Use of reporter transposons for tagging and detection of Mycobacterium sp. strain 1B in PAH-contaminated soil

An environmental Mycobacterium able to degrade phenanthrene, pyrene and fluoranthene was transformed with an IS1096-based transposon marker system. Electroporation and subsequent delivery of the transposon enabled formation of constitutive lacZ transformants, with similar growth rates on pyrene and R2A media to the parental strain. A semi-selective medium was developed to recover and detect colonies of the transformed strain after inoculation into polycyclic aromatic hydrocarbon-contaminated soil. Microcosm experiments involving inoculation of the tagged Mycobacterium strain into a historically PAH-contaminated soil indicated survival when an appropriate carbon source was available. The results reported show that transposon systems developed for clinical mycobacterial isolates are also applicable for use in environmental isolates. The results also show that inoculated Mycobacterium strains could survive for at least 100 days at 10⁶–10⁷ cfu g⁻¹ in the PAH-contaminated soil tested here.     

Kinetic properties of a commercial and a native inoculum for aerobic milk fat degradation

The aerobic fat biodegradation potential and growth characteristics of a commercial and a native inoculum (activated sludge from a dairy wastewater treatment pond), were evaluated. Batch tests were conducted with a medium based on butter oil, as the sole source of carbon, and mineral salts. Residual fat, biomass and CO(2) production were measured. Overall fat removal values were above 78% for both inocula. The growth kinetics of the commercial and native inocula followed Haldane and Monod models respectively. Both inocula showed a similar behaviour when butter oil concentration was under 360 mg/l; at higher values, the difference between the growth rates increased as a consequence of the inhibition exhibited by the commercial inoculum. The selection of an inoculum for bioaugmentation of bioreactors in the wastewater treatment requires a comprehensive knowledge of their degradation ability and tolerance to fluctuating compounds and of the operational conditions that will be utilized.     

Inoculation of grass and tree seedlings used for reclaiming eroded areas in Iceland with mycorrhizal fungi

The objective of this study was to investigate the response of plant species used for reclamation of eroded areas in Iceland to inoculation with mycorrhizal fungi. In a greenhouse trial,Leymus arenarius andDeschampsia beringensis were grown in pots with volcanic ash collected from a site near the Mt. Hekla volcano in Iceland and were inoculated with arbuscular mycorrhizal fungi (AMF) isolatesGlomus mosseae BEG25 orGlomus intraradices BEG75. In two field experiments conducted on volcanic tephra fields near Mt. Hekla, a native soil inoculum or commercial inocula TerraVital-D and Terra Vital-G Ecto Mix were compared for efficacy onL. arenarius andBetula pubescens. After four months of growth, the presence of AMF in the pot experiment significantly increased the capacity of grass root systems to bind soil particles. In the field, inoculation significantly increased the number ofL. arenarius plants, which emerged from seed and their subsequent survival and growth. Seedlings ofB. pubescens grew best following inoculation with ectomycorrhizal fungal (ECMF) inoculum and a subsequent application of inorganic NP-fertilizer. The addition of native soil inoculum had almost no effect on growth of either grass or trees. Our results indicate that reclamation of eroded areas in Iceland could benefit from the use of a ppropriate mycorrhizal fungi, which might improve plant establishment and growth and increase soil aggregation and stability.     

Enhancing bioremediation of diesel-fuel-contaminated soil in a boreal climate: Comparison of biostimulation and bioaugmentation

Cold conditions delay bioremediation of oil hydrocarbons, but other bottlenecks also affect the outcome. Means to stimulate biodegradation of diesel oil hydrocarbons in contaminated soil were compared. Different combinations of nutrients, bulking agent, aeration, and microbial inocula were examined in lab simulations, and effective combinations were tested in field conditions. Bacterial communities were investigated by cloning and sequencing 16S-rRNA genes. Efficient degradation was attained when slow-release nutrients and aeration were used simultaneously. Bacterial inocula did not advance soil remediation, nor did they have any lasting effect on bacterial densities. Bacteria belonging to Proteobacteria were dominant in all cases. In the field test, a bulking agent promoting air passage through the soil ensured sufficient aeration, while forced air decreased the soil moisture excessively. We concluded that biostimulation via optimization of nitrogen and oxygen supply significantly improved bioremediation of oil-contaminated soil, while bioaugmentation had no additional effect.     

Biodegradation of Polycyclic Aromatic Hydrocarbons (PAHs) by Trichoderma reesei FS10-C and Effect of Bioaugmentation on an Aged PAH-Contaminated Soil

ABSTRACT?The co-metabolism of benzo[a]pyrene (B[a]P) and the capacity of the fungus Trichoderma reesei FS10-C to bioremediate an aged polycyclic aromatic hydrocarbon (PAH)-contaminated soil were investigated. The fungal isolate removed about 54% of B[a]P (20 mg L^?1) after 12 days of incubation with glucose (10 g L^?1) supplementation as a co-metabolic substrate. Bioaugmented microcosms showed a 25% decrease in total PAH concentrations in soil after 28 days, and the degradation percentages of 3-, 4-, and 5(+6)-ring PAHs were 36%, 35%, and 25%, respectively. In addition, bioaugmented microcosms exhibited higher dehydrogenase (DHA) and fluorescein diacetate hydrolysis (FDAH) activities and increased average well-color development (AWCD), Shannon-Weaver index (H), and Simpson index (D) significantly. Principal component analysis (PCA) also distinguished clear differentiation between treatments, indicating that bioaugmentation restored the microbiological function of the PAH-contaminated soil. The results suggest that bioaugmentation by T. reesei FS10-C might be a promising bioremediation strategy for aged PAH-contaminated soils.     

Effects of preculture variability on clavulanic acid fermentation.

The production profile of clavulanic acid by Streptomyces clavuligerus was shown to be strongly dependent on inoculum activity. Two sets of fermentations (A and B) were investigated at industrial pilot-plant scale using complex media. Type A fermentations were inoculated using late exponential growth phase mycelia. Type B fermentations were inoculated using mycelia harvested at stationary phase. Productivities throughout type A fermentations were consistently higher than type B, reaching a maximum at about 70 h and then decaying to the same final productivities at 140 h of type B runs. Several scheduling alternatives, based on combinations of the two inocula types and different fermentation lengths, were compared in terms of the overall process economics (fermentation and downstream). An increase of ca. 22% on the overall process profit is predicted using late exponential growth phase inocula and a fermentation duration of only 96 h. A new operating strategy was thus proposed for inoculum production based on the control of preculture activity using off-gas analysis. This method ensures higher productivity and better batch-to-batch reproducibility of clavulanic acid fermentations than traditional methods based on constant age inocula.     

Media and Fermentation Processes for the Rapid Production of High Concentrations of Stable Blastospores of the Bioinsecticidal Fungus Paecilomyces fumosoroseus

In shake flask and fermentor studies, various media components and culture inocula were tested to improve P. fumosoroseus spore production rates, yield and stability. To evaluate inoculum potential and inoculum scale-up for fermentor studies, conidia and liquid culture-produced spores of various strains of P. fumosoroseus were compared as inoculum. Inoculation of liquid cultures with blastospores at concentrations of at least 1×10⁶ spores mL^-1 resulted in the rapid production of high concentrations of blastospores (∼1×10⁹ spores mL^-1, 48 h fermentation time) for all strains tested. The rapid germination rate of blastospores (90% after 6 h incubation) compared to conidia (>90% after 16 h incubation) and the use of higher inoculum rates reduced the fermentation time from 96 to 48 h for maximal spore yields. A comparison of various complex nitrogen sources showed that liquid media supplemented with acid hydrolyzed casein or yeast extract supported the production of high concentrations of blastospores that were significantly more desiccation-tolerant (79-82% survival after drying) when compared to blastospores produced in media supplemented with other nitrogen sources (12-50% survival after drying). For rapid spore production, requirements for trace metals and vitamin supplementation were dependent on the type of hydrolyzed casein used in the medium. Fermentor studies with two strains of P. fumosoroseus showed that high concentrations (1.3-1.8×10⁹ spores mL^-1) of desiccation-tolerant blastospores could be produced in 48-h fermentations. These studies have demonstrated that the infective spores of various strains of the fungal bioinsecticide Paecilomyces fumosoroseus can be rapidly produced using deep-tank, liquid culture fermentation techniques.     

Ex situ slurry phase bioremediation of chrysene contaminated soil with the function of metabolic function: process evaluation by data enveloping analysis (DEA) and Taguchi design of experimental methodology (DOE)

Bioremediation of chrysene in soil matrix was evaluated in soil slurry phase bioreactor in conjugation with metabolic functions (aerobic, anoxic and anaerobic), microenvironment (single and mixed) conditions and nature of mixed consortia (native/resident mixed microflora and bioaugmented inoculum). Twelve experiments were operated independently in agitated-batch reactor keeping all other operating conditions constant (substrate loading rate--0.084 g chrysene/kg soil-day; soil loading rate--10 kg soil/m(3)-day (3:25 soil water ratio); operating temperature--35+/-2 degrees C). Data envelopment analysis (DEA) procedure was employed to analyze the performance of experimental variations in terms of chrysene degradation and pH. The efficacy of anoxic metabolism over the corresponding aerobic and anaerobic metabolic functions was documented. Aerobic metabolic function showed effective degradation capability under mixed microenvironment after augmentation with anaerobic inoculum. Anaerobic metabolic function showed lowest degradation potential. Application of bioaugmentation showed positive influence on the chrysene degradation rate. Design of experimental methodology (DOE) by Taguchi approach was applied to evaluate the effect of four selected factors (native soil microflora, microenvironment, metabolic function and bioaugmentation) on the chrysene degradation process. The optimized factors derived from analysis depicted the requirement of native soil microflora under anoxic metabolic function using mixed microenvironment after augmenting with anaerobic inoculum for achieving effective chrysene degradation efficacy.     

Isolation and characterization of indigenous soil bacteria for bioaugmentation of PAH contaminated soil of semiarid Patagonia, Argentina

The aim of this work was to isolate PAH degrading-bacteria from contaminated Patagonia soil with the ability to tolerate the usual environmental stresses (oligotrophic and dryness conditions). Two approaches were utilized to obtain PAH-degrading bacteria from the Patagonian soil. With a traditional enrichment approach only the PAH- degrading strain 36 was isolated. Using a direct isolation approach three PAH-degrading strains (1A, 22A and 22B) were isolated. The phylogenetic analysis revealed that all isolates belonged to Sphingomonas genus. The PAH degrading activity and the resistance to stress conditions of the strains were determined and compared with those of the exogenous PAH-degrading Sphingomonas paucimobilis 20006FA. The strains 1A, 22A and 36 were phylogenetically closely related between them and with the strain 20006FA. The strain 22B, that showed a different phylogenetic position, was more resistant to C-starvation and drying conditions than other Patagonian strains. The effect of the inoculation of these strains on phenanthrene-induced mineralization and elimination was studied in Patagonian soil artificially contaminated, at different environmental conditions. The results suggest that strain 22B is the most suitable strain for bioaugmentation in PAH-contaminated soils of Central Patagonia, due to its adaptation to the usual environmental conditions. Our results show the importance of a detailed physiological characterization of isolates for autochthonous bioaugmentation strategies success.     

Production of staphylococcal enterotoxin in mixed cultures

Two Staphylococcus aureus strains were grown in brain-heart infusion (BHI) broth and a meat medium with Bacillus cereus, Streptococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa. Both S. aureus strains grew well and produced enterotoxin in the presence of S. faecalis in BHI broth; however, enterotoxin production was observable in the meat medium only when the S. aureus inoculum was greater than the S. faecalis inoculum. S. aureus FRI-100 grown with B. cereus produced enterotoxin in both media only when the S. aureus inoculum was much higher than the B. cereus inoculum (10 versus 10(4) CFU), whereas S. aureus FRI-196E produced enterotoxin in both media at all inoculum combinations except in the meat medium, when the inocula of the two organisms were the same. S. aureus grown with E. coli in BHI broth produced enterotoxin at all inoculum combinations except when the E. coli inoculum was greater than the S. aureus inoculum; however, in the meat medium, enterotoxin was produced only when the S. aureus inoculum was much greater than the E. coli inoculum (10 versus 10(4) CFU), S. aureus FRI-100 grown with P. aeruginosa in either medium produced enterotoxin only when the S. aureus inoculum was much greater than the P. aeruginosa inoculum (10 versus 10(3) or 10(4) CFU). It can be concluded from these results that enterotoxin production is unlikely in mixed cultures unless the staphylococci outnumber the other contaminating organisms.     

Production of staphylococcal enterotoxin in mixed cultures.

Two Staphylococcus aureus strains were grown in brain-heart infusion (BHI) broth and a meat medium with Bacillus cereus, Streptococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa. Both S. aureus strains grew well and produced enterotoxin in the presence of S. faecalis in BHI broth; however, enterotoxin production was observable in the meat medium only when the S. aureus inoculum was greater than the S. faecalis inoculum. S. aureus FRI-100 grown with B. cereus produced enterotoxin in both media only when the S. aureus inoculum was much higher than the B. cereus inoculum (10 versus 10(4) CFU), whereas S. aureus FRI-196E produced enterotoxin in both media at all inoculum combinations except in the meat medium, when the inocula of the two organisms were the same. S. aureus grown with E. coli in BHI broth produced enterotoxin at all inoculum combinations except when the E. coli inoculum was greater than the S. aureus inoculum; however, in the meat medium, enterotoxin was produced only when the S. aureus inoculum was much greater than the E. coli inoculum (10 versus 10(4) CFU), S. aureus FRI-100 grown with P. aeruginosa in either medium produced enterotoxin only when the S. aureus inoculum was much greater than the P. aeruginosa inoculum (10 versus 10(3) or 10(4) CFU). It can be concluded from these results that enterotoxin production is unlikely in mixed cultures unless the staphylococci outnumber the other contaminating organisms.     

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.     

Microcycle Conidiation and Spore-Carrying Capacity of Colletotrichum gloeosporioides on Solid Media

The effect of spore inoculum density, medium concentration, and temperature on slime-spot formation, spore yield, and mycelium production by Colletotrichum gloeosporioides on agar media were studied with a simple microplate assay. A steady-state spore yield (spore-carrying capacity) independent of inoculum density was reached only on media that supported good fungal growth and sporulation. The spore-carrying capacity was reached earlier, the denser the inoculum. On standard mycological media a high inoculum density (2.5 × 10⁶ spores per ml) resulted in a slimy mass of conidia forming a slime spot, a phenomenon associated with greatly reduced mycelium formation and indicative of microcycle conidiation. In contrast, for a similar inoculum density, enhanced mycelial growth preceded sporulation and overrode slime-spot formation on highly concentrated media; a very low medium concentration resulted in much less mycelium, but spore production was also decreased. Exposure to suboptimal growth temperatures of 36 to 48°C for up to 8 days did not induce microcycle conidiation from inocula that did not form a slime spot at 28°C.     

Feedbacks of Soil Inoculum of Mycorrhizal Fungi Altered by N Deposition on the Growth of a Native Shrub and an Invasive Annual Grass

Anthropogenic nitrogen (N) deposition causes shifts in vegetation types as well as species composition of arbuscular mycorrhizal (AM) fungi and other soil microorganisms. A greenhouse experiment was done to determine whether there are feedbacks between N-altered soil inoculum and growth of a dominant native shrub and an invasive grass species in southern California. The region is experiencing large-scale loss of Artemisia californica shrublands and replacement by invasive annual grasses under N deposition. Artemisia californica and Bromus madritensis ssp. rubens were grown with soil inoculum from experimental plots in a low N deposition site that had (1) N-fertilized and (2) unfertilized soil used for inoculum, as well as (3) high-N soil inoculum from a site exposed to atmospheric N deposition for four decades. All treatments plus a nonmycorrhizal control were given two levels of N fertilizer solution. A. californica biomass was reduced by each of the three inocula compared to uninoculated controls under at least one of the two N fertilizer solutions. The␣inoculum from the N-deposition site caused the greatest growth depressions. By contrast, B.␣madritensis biomass increased with each of the three inocula under at least one, or both, of the N solutions. The different growth responses of the two plant species may be related to the types of AM fungal colonization. B. madritensis was mainly colonized by a fine mycorrhizal endophyte, while A. californica had primarily coarse endophytes. Furthermore, A. californica had a high level of septate, nonmycorrhizal root endophytes, while B. madritensis overall had low levels of these endophytes. The negative biomass response of A. californica seedlings to high N-deposition inoculum may in part explain its decline; a microbially-mediated negative feedback may occur in this system that causes poor␣seedling growth and establishment of A.␣californica in sites subject to N deposition and B. madritensis invasion.     

Ectomycorrhizal synthesis on seedlings of Afzelia quanzensis Welw. using various types of inoculum

Ectomycorrhizal synthesis on seedlings of Afzelia quanzensis was initiated in the greenhouse and in the field using basidiospores or soil inoculum originating from fungi associated with adult trees of A. quanzensis, Brachystegia microphylla, B. spiciformis, and Julbernardia globiflora. Of the spore inocula used, only a Pisolithus sp. associated with adult A. quanzensis formed mycorrhizae. Seedlings raised in contact with all soil inocula formed mycorrhizae; however, the mycorrhizal types formed differed between soil inoculum used in the greenhouse and soil inoculum directly used in the field. A. quanzensis has a low specificity for mycorrhizal association. The concepts of ectomycorrhizal succession are also applicable to African savanna ecosystems.     

Role of inoculum preparation and density on the bioremediation of 2,4-D-contaminated soil by bioaugmentation

The effect of inoculum preparation and density on the efficiency of remediation of 2,4-dichlorophenoxyacetic acid (2,4-D) by bioaugmentation was studied in non-sterile soil. A 2,4-D-degrading Pseudomonas cepacia strain (designated BRI6001) was used initially in liquid culture to determine the effects of pre-growth induction and of inoculum density. The time for complete 2,4-D degradation was reduced by 0.5 day for each log increase of inoculum density. In mixed (BRI6001 and soil bacteria) liquid cultures, a competition effect for 2,4-D became apparent at low inoculum levels (less than 10 10⁵ cfu/ml BRI6001 for 10⁸ cfu/ml soil bacteria) but only when the soil bacteria included indigenous 2,4-D degraders. In static non-sterile soil, the effect of inoculum density on 2,4-D degradation was comparable to that in liquid culture but only at high inoculation levels. At lower levels, a biological effect for 2,4-D degradation became apparent, as was observed in mixed liquid cultures, whereas at intermediate levels, a combination of biological, physical and chemical factors decreased the efficiency of bioaugmentation. The acclimation period for 2,4-D degradation in soil bioaugmented with BRI6001 reflected mainly the time required for cell induction and, presumably, for overcoming the physical limitation of diffusion of both 2,4-D and added bacteria in the soil matrix. Correspondence to: R. SamsonISSUED AS NRCC 33848     

Maximizing the expression of recombinant proteins in Escheria coli by manipulation of culture conditions

The expression of hybrid proteins β-galactosidase-human insulin chain A (constitutive), and β-galactosidase-human insulin chain A (constitutive), and β-galactosidase-human insulin chain B (inducible) was studied. The main aspects covered included plasmid stability and optimization of expression levels. In both systems, the ampicillin used for selective pressure exerted its action for only a few minutes during culture, hardly affecting plasmid segregation trends. Without the antibiotic, segregants were very low and reached a level of 10% only after seven sub-cultures. Expression levels in the A chain system were closely related to the biomass production. Maximum levels were reached developing the inoculum in minimal media, as well as by balancing and statistically optimizing the medium. The B chain system appeared to have higher plasmid stability than the A chain one. By optimizing the medium, similar induction levels to those obtained by using IPTG were attained using lactose as the main carbon source. Hybrid production was inversely related to the cell/glucose yield. In both systems, sub-culturing the bacteria in minimal medium increased substantially the production of hybrid proteins. Sub-culturing in rich medium with small amounts of ampicillin had the opposite effect. It seems that plasmid copy number dynamics could be playing an important role in this phenomenon.     

Production, Survival and Evaluation of Liquid Culture-produced Inocula of Coniothyrium minitans Against Sclerotinia sclerotiorum

Growth of Coniothyrium minitans on potato dextrose broth was compared with that on an inexpensive molasses-yeast liquid medium at 18-22°C in static culture. Biomass and conidial production were, in general, similar, although the rate of biomass production was quicker and conidial production was slightly greater per unit volume of medium in the molasses-yeast medium. Air-dried biomass from molasses-yeast liquid culture containing mycelia, pycnidia and conidia of C. minitans was mixed (12%, w/w) with kaolin to give a kaolin-biomass dust. The ability of C. minitans to survive and subsequently infect and reduce the viability of sclerotia of Sclerotinia sclerotiorum from this kaolin-biomass dust was found to be little affected by storage for 48 weeks between 4 and 15°C but was decreased by higher storage temperatures. The kaolin-biomass dust preparation did not differ from a standard maizemeal-perlite inoculum of C. minitans in its ability to infect sclerotia of S. sclerotiorum or reduce their viability or carpogenic germination in glasshouse and field pot bioassays. Further, when either inoculum was applied once to glasshouse soil naturally infested with S. sclerotiorum prior to planting three successive crops of lettuce, the pattern of disease control, reduction of sclerotial numbers/ plot, infection of sclerotia, reduction of sclerotial viability and survival in soil were similar for both inocula. The potential for the commercial development of liquid-culture-produced inocula of C. minitans is discussed.     

Perspectives and vision for strain selection in bioaugmentation

Notwithstanding the phenomenally large and ever-increasing resource of pollutant-degrading microbial isolates in laboratories around the globe, inoculum survival remains the 'Achilles' heel' for bioaugmentation of contaminated land. Considerable effort has been invested into inoculum strain selection to facilitate pollutant biodegradation, ranging from the isolation of 'superbugs,' which are microorganisms highly resilient to environmental stresses, harboring catabolically superior pollutant-degrading enzymes, to the other extreme in 'priming', where pollutant degradation is carried out through the addition of soil enriched with an undefined consortium of pollutant-degrading microorganisms.     

Effect of arbuscular mycorrhizal fungal isolates and organic manure on growth and mycorrhization of micropropagated Dendrocalamus asper plantlets and on spore production in their rhizosphere

The effect of three different inocula of arbuscular mycorrhizal (AM) fungi was studied on the growth, biomass, P uptake, root colonization and AM spore production in tisssue culture-raised Dendrocalamus asper (bamboo) plantlets in two types of soil: (1) sand?:?soil 1?:?1 (2) sand?:?soil?:?organic manure 1?:?1?:?0.5 (v/v). The first two inocula were isolated from the bamboo rhizosphere and the third from teak rhizosphere soil. After 12 months, significant positive effects of inoculum on shoot P concentration, root colonization and spore production were observed in the sand?:?soil medium. In the organic manure-amended medium, these parameters further improved. Amendment with organic manure highly influenced spore production (5.3 to 17.8 fold increase) and enhanced height and dry biomass of D. asper plantlets.