Quantification of Gordona amarae Strains in Foaming Activated Sludge and Anaerobic Digester Systems with Oligonucleotide Hybridization Probes

Previous studies have shown the predominance of mycolic acid-containing filamentous actinomycetes (mycolata) in foam layers in activated sludge systems. Gordona (formerly Nocardia) amarae often is considered the major representative of this group in activated sludge foam. In this study, small-subunit rRNA genes of four G. amarae strains were sequenced, and the resulting sequences were compared to the sequence of G. amarae type strain SE-6. Comparative sequence analysis showed that the five strains used represent two lines of evolutionary descent; group 1 consists of strains NM23 and ASAC1, and group 2 contains strains SE-6, SE-102, and ASF3. The following three oligonucleotide probes were designed: a species-specific probe for G. amarae, a probe specific for group 1, and a probe targeting group 2. The probes were characterized by dissociation temperature and specificity studies, and the species-specific probe was evaluated for use in fluorescent in situ hybridizations. By using the group-specific probes, it was possible to place additional G. amarae isolates in their respective groups. The probes were used along with previously designed probes in membrane hybridizations to determine the abundance of G. amarae, group 1, group 2, bacterial, mycolata, and Gordona rRNAs in samples obtained from foaming activated sludge systems in California, Illinois, and Wisconsin. The target groups were present in significantly greater concentrations in activated sludge foam than in mixed liquor and persisted in anaerobic digesters. Hybridization results indicated that the presence of certain G. amarae strains may be regional or treatment plant specific and that previously uncharacterized G. amarae strains may be present in some systems.     

Comparative studies on utilization of fatty acids and hydrocarbons in Nocardia amarae and Rhodococcus spp.

Comparative investigations on the substrate utilization of Nocardia amarae and Rhodococcus spp. (R. rhodochrous and R. erythropolis) were carried out using various fatty acids and paraffinic hydrocarbons. Upon calculation and comparison of their specific growth rates (μ) during the logarithmic phase, it was shown that the lower fatty acids (C2–C5) were utilized preferably and the highest μ value was obtained in octadecane (C18) for all three strains. The initial total organic carbon (TOC) concentration affecting the growth of the 3 strains was investigated, using octadecane as the carbon source. From a comparison of the kinetic parameters of the 3 strains with those obtained in the past studies, it was found that Rhodococcus spp. had higher growth rate and lower affinity for octadecane than N. amarae.     

Amyloid-Like Adhesins Produced by Floc-Forming and Filamentous Bacteria in Activated Sludge

Amyloid proteins (fimbriae or other microbial surface-associated structures) are expressed by many types of bacteria, not yet identified, in biofilms from various habitats, where they likely are of key importance to biofilm formation and biofilm properties. As these amyloids are potentially of great importance to the floc properties in activated sludge wastewater treatment plants (WWTP), the abundance of amyloid adhesins in activated sludge flocs from different WWTP and the identity of bacteria producing these were investigated. Amyloid adhesins were quantified using a combination of conformationally specific antibodies targeting amyloid fibrils, propidium iodide to target all fixed bacterial cells, confocal laser scanning microscopy, and digital image analysis. The biovolume fraction containing amyloid adhesins ranged from 10 to 40% in activated sludge from 10 different WWTP. The identity of bacteria producing amyloid adhesins was determined using fluorescence in situ hybridization with oligonucleotide probes in combination with antibodies or thioflavin T staining. Among the microcolony-forming bacteria, amyloids were primarily detected among Alpha- and Betaproteobacteria and Actinobacteria. A more detailed analysis revealed that many denitrifiers (from Thauera, Azoarcus, Zoogloea, and Aquaspirillum-related organisms) and Actinobacteria-related polyphosphate-accumulating organisms most likely produced amyloid adhesins, whereas nitrifiers did not. Many filamentous bacteria also expressed amyloid adhesins, including several Alphaproteobacteria (e.g., Meganema perideroedes), some Betaproteobacteria (e.g., Aquaspirillum-related filaments), Gammaproteobacteria (Thiothrix), Bacteroidetes, Chloroflexi (e.g., Eikelboom type 1851), and some foam-forming Actinobacteria (e.g., Gordonia amarae). The results show that amyloid adhesins were an abundant component of activated sludge extracellular polymeric substances and seem to have unexpected, divers functions.     

Cultivation of Denitrifying Bacteria: Optimization of Isolation Conditions and Diversity Study

An evolutionary algorithm was applied to study the complex interactions between medium parameters and their effects on the isolation of denitrifying bacteria, both in number and in diversity. Growth media with a pH of 7 and a nitrogen concentration of 3 mM, supplemented with 1 ml of vitamin solution but not with sodium chloride or riboflavin, were the most successful for the isolation of denitrifiers from activated sludge. The use of ethanol or succinate as a carbon source and a molar C/N ratio of 2.5, 20, or 25 were also favorable. After testing of 60 different medium parameter combinations and comparison with each other as well as with the standard medium Trypticase soy agar supplemented with nitrate, three growth media were highly suitable for the cultivation of denitrifying bacteria. All evaluated isolation conditions were used to study the cultivable denitrifier diversity of activated sludge from a municipal wastewater treatment plant. One hundred ninety-nine denitrifiers were isolated, the majority of which belonged to the Betaproteobacteria (50.4%) and the Alphaproteobacteria (36.8%). Representatives of Gammaproteobacteria (5.6%), Epsilonproteobacteria (2%), and Firmicutes (4%) and one isolate of the Bacteroidetes were also found. This study revealed a much more diverse denitrifying community than that previously described in cultivation-dependent research on activated sludge.     

‘Morphological shifts’ in filamentous bacteria isolated from activated sludge processes

Summary This study aimed at isolating filamentous bacteria from full-scale activated sludge processes and studying them in pure culture. Three cultures were isolated using conventional microbiological techniques. The isolates were positively identified as Gordonia amarae, Thiothrix nivea and Type 1863/Acinetobacter spp., using fluorescent in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes. However, a ‘morphological shift’ from filamentous to single-cell form was observed in pure culture. The application of fluorescent in situ hybridization (FISH) showed filamentous bacteria to be much more diverse in their ability to adapt to their changing enviroments. Pure culture studies of filamentous bacteria form the basis for application in full-scale activated sludge plants. It therefore remains important that the taxonomic status of filamentous bacteria be determined.     

Induction of Excessive Deoxyribonucleic Acid Synthesis in Escherichia coli by Nalidixic Acid

Prior treatment of Escherichia coli with nalidixic acid in nutritionally complete medium altered the subsequent pattern of deoxyribonucleic acid (DNA) synthesis normally observed in nutritionally deficient medium. Transfer of E. coli 15 TAU to an amino acid- and pyrimidine-deficient medium usually resulted in a 40 to 50% increase in DNA content. Previous treatment with nalidixic acid caused a 200 to 300% increase in DNA content under these conditions. The extent of this DNA synthesis depended on the duration of prior exposure to nalidixic acid. The maximal rate of synthesis was obtained after a 40- to 60-min exposure to nalidixic acid and was two to three times that of the control. The induction of this excessive DNA synthesis was prevented by chloramphenicol or phenethyl alcohol, but the synthesis of this DNA was only partially sensitive to these agents. With E. coli TAU-bar, the rate of DNA synthesis, after removal of nalidixic acid, was similar to that of E. coli 15 TAU, but the maximal amount of DNA synthesized was 180 to 185% of that initially present. Cesium chloride density gradient analysis demonstrated that DNA synthesis after removal of nalidixic acid occurs by a semiconservative mode of replication. The density distribution of this DNA was similar to that obtained after thymine starvation. These results suggest that nalidixic acid treatment may induce additional sites for DNA synthesis in E.coli.     

Evaluation of Standard and Modified M-FC, MacConkey, and Teepol Media for Membrane Filtration Counting of Fecal Coliforms in Water

MacConkey agar, standard M-FC agar, M-FC agar without rosolic acid, M-FC agar with a resuscitation top layer, Teepol agar, and pads saturated with Teepol broth, were evaluated as growth media for membrane filtration counting of fecal coliform bacteria in water. In comparative tests on 312 samples of water from a wide variety of sources, including chlorinated effluents, M-FC agar without rosolic acid proved the medium of choice because it generally yielded the highest counts, was readily obtainable, easy to prepare and handle, and yielded clearly recognizable fecal coliform colonies. Identification of 1,139 fecal coliform isolates showed that fecal coliform tests cannot be used to enumerate Escherichia coli because the incidence of E. coli among fecal coliforms varied from an average of 51% for river water to 93% for an activated sludge effluent after chlorination. The incidence of Klebsiella pneumoniae among fecal coliforms varied from an average of 4% for the activated sludge effluent after chlorination to 32% for the river water. The advantages of a standard membrane filtration procedure for routine counting of fecal coliforms in water using M-FC agar without rosolic acid as growth medium, in the absence of preincubation or resuscitation steps, are outlined.     

Direct Shoot Bud Formation and Tuberization from Aseptically Cultured Root Tubers of Calla Lily (Zantedeschia aethiopica L)

We describe here the development of a micropropagation protocol for mass multiplication of Zantedeschia aethiopica by using root tubers as explant. The surface sterilized root tubers produced five to six shoot-buds on semi-solid Murashige and Skoog’s (MS) medium with 10.0 mg l^?1 of 6-benzylaminopurine (BAP) and additives (50.0 mg l^?1 of ascorbic acid; 25.0 mg l^?1 each of adenine sulphate, L-arginine and citric acid). The cultures were multiplied by sub-culture of individual shoot bud produced in vitro and clumps of shoot buds generated in vitro in cultures on MS medium containing 3.0 mg l^?1 of BAP and additives. Further multiplication of propagules was achieved through tuber formation along with amplifying shoots on MS medium with 5.0 mg l^?1 of BAP. The micropropagated shoots were rooted both in vitro as well as ex vitro. Cent percent of the cloned shoots rooted in vitro within 15–18 days on hormone-free 1/2 strength MS salts with 200.0 mg l^?1 of activated charcoal. Alternatively 95–100% shoots rooted ex vitro under greenhouse conditions on soilrite after pulse-treatment with 500.0 mg l^?1 of Indole-3-butyric acid (IBA) or β-naphthoxyacetic acid (NOA) for 300 sec. The cloned plants were hardened in the greenhouse. The hardened plants were transplanted to soil for further acclimatization.     

Use of Epifluorescence Microscopy in Studies of the Germination and Recovery of Thermoactinomycetes

Spore germination and growth of thermoactinomycetes were observed by epifluorescence microscopy. Each of the principal stages in germination was recognized and found to correspond to changes in phase-contrast appearance commonly monitored during endospore germination. The effects of novobiocin and nalidixic acid on germination of Thermoactinomyces vulgaris and T. thalpophilus were studied by using epifluorescence microscopy. Outgrowth but not initiation was inhibited by 100 μg of nalidixic acid per ml and 50 μg of novobiocin per ml. When each of the compounds, at various concentrations, was incorporated into a medium for thermoactinomycete recovery, the antibiotics were found to inhibit colony development. Samples of water and sediment incubated in a growth medium containing novobiocin and selective for thermoactinomycete species were examined by epifluorescence microscopy for total numbers of outgrown spores. Direct viable counts of outgrown spores indicated that the standard plate count enumerated less than 10% of the viable population of thermoactinomycetes.     

Triploid plant regeneration from mature endosperms of Sapium sebiferum

Sapium sebiferum is a potential bioenergy plant that can be cultivated under various soil, water and climate conditions for both oil-rich seeds and woody biomass. An efficient protocol for regenerating triploid plants of S. sebiferum was established using mature endosperms as explants. Green and compact calli were induced from endosperms within 30 days on Murashige and Skoog medium (MS) containing 1.0–5.0 mg/l 6-benzylaminopurine (BAP) or in combination with 0.2 mg/l α-naphthalene acetic acid (NAA). Within 45 days after endosperm-derived calli were cut into pieces and cultured on media supplemented with 1.0–2.0 mg/l BAP alone or plus 0.1 mg/l NAA, more than 60 % of the callus explants initiated adventitious buds. The buds elongated into shoots after transfer onto a MS medium containing 0.1 mg/l BAP and 1.0 g/l activated charcoal. Approximate 80 % of shoots rooted on a MS medium amended with 1.0 g/l activated charcoal and 1.0 or 2.0 mg/l indole-3-butyric acid within 30 days. The triploidy of the endosperm-derived plantlets was confirmed by flow cytometric analysis, and the triploid plants grew normally after transplantation.     

Combined Molecular and Conventional Analyses of Nitrifying Bacterium Diversity in Activated Sludge: Nitrosococcus mobilis and Nitrospira-Like Bacteria as Dominant Populations

The ammonia-oxidizing and nitrite-oxidizing bacterial populations occurring in the nitrifying activated sludge of an industrial wastewater treatment plant receiving sewage with high ammonia concentrations were studied by use of a polyphasic approach. In situ hybridization with a set of hierarchical 16S rRNA-targeted probes for ammonia-oxidizing bacteria revealed the dominance of Nitrosococcus mobilis-like bacteria. The phylogenetic affiliation suggested by fluorescent in situ hybridization (FISH) was confirmed by isolation of N. mobilis as the numerically dominant ammonia oxidizer and subsequent comparative 16S rRNA gene (rDNA) sequence and DNA-DNA hybridization analyses. For molecular fine-scale analysis of the ammonia-oxidizing population, a partial stretch of the gene encoding the active-site polypeptide of ammonia monooxygenase (amoA) was amplified from total DNA extracted from ammonia oxidizer isolates and from activated sludge. However, comparative sequence analysis of 13 amoA clone sequences from activated sludge demonstrated that these sequences were highly similar to each other and to the corresponding amoA gene fragments of Nitrosomonas europaea Nm50 and the N. mobilis isolate. The unexpected high sequence similarity between the amoA gene fragments of the N. mobilis isolate and N. europaea indicates a possible lateral gene transfer event. Although a Nitrobacter strain was isolated, members of the nitrite-oxidizing genus Nitrobacter were not detectable in the activated sludge by in situ hybridization. Therefore, we used the rRNA approach to investigate the abundance of other well-known nitrite-oxidizing bacterial genera. Three different methods were used for DNA extraction from the activated sludge. For each DNA preparation, almost full-length genes encoding small-subunit rRNA were separately amplified and used to generate three 16S rDNA libraries. By comparative sequence analysis, 2 of 60 randomly selected clones could be assigned to the nitrite-oxidizing bacteria of the genus Nitrospira. Based on these clone sequences, a specific 16S rRNA-targeted probe was developed. FISH of the activated sludge with this probe demonstrated that Nitrospira-like bacteria were present in significant numbers (9% of the total bacterial counts) and frequently occurred in coaggregated microcolonies with N. mobilis.     

The plant activator BTH promotes Ornithogalum dubium and O. thyrsoides differentiation and regeneration in vitro

Benzothiadiazole (BTH) is a structural analogue of salicylic acid (SA) which is widely recognized for its role in elicitation of systemic acquired resistance in a broad range of plant species. Here, BTH was applied to cell cultures of the bulbous ornamental plants Ornithogalum dubium and O. thyrsoides, showing a strong effect on rates of differentiation and morphogenesis. Morphogenic cell clusters in liquid Murashige and Skoog (MS) medium containing 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) were used for all treatments. The calluses were washed thoroughly and activated with increasing concentrations of BTH. Following the induction, calli were grown on a solid MS medium without growth regulators (MS) or on a comparable media with NAA and BAP (M-206). The calli treated with BTH displayed a dose dependent increase in formation of meristematic centres followed by enhanced shoot formation compared to controls. Microscopic analyses revealed increased differentiation to cell organelles and a strengthening of the cell wall. A stronger response to BTH was observed in MS than in M-206 medium. A similar effect on calli differentiation was obtained by three weeks darkness followed by light exposure. The dark/light positive effect on differentiation was further augmented by BTH in a synergistic fashion. It is suggested that BTH enhances the rates of morphogenesis in Ornithogalum cultures by triggering a plant regulator-like activity.     

Plant regeneration of non-toxic Jatropha curcas—impacts of plant growth regulators, source and type of explants

Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel plant, however, oil and deoiled cake are toxic. A non-toxic variety of J. curcas is reported from Mexico. The present investigation explores the effects of different plant growth regulators (PGRs) viz. 6-benzyl aminopurine (BAP) or thidiazuron (TDZ) individually and in combination with indole-3-butyric acid (IBA), on regeneration from in vitro and field-grown mature leaf explants, in vitro and glasshouse-grown seedlings cotyledonary leaf explants of non-toxic J. curcas. In all the tested parameters maximum regeneration efficiency (81.07%) and the number of shoot buds per explants (20.17) was observed on 9.08 μM TDZ containing Murashige and Skoog’s (MS) medium from in vitro cotyledonary leaf explants. The regenerated shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM BAP and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with 2.25 μM BAP and 8.5 μM IAA. Rooting was achieved when the basal cut end of elongated shoots were dipped in half strength MS liquid medium containing different concentrations and combinations of IBA, IAA and NAA for four days followed by transfer to growth regulators free half strength MS medium supplemented 0.25 mg/l activated charcoal. The rooted plants could be established in soil with more than 90% survival rate.     

Microbial biogeography across a full-scale wastewater treatment plant transect: evidence for immigration between coupled processes

Wastewater treatment plants use a variety of bioreactor types and configurations to remove organic matter and nutrients. Little is known regarding the effects of different configurations and within-plant immigration on microbial community dynamics. Previously, we found that the structure of ammonia-oxidizing bacterial (AOB) communities in a full-scale dispersed growth activated sludge bioreactor correlated strongly with levels of NO₂ ^? entering the reactor from an upstream trickling filter. Here, to further examine this puzzling association, we profile within-plant microbial biogeography (spatial variation) and test the hypothesis that substantial microbial immigration occurs along a transect (raw influent, trickling filter biofilm, trickling filter effluent, and activated sludge) at the same full-scale wastewater treatment plant. AOB amoA gene abundance increased >30-fold between influent and trickling filter effluent concomitant with NO₂ ^? production, indicating unexpected growth and activity of AOB within the trickling filter. Nitrosomonas europaea was the dominant AOB phylotype in trickling filter biofilm and effluent, while a distinct “Nitrosomonas-like” lineage dominated in activated sludge. Prior time series indicated that this “Nitrosomonas-like” lineage was dominant when NO₂ ^? levels in the trickling filter effluent (i.e., activated sludge influent) were low, while N. europaea became dominant in the activated sludge when NO₂ ^? levels were high. This is consistent with the hypothesis that NO₂ ^? production may cooccur with biofilm sloughing, releasing N. europaea from the trickling filter into the activated sludge bioreactor. Phylogenetic microarray (PhyloChip) analyses revealed significant spatial variation in taxonomic diversity, including a large excess of methanogens in the trickling filter relative to activated sludge and attenuation of Enterobacteriaceae across the transect, and demonstrated transport of a highly diverse microbial community via the trickling filter effluent to the activated sludge bioreactor. Our results provide compelling evidence that substantial immigration between coupled process units occurs and may exert significant influence over microbial community dynamics within staged bioreactors.     

The influence of bud length,age of the tree and culture media on androgenesis induction inAesculus carnea Hayne anther culture

Statistical analyses of the data revealed very significant differences in androgenesis induction ofA. carnea Hayne anther culture depending on the bud length, nutrient medium composition and age of the parental tree. Significant mutual influence of all these factors was also observed. The highest number of androgenic anthers was obtained when 4 mm long buds were used. Older trees (60 and 100 yrs) gave a higher number of androgenic anthers than the younger ones (20 and 40 yrs). MS medium supplemented with 2,4-d and Kin (1 mg l^–1, each) was the most favourable for androgenesis induction. Pollen embryos (haploids and aneuploids) were formed by the division of uninuclear microspores.The highest percentage of germinated embryos and further synchronous development of the shoot and root was achieved in MS medium supplemented with IAA, GA₃ (1 mg l^–1) and activated charcoal (1%). When other germination media were used, malformations of androgenic embryos were observed.Abbreviations AC activated charcoal - H casein hydrolysate - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BAP 6-benzylaminopurine - GA₃ gibberelic acid - Kin 6-furfurylaminopurine - MS Murashige and Skoog - T thidiazurone - N phenyl-N'-1,2,3-thiadiazol-5-ylurea - Z zeatin-6-(4-hydroxy-3-methyl-trans-2-butenylamino)purine     

Effect of abscisic acid and cytokinins on cultured zygotic embryos of Coffea arabicacv.Cauvery (Catimor)

Immature zygotic embryos of Coffea arabica L.cv.Cauvery (Catimor) were sequentially cultured on different modifications of Murashige and Skoog's (MS) medium to test the effect of abscisic acid and cytokinins. The type of response depended on the medium strength, the growth regulator combinations as well as the period of initial culture in both abscisic acid or cytokinin supplemented media. Increasing concentration of abscisic acid from 0.4 to 18.9 μM enhanced the quiescence of the zygotic embryos. All the cytokinins promoted germination but Kinetin and isopentenyladenine (2-IP) were less effective than benzyl amino purine (BAP). The maximum mature embryos were obtained when immature embryos were cultured initially for 30 days on full strength MS medium with 3.8 μM ABA, followed by 60 days on half strength MS medium with 0.1 μM BAP and finally on half strength MS media with out growth regulator for next 60 days.     

Interaction of Operational and Physicochemical Factors Leading to Gordonia amarae-Like Foaming in an Incompletely Nitrifying Activated Sludge Plant

The overgrowth of Gordonia amarae-like bacteria in the mixed liquor of an incompletely nitrifying water reclamation plant was inversely correlated with temperature (r = −0.78; P < 0.005) and positively correlated with the solids retention time (SRT) obtained a week prior to sampling (r = 0.67; P < 0.005). Drops followed by spikes in the food-to-mass ratio (0.18 to 0.52) and biochemical oxygen demand concentrations in primary effluent (94 to 298 mg liter⁻¹) occurred at the initiation of G. amarae-like bacterial growth. The total bacterial concentration did not increase as concentrations of G. amarae-like cells increased, but total bacterial cell concentrations fluctuated in a manner similar to that of G. amarae-like bacteria in the pseudo-steady state. The ammonium ion removal rate (percent) was inversely related to G. amarae-like cell concentrations during accelerated growth and washout phases. The dissolved oxygen concentration decreased as the G. amarae-like cell concentration decreased. The concentrations of G. amarae-like cells peaked (2.47 × 10⁹ cells liter⁻¹) approximately 1.5 months prior to foaming. Foaming occurred during the late pseudo-steady-state phase, when temperature declines reversed. These findings suggested that temperature changes triggered operational and physicochemical changes favorable to the growth of G. amarae-like bacteria. Fine-scale quantitative PCR (qPCR) monitoring at weekly intervals allowed a better understanding of the factors affecting this organism and indicated that frequent sampling was required to obtain statistical significance with factors changing as the concentrations of this organism increased. Furthermore, the early identification of G. amarae-like cells when they are confined to mixed liquor (10⁷ cells liter⁻¹) allows management strategies to prevent foaming.     

Implicating the Glutathione-Gated Potassium Efflux System as a Cause of Electrophile-Induced Activated Sludge Deflocculation

The glutathione-gated K⁺ efflux (GGKE) system represents a protective microbial stress response that is activated by electrophilic or thiol-reactive stressors. It was hypothesized that efflux of cytoplasmic K⁺ occurs in activated sludge communities in response to shock loads of industrially relevant electrophilic chemicals and results in significant deflocculation. Novosphingobium capsulatum, a bacterium consistent with others found in activated sludge treatment systems, responded to electrophilic thiol reactants with rapid efflux of up to 80% of its cytoplasmic K⁺ pool. Furthermore, N. capsulatum and activated sludge cultures exhibited dynamic efflux-uptake-efflux responses very similar to those observed by others in Escherichia coli K-12 exposed to the electrophilic stressors N-ethylmaleimide and 1-chloro-2,4-dinitrobenzene and the reducing agent dithiothreitol. Fluorescent LIVE/DEAD stains were used to show that cell lysis was not the cause of electrophile-induced K⁺ efflux. Nigericin was used to artificially stimulate K⁺ efflux from N. capsulatum and activated sludge cultures as a comparison to electrophile-induced K⁺ efflux and showed that cytoplasmic K⁺ efflux by both means corresponded with activated sludge deflocculation. These results parallel those of previous studies with pure cultures in which GGKE was shown to cause cytoplasmic K⁺ efflux and implicate the GGKE system as a probable causal mechanism for electrophile-induced, activated sludge deflocculation. Calculations support the notion that shock loads of electrophilic chemicals result in very high K⁺ concentrations within the activated sludge floc structure, and these K⁺ levels are comparable to that which caused deflocculation by external (nonphysiological) KCl addition.     

High-Frequency Regeneration by Abscisic Acid (ABA) from Petiole Callus of Jatropha curcas

Jatropha curcas L. is attaining worldwide interest as an important biofuel crop. Experiments were conducted to improve the prevailing micropropagation technique as well as to develop a new ex vitro rooting method for J. curcas plant regeneration. Regeneration and ex vitro rooting efficiency was enhanced by augmenting the culture medium with abscisic acid (ABA). Different concentrations of 6-benzylaminopurine (BAP) and indole-3-butyric acid (IBA) were tested for callus generation from both in vitro and in vivo explants (leaf and petiole) on Murashige and Skoog (MS) medium. The best regenerative callus was achieved on MS medium supplemented with BAP (4.44 μM) and IBA (2.45 μM) from in vitro-cultured petioles. Highest regeneration (91%) was achieved by culturing petiole callus on MS medium supplemented with BAP (8.88 μM), IBA (0.49 μM), and ABA (1.9 μM), whereas 61% regeneration was obtained from in vitro leaf callus. Shoot proliferation and elongation was achieved on BAP (2.22 μM) and IAA (8.56 μM) with 10–13 shoots per explants. Highest rooting (65%) was achieved from M1 shoots (BAP, IAA, and ABA) on MS medium supplemented with IBA (2.45 μM), naphthaleneacetic acid NAA (0.54 μM), and 0.02% activated charcoal. Ex vitro rooting of 1-mo-old M1 shoots obtained from the charcoal-containing medium resulted optimum rooting (>72%) when transferred to polybags containing sterile sand. The plantlets were successfully acclimatized in soil with more than 98% survival rate in the greenhouse.     

Tissue culture of forest trees: Clonal multiplication of Eucalyptus grandis L.

Axillary shoot proliferation was obtained using explants of Eucalyptus grandis L. juvenile and mature stages on a defined medium. Murashige and Skoog medium (MS) supplemented with benzyladenine (BA), naphthalene acetic acid (NAA) and additional thiamine. Excised shoots were induced to root on a sequence of three media: (1) White's medium containing indoleacetic acid (IAA), NAA and indole butyric acid; (IBA), (2) half-strength MS medium with charcoal and (3) half-strength MS liquid medium. The two types of explants differed in rooting response, with juvenile-derived shoots giving 60% rooting and adult-derived ones only 35%. Thus, the factors limiting cloning of selected trees in vitro are determined to be those controlling rooting of shoots in E. grandis.