Treatment of low strength soluble wastewaters in UASB reactors

Low strength wastewaters can be those with chemical oxygen demand (COD) below 2,000 mg/l. The anaerobic treatment of such wastewaters has not been fully explored so far. The suboptimal reaction rates with low substrate concentrations, and the presence of dissolved oxygen in the influent are regarded as possible constraints. In this study, the treatment of low strength soluble wastewaters containing ethanol or whey was studied in lab-scale upflow anaerobic sludged bed (UASB) reactors at 30°C. The high treatment performance obtained demonstrates that UASB reactors are viable for treating both types of wastewaters at low COD concentrations. The treatment of the ethanol containing wastewater resulted in COD removal efficiencies exceeding 95% at organic loading rates (OLR) between 0.3 to 6.8 g COD/l-d with influent concentrations in the range of 422 to 943 mg COD/l. In the case of the more complex whey containing wastewater, COD removal efficiencies exceeded 86% at OLRs up to 3.9 g COD/l·, as long as the COD influent was above 630 mg/l. Lowering the COD influent resulted in decreased efficiency with sharper decrease at values below 200 mg/l. Acidification instead of methanogenesis was found to be the rate limiting step in the COD removal at low concentrations, which was not the case when treating ethanol. The effect of dissolved oxygen in the influent as a potential danger in anaerobic treatment was investigated in reactors fed with and without dissolved oxygen. Compared with the control reactor, the reactor receiving oxygen showed no detrimental effects in the treatment performance. Thus, the presence of dissolved oxygen in dilute wastewaters is expected to be of minor importance in practice.     

Anaerobic treatment of distillery spent wash - a study on upflow anaerobic fixed film bioreactor

Anaerobic digestion of wastewater from a distillery industry having very high COD (1,10,000-1,90,000 mg/L) and BOD (50,000-60,000 mg/L) was studied in a continuously fed, up flow fixed film column reactor using different support materials such as charcoal, coconut coir and nylon fibers under varying hydraulic retention time and organic loading rates. The seed consortium was prepared by enrichment with distillery spent wash in a conventional type reactor having working capacity of 3 L and was used for charging the anaerobic column reactor. Amongst the various support materials studied the reactor having coconut coir could treat distillery spent wash at 8d hydraulic retention time with organic loading rate of 23.25 kg COD m(-3)d(-1) leading to 64% COD reduction with biogas production of 7.2 m3 m(-3)d(-1) having high methane yield without any pretreatment or neutralization of the distillery spent wash. This study indicates fixed film biomethanation of distillery spent wash using coconut coir as the support material appears to be a cost effective and promising technology for mitigating the problems caused by distillery effluent.     

Biomethanation of Spent Wash: Bacterial Pretreatment to Remove Heavy Metals by Adsorption

In a semicontinuous fermentation system, biomethanation of diluted spent wash (DW, initial COD 25–27 g/l) resulted in only 0.2 l/l of methane production in 20 d. Two capsular Gram-negative strains of bacteria were used for adsorption of heavy metals such as lead, copper, and zinc from spent wash. Strain I removed 64 % of the lead in 1 h and 82% of the copper in 2 h, while strain II removed 76% of the zinc in 2 h. The diluted spent wash from which heavy metals were removed was supplemented with synthetic medium and an acidophilic strain of Candida sp. This treatment improved methanogenesis. In 12 d, 4.9 l/l of biogas containing 63% methane was produced.     

Characteristics of granular methanogenic sludge grown on phenol synthetic medium and methanogenic fermentation of phenolic wastewater in a UASB reactor

The growth of granules on a phenol synthetic medium and the methanogenic fermentation of industrial phenolic wastewater from a steel factory in an upflow anaerobic sludge blanket (UASB) reactor were investigated. Total granular sludge concentration retained in the UASB reactor was 6.7 g MLSS/l (6.0 g MLVSS/l) during the 10 months' operation on the phenol synthetic medium. This realized a maximum phenol removal rate of 2.2 g/l·d (phenol concentration of influent = 500 mg/l), which corresponded to 5.2 g COD/l·d at space velocity (SV) of 4.4 d⁻¹. The granules formed were of relatively small size ranging from 0.61 to 0.77 mm, and had a relatively low density of 0.013–0.023 g MLVSS/cm³ and low specific gravity (1.11) due to very low ash content (8.7–11.9%). Electron microscopic analysis showed that Methanothrix spp. appeared dominantly on the granule surface as well as within it. The specific metabolic activities of bacterial trophic groups were the highest for H₂ followed by acetate, benzoate, phenol, and propionate. In the case of industrial phenolic wastewater, although phenol efficiency was only 50% at SV of 0.4 d⁻¹, when the wastewater was diluted twofold and the treated wastewater was recycled at SV of 7.3 d⁻¹, the removal efficiencies of phenol and CODcr were restored to 90% (influent=400 mg/l) and 80% (influent=5,000 mg/l), respectively. It was suggested that recycling of the treated wastewater might be improved by partly degrading unknown toxic compounds contained in phenolic wastewater.     

Characteristics of granular methanogenic sludge grown on glucose in a UASB reactor

The formation of granules grown on glucose in an upflow anaerobic sludge blanket (UASB) reactor was investigated. Total granular sludge concentration retained in the UASB reactor was 34.5 g MLSS/l (30.0 g MLVSS/l) during 240 d operation on glucose minimum medium with the supplementation of 1.07 g NaHCO₃ per 1 g glucose. This realized a high-rate methanogenic fermentation of glucose of 17.6 g COD/l-reactor-d at 3.4 d⁻¹ of space velocity. The granules formed were relatively small, ranging mainly from 0.4 to 0.5 mm, had a relatively low cell density of 0.0542–0.0560 g MLVSS/ml, and had low specific gravity (0.97–1.19) due to very low ash content (11–13%). Electron microscopic analysis showed that Methanothrix spp. appeared dominant over the granules. The specific metabolic activities of bacterial trophic groups were the highest for H₂ followed by glucose, acetate, and propionate.     

Characteristics of methanogenic granules grown on propionate in an upflow anaerobic sludge blanket (UASB) reactor

Granulation of a propionate-degrading consortia was performed with a mesophilic propionate-acclimatized sludge in an upflow anaerobic sludge blanket (UASB) reactor. The granules formed were relatively small, ranging mainly from 0.3 to 0.6 mm in diameter, but had an excellent sedimentation velocity due to a high specific gravity of 1.355 g/cm³ (ash content, 48.2%). The ash consisted mainly of calcium (30.2%), phosphorus (19.7%), and magnesium (3.95%) forming plate crystals in the granules. The populations of three bacterial trophic groups present in the granules, propionate-degraders, hydrogenotrophic and aceticlastic methanogens were 5.6 × 10⁸, 1.6 × 10¹⁰, and 2 × 10⁹ (in most probable number/g mixed-liquor volatile suspended solids [MLVSS]), respectively, while the specific utilization rates of propionate, hydrogen, and acetate of the granules were 9.4, 850, and 20.9 (mmol/g MLVSS·d), respectively. Electron microscopic analysis showed that Methanothrix spp. appeared dominant over the granules. Total granular sludge concentration retained in the UASB reactor during 178 d of operation was 80.0 g mixed-liquor suspended solids (MLSS)/l-reactor, corresponding to 41.4 g MLVSS/l-reactor, which realized a high-rate methanogenic fermentation of propionate of 85 g chemical oxygen demand (COD)/l-reactor·d.     

Propagation of Date Palm (Phoenix dactylifera L.) in vitro

Adventitious plantlets were obtained from lateral buds, shoottips, embryos, and pieces of stem and rachilla tissue of Phoenixdactylifera L. cultured on a modified Murashige and Skoog mediumcontaining 3 mg l^–1 N-(²-isopenty)adenine 0?1–100mg l^–1 -naphthaleneacetic acid or 2,4-dichlorophenoxyaceticacid, and 3 g l^–1 activated charcoal. Additions of auxinswere necessary to induce explants to produce callus, adventitiousplantlets, and roots. Plantlets were obtained from explantscultured 3–4 months in vitro. No difference in growthresponses between male and female explants was observed duringculture. Complex addenda of activated charcoal and polyvinylpyrrolidonewere tested in the nutrient media at various concentrationsto prevent explant browning. Activated charcoal fostered satisfactorygrowth by reducing the browning and inhibition of growth ofexplants.     

Anaerobic treatment of starch-containing wastewater using a plate-column reactor

An anaerobic plate-column reactor, developed to retain a high concentration of biomass, was studied using starch-containing synthetic wastewaters with regard to its start-up profile and the effects of TOC-loading rate, hydraulic retention time (HRT), and temperature in a steasy state. Each operation was started up at an initial biomass concentration of ca. 0.5 mg-N/ml (ca. 5 mg-VSS/ml), 20°C, an HRT of 30 h, and a TOC-loading rate of 0.8 g/l/d. The removal defficiency of dissolved organic carbon exceeded 90% after 29 d. The efficiency reached a steady state at 98% removal after 116 d. The biomass concentration in the reactor was 2.3 mg-N/ml after 154 d. Circulation of effluent at a ratio of 1 gave a lower removal efficiency and a lower biomass concentration than the same reactor without circulation. The effect of TOC-loading rate was studied at an HRT of 30 h, 20°C, and removal efficiencies were found 91% at 1.6 g/l/d and 77% at 3.2 g/l/d. The effect of HRT was studied at a TOC-loading rate of 0.8 g/l/d, 20°C, and removal efficiencies 91% at an HRT of 12 h and 72% at an HRT of 6 h.     

Two-stage anaerobic treatment of dairy wastewater using HUASB with PUF and PVC carrier

In the present study, an attempt has been made to treat dairy wastewater entirely via anaerobic treatment over a period of 215 days, using two-stage Hybrid Upflow Anaerobic Sludge Blanket (HUASB) reactors, which offer the advantages associated both with fixed film and upflow sludge blanket treatments. A HUASB with polyurethane foam cubes was used for stage I, and a HUASB utilizing PVC-cut rings was used for stage II. The output from stage I was used as the input for stage II. The two-stage reactor was operated at an organic loading rate that varied from 10.7 to 21.4 kg COD m³/d for a period of 215 days, including the start-up period. The ideal organic loading rate for the two-stage reactor was 19.2 kg COD/m³/d. A further 21.4 kg COD m³/d increase in the organic loading rate resulted in the souring of the reactor function in stage I, which consequently reduced the overall reactor performance. Combined COD removal during the stable operation period (10.7 to 19.2 kg COD m³/d) occurred in a range between 97 and 99%. The methane content in the biogas varied from 65 to 70% in stage I, and from 63 to 66% in stage II. The two-stage anaerobic treatment using HUASB with PUF and PVC described in this work is expected to constitute a better alternative for the complete treatment of dairy wastewater than high-rate anaerobic, anaerobic/aerobic, and two-phase anaerobic treatment methods.     

Microbial conversion of distillery waste to bioenergy: Effects of media enrichment with low chain fatty acids and Candida sp.

During microbial methanogensis of diluted distillery waste or spent wash (initial COD 25,000 mg/l) in the presence of sodium acetate, sodium propionate, or sodium butyrate at the concentration of 2000 mg/l, biogas containing 22.0% to 39.4% methane was produced in 20 days in a semicontinuous fermentation system. The analysis of the volatile fatty acid spectrum of the effluent showed accumulation of 46.3% branched chain fatty acids. When the fermentation medium was supplemented with modified Smith and Mah (SM) medium containing electrolytes and 1% sodium acetate, production of methane went up to 59.0% in 18 days. With the addition of a strain of Candida sp. the coculture produced about the same volume of methane (61%) but the time required was reduced (14 days). COD (16600.8 mg/l) was reduced and the effluent contained only 3% branched chain fatty acids adn 96% straight chain fatty acids. Fortification with the sodium salts of three branched-chain fatty acids as sources of carbon in SM medium (alone) reduced methane production. Only 0.0% to 14.2% methane was recorded. By combining these acids with straight chain fatty acids in SM medium, methane production increased significantly (about 4-fold) in proportion to the concentration of straight chain fatty acids added.     

Anaerobic treatment of wastewater with high salt content from a pickled-plum manufacturing process

Anaerobic treatment of wastewater with a high salt content generated during a pickled-plum manufacturing process (TOC, 14g/l; ash, 150g/l; pH 2.7, hereafter called pickled-plum effluent) was investigated for its effect on the high salt content of the wastewater. The synthetic wastewater, including NaCl up to 30g/l, was treated anaerobically by a draw and fill method (treatment temperature, 37°C; volumetric loading rate of organic matter, 2g/l·d). The TOC removal efficiency and rate of gas evolution then gradually decreased as salt content increased, although stable operation was maintained. At NaCl concentrations above 30g/l, TOC removal efficiency decreased rapidly and stable operation could not be maintained. Five-fold-diluted pickled-plum effluent was treated by the same method at a volumetric TOC loading rate of 2.9g/l·d with a TOC removal efficiency of 71%. Five-fold-diluted pickled-plum effluent was also treated in an anaerobic fluidized-bed reactor (AFBR) at a maximum volumetric TOC loading rate of 3.0g/l·d, which gave almost the same results as the draw and fill method. However, ten-fold-diluted pickled-plum effluent could be treated in the AFBR at a maximum volumetric TOC loading rate of 11.1g/l·d with a TOC removal efficiency of 84.6%. The red pigment in the pickled-plum effluent was completely decolorized by the anaerobic treatment.     

Treatment of chlorine bleaching effluent using a white-rot fungus

This study was undertaken to better understand the reactions related to fungal treatment of pulp bleaching effluents. Color, COD (chemical oxygen demand), glucose, chloride and ammonium concentration were monitored during the course of treatment of alkali extraction stage liquor (E1) with a white-rot fungus Phanerochaete chrysosporium.The color removal rate was independent on the initial glucose concentration. The earlier used glucose concentration of 10 g l⁻¹ was found to be unnecessary high as the residual glucose accounted for about 50% of the final COD of the effluent. The lowest practical glucose concentration was 2 g l⁻¹. Below this the fungus lost its decolorizing activity in a few days. The lignin-related COD decreased 32% and up to 16.5 mM inorganic chloride was liberated (being 34% increase from the original concentration) from the chlorinated organic material in the effluent in 2 d. The observed rapid depletion of added ammonium nitrogen is believed to indicate a switch of a part of the mycelium to primary growth which leads to higher activity and longer active period of time due to renewal of the cells.     

Evaluation of ethanol production by a new isolate of yeast during fermentation in synthetic medium and sugarcane bagasse hemicellulosic hydrolysate

A new xylose fermenting yeast was isolated from over-ripe banana by enrichment in xylose-containing medium. The phylogenetic analysis of ITS1-5.8S-ITS2 region sequences of ribosomal RNA of isolate BY2 revealed that it shows affiliation to genus Pichia and clades with Pichia caribbica. In batch fermentation, Pichia strain BY2 fermented xylose, producing 15 g l^?1 ethanol from 30 g l^?1 xylose under shaking conditions at 28°C, with ethanol yield of 0.5 g g^?1 and volumetric productivity of 0.31 g l^?1 h^?1. The optimum pH range for ethanol production from xylose by Pichia strain BY2 was 5–7. Pichia strain BY2 also produced 6.08 g l^?1 ethanol from 30 g l^?1 arabinose. Pichia strain BY2 can utilize sugarcane bagasse hemicellulose acid hydrolysate for alcohol production, efficiency of fermentation was improved by neutralization, and sequential use of activated charcoal adsorption method. Percent total sugar utilized and ethanol yield for the untreated hydrolysate was 17.14% w/v and 0.33 g g^?1, respectively, compared with 66.79% w/v and 0.45 g g^?1, respectively, for treated hemicellulose acid hydrolysate. This new yeast isolate showed ethanol yield of 0.45 g g^?1 and volumetric productivity of 0.33 g l^?1 h^?1 from sugarcane bagasse hemicellulose hydrolysate detoxified by neutralization and activated charcoal treatment, and has potential application in practical process of ethanol production from lignocellulosic hydrolysate.     

Differential responses to somatic embryogenesis of different genotypes of Brazilian oil palm (Elaeis guineensis Jacq.)

To assess the potential of different genotypes of Brazilian oil palm (Elaeis guineensis Jacq.) to somatic embryogenesis and somatic embryo proliferation, mature zygotic embryos of nine commercial genotypes of E. guineensis (BRSC2001, BRSC2328, BRSC2301, BRSC3701, BRSCM1115, BRSC7201, BRSC2528, BRSC2501, and BRSCN1637) were used. Explants were incubated on Murashige and Skoog (MS) supplemented with 450 μM picloram, 3.0 % sucrose, 500 mg l^?1 glutamine, and 2.5 g l^?1 activated charcoal, and gelled with 2.5 g l^?1 Phytagel. After induction, for differentiation and maturation, the embryogenic calli (ECs) were transferred into fresh medium supplemented with 0.6 μM naphthaleneacetic acid (NAA) and 12.30 μM 2-isopentenyladenine (2iP) or 40 μM picloram in combination with 0.3 g l^?1 activated charcoal, and 500 mg l^?1 glutamine. Somatic embryos were converted into plants on MS medium with macro- and micro-nutrients at half strength, 2 % sucrose, and 2.5 g l^?1 activated charcoal, and gelled with 2.5 g l^?1 Phytagel. In general, zygotic embryos swelled after 14 days. Primary calli, which were observed in all the genotypes after 45–60 days of culture, eventually progressed to ECs at 90 days. At this time, scanning electron microscopy (SEM) analysis showed cellular differences between compact and friable calli. After 150 days in the induction phase, the ECs with proembryos that were transferred to the medium for differentiation and maturation, differentiated asynchronically into somatic embryos at globular and torpedo stages. The results showed that BRSC2328 and BRSCM1115 had the highest potential for EC formation (90–100 %) and somatic embryo differentiation (40.7 and 52.5 somatic embryos per callus, respectively) when compared to other genotypes. After approximately 90 days of culture on MS basal medium without growth regulators, protrusion of the leaf primordia was observed, characterizing the onset of germination of the somatic embryos into plants.     

Liquefaction and gasification during anaerobic digestion of coffee waste by two-phase methane fermentation with slurry-state liquefaction

In order to improve the gas evolution rate during anaerobic digestion of coffee waste by two-phase methane fermentation with slurry-state liquefaction, the liquefaction and gasification processes were separately investigated. In the liquefaction process (including the acidification process), treatment at a pH above 6 had no major effects on the liquefaction and acidification rates. However, the VFA production rates were 880 and 320 mg/l·d during mesophilic (37°C) and thermophilic (53°C) liquefaction, respectively. Mesophilic conditions were superior to thermophilic conditions in the liquefaction. With respect to the gasification process, a high TOC volumetric loading rate of 21 g/l·d was achieved during thermophilic gasification. However, the mesophilic gasification did not yield stable data, even at a low TOC volumetric loading rate of 2 g/l·d. The gas yield was 1.7 l/g TOC consumed during thermophilic gasification. The thermophilic liquefaction and thermophilic gasification reactors were connected in series and a two-phase experiment was conducted with the reactors at various volumetric ratios. The maximum gas evolution rate of 1.43 l/l·d was achieved with a combination of a gasification reactor with a 0.45l working volume and liquefaction reactor with a 2l working volume. This rate was 1.7 times higher than the rate obtained in a previous study.     

In Vitro Propagation and Reintroduction of the Endangered Renanthera imschootiana Rolfe

Renanthera imschootiana Rolfe is an endangered tropical epiphytic orchid that is threatened with extinction due to over-collection and the loss of suitable habitats. In vitro propagation is a useful way to mass produce plants for re-establishment in the wild and for commercial propagation. Seeds collected 150 days after pollination (DAP) were the optimum stage for in vitro culture. Seed germination reached 93.1% on quarter-strength MS (i.e., MS containing a quarter of macro- and micronutrients) medium containing 0.5 mg l⁻¹ α-naphthaleneacetic acid (NAA), 20% coconut water (CW), 1.0 g l⁻¹ peptone, 10 g l⁻¹ sucrose and 1.0 g l⁻¹ activated charcoal (AC). Quarter-strength MS medium supplemented with 1.0 mg l⁻¹ BA, 0.5 mg l⁻¹ NAA, 1.0 g l⁻¹ peptone, 10 g l⁻¹ sucrose and 20% CW was suitable for the sub-culture of protocorm-like bodies (PLBs) in which the PLB proliferation ratio was 2.88. Quarter-strength MS medium containing 1.0 mg l⁻¹ NAA, 1.0 g l⁻¹ peptone, 100 g l⁻¹ banana homogenate (BH), and 1.0 g l⁻¹ AC was suitable for plantlet formation and 95.67% of plantlets developed from PLBs within 60 days of culture. Hyponex N016 medium supplemented with 0.5 mg l⁻¹ NAA, 1.0 g l⁻¹ peptone, 20 g l⁻¹ sucrose, 150 g l⁻¹ BH, and 1.0 g l⁻¹ AC was suitable for the in vitro growth of plantlets about 2-cm in height. Plantlets 3-cm in height or taller were transplanted to Chilean sphagnum moss, and 95% of plantlets survived after 60 days in a greenhouse. Three hundred transplanted of seedlings 360-days old were reintroduced into three natural habitats. Highest percentage survival (79.67%) was observed in Yuanjiang Nature Reserve two years after reintroduction, followed by Huolu Mountain forest park (71.33%). This protocol is an efficient means for the large-scale propagation and in vitro and in vivo germplasm conservation of R. imschootiana.     

Dyestuff wastewater treatment using chemical oxidation,physical adsorption and fixed bed biofilm process

Fenton’s oxidation and activated carbon adsorption were examined as pretreatment processes for dyestuff wastewater having high salinity, colour, and non-biodegradable organic concentrations. In this work, each wastewater stream produced by individual production processes was classified as streams R1, R2, and R3. The stream having a value of BOD₅/COD lower than 0.4 was pretreated by Fenton’s oxidation or activated carbon adsorption to increase the ratio of BOD₅/COD which indicates biodegradability. For Fenton’s oxidation with one stream having a value of BOD₅/COD lower than 0.4, the optimal reaction pH was 3.0 and the minimum dosing concentration (mg l⁻¹) of H₂O₂:FeSO₄·7H₂O was 700:3500. Stream R3, which consisted mainly of methanol was efficiently treated by activated carbon adsorption. The ratio of BOD₅/COD was also increased to 0.432 and 0.31 from 0.06 in Fenton’s oxidation and activated carbon adsorption, respectively. A biological treatment system using a fixed bed reactor was also investigated to enhance biological treatment efficiency at various hydraulic retention times, pretreatment conditions by Fenton’s reagent and salt concentrations by dyestuff wastewater. In addition, the efficiency of Fenton’s oxidation as a post-treatment system was also investigated to present a total treatment process of dyestuff wastewater. As the influent COD and salinity were increased, the effluent SS and COD were consequently increased. However, as the microorganisms became adapted to the changed influent condition, the treatment efficiency of the fixed bed reactor quickly recovered under the high COD and salinity since the microorganisms were well adapted to toxic influent conditions. A wastewater treatment process consisting of chemical oxidation, activated carbon adsorption, fixed bed biofilm process and Fenton’s oxidation as a post-treatment system can be useful to treat dyestuff wastewater having high salinity, colour, and non-biodegradable organic concentration.     

Improved production of biosurfactant by a <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> mutant using vegetable oil refinery wastes

Biosurfactant production by Pseudomonas aeruginosa EBN-8 mutant was studied in shake flasks on separate wastes from canola, soybean and corn oil refineries. Of the substrates tested, canola oil refinery waste (COD=20 g l⁻¹) supplemented with sodium nitrate (at COD/N=20) showed the best microbial growth (4.50 g l⁻¹) and rhamnolipid production (8.50 g l⁻¹), at 10 d of incubation with the specific growth rate of 0.316 h⁻¹ and specific product yield of 0.597 g g⁻¹ h. Its cell-free supernatant showed the critical micelle dilution (CMD) of 150 and surface tension (ST) of 28.5 mN m⁻¹.     

Anaerobic treatment of distillery wastewater from barley-Shochu making by UASB

Distillery wastewater from barley-shochu making was anaerobically treated by a single upflow anaerobic sludge blanket (UASB), and stable operation at a volumetric TOC loading rate of 3 g/l·d could be attained by diluting raw wastewater to give a TOC concentration of less than 6,000 mg/l (18,000 mg/l as CODer). When the distribution of the concentrations of aliphatic acids at various levels within the reactor was investigated, it was found that the methanogenic gasification reaction did not occur in the sludge blanket of the reactor. Distillery wastewater from barley-shochu making with various TOC concentrations was preliminary adjusted to pH 7 and treated by a single passage without circulation of effluent to the bottom of the reactor. As a result, a maximum TOC removal rate (V_max) of 28.5 g/l·d and a saturation constant (K_s) of 0.63 g/l were obtained.     

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.