 vs 15/24, P less than 0.01 Fisher's exact test). Bronchoalveolar lavage of uninfected elastase-treated hamsters yielded twice as many cells per animal as uninfected controls (P less than 0.0001, paired t test), and the cells contained a higher percentage of polymorphonuclear leukocytes (37.8% vs 3.8%, P less than 0.0001). Lavage cells from both groups of animals were equally efficient per cell at killing opsonized S. aureus in an in vitro bactericidal assay. Hamsters with elastase-induced emphysema were resistant to infection with S. aureus alone despite marked structural abnormalities in the lung, possibly due in part to increased numbers of resident phagocytic cells. After infection with S. aureus in mucin as a virulence enhancing factor emphysematous animals had impaired clearance and decreased survival compared to controls.     

Water temperature and stratification depth independently shift cardinal events during plankton spring succession

In deep temperate lakes, the beginning of the growing season is triggered by thermal stratification, which alleviates light limitation of planktonic producers in the surface layer and prevents heat loss to deeper strata. The sequence of subsequent phenological events (phytoplankton spring bloom, grazer peak, clearwater phase) results in part from coupled phytoplankton–grazer interactions. Disentangling the separate, direct effects of correlated climatic drivers (stratification‐dependent underwater light climate vs. water temperature) from their indirect effects mediated through trophic feedbacks is impossible using observational field data, which challenges our understanding of global warming effects on seasonal plankton dynamics. We therefore manipulated water temperature and stratification depth independently in experimental field mesocosms containing ambient microplankton and inocula of the resident grazer Daphnia hyalina. Higher light availability in shallower surface layers accelerated primary production, warming accelerated consumption and growth of Daphnia, and both factors speeded up successional dynamics driven by trophic feedbacks. Specifically, phytoplankton peaked and decreased earlier and Daphnia populations increased and peaked earlier at both shallower stratification and higher temperature. The timing of ciliate dynamics was unrelated to both factors. Volumetric peak densities of phytoplankton, ciliates and Daphnia in the surface layer were also unaffected by temperature but declined with stratification depth in parallel with light availability. The latter relationship vanished, however, when population sizes were integrated over the entire water column. Overall our results suggest that, integrated over the entire water column of a deep lake, surface warming and shallower stratification independently speed up spring successional events, whereas the magnitudes of phytoplankton and zooplankton spring peaks are less sensitive to these factors. Therefore, accelerated dynamics under warming need not lead to a trophic mismatch (given similar grazer inocula at the time of stratification). We emphasize that entire water column dynamics must be studied to estimate global warming effects on lake ecosystems.