Desired pH Research Articles

Effects of reduced pH on the growth and survival of postlarvae of the donkey’s ear abalone, Haliotis asinina (L.)

High atmospheric CO2 levels primarily from burning fossil fuels have increased CO2 concentration in the surface water of the ocean, this in turn has resulted in a drop in seawater pH known as “ocean acidification” (OA) and presents a potential threat to calcifying marine organisms. Up to the present, however, the impacts of OA on the early developmental stages of tropical abalone were not known. For the first time in the Philippines, we investigated the direct effects of reduced pH on the growth and survival of the postlarvae of the donkey’s ear abalone, Haliotis asinina L. H. asinina is the only commercial species of haliotid in the country. We reduced the ambient pH (7.99) of experimental seawater by bubbling food-grade CO2 to obtain the desired pH levels as expected by the year 2100. Survival of H. asinina postlarvae was negatively affected by reduced pH. Mean survival of postlarvae significantly decreased from 86.3 % at ambient conditions to 47.2 and 18.3 % at pH 7.62 and 7.42, respectively, after 20 days (P 0.05). Mean shell lengths of postlarvae also differed significantly among treatments and across exposure period (P = 0.000 for both). Postlarvae reared at pH 7.62 and pH 7.42 exhibited smaller shell size (138 and 124 µm, respectively) compared to those at ambient conditions (175 µm). Postlarvae reared at pH 7.81 showed larger shell length (162 µm) compared to the postlarvae reared at pH 7.42 (124 µm). Across time, significant increase in shell size from day 5 to day 20 was observed only in postlarvae reared at ambient conditions (P = 0.000) and in the pH 7.81 treatment (P = 0.006). Daily growth rate decreased significantly from 2.83 µm day−1 (ambient) to 0.95 and 0.30 µm day−1 at pH 7.62 and 7.42, respectively. Our results suggest that growth and survival of postlarval H. asinina was found to be sensitive to reduced pH of seawater. This implies that future decrease in pH unit of the world’s oceans will most likely threaten the natural population of this economically important species.

Rheological and structural properties of differently acidified and renneted milk gels

In this study we assessed the rheological and structural properties of differently acidified and renneted milk gels by controlling pH value and renneting extent. Skim milk were exactly renneted to 4 extents (20, 35, 55, and 74%) and then direct acidified to the desired pH (4.8, 5.0, 5.2, 5.5, 5.8, and 6.2), respectively. Rheological properties were assessed by dynamic rheological measurements, structural properties were studied by spontaneous whey separation and confocal laser scanning micrograph, and protein interactions were studied by dissociation test. Results showed that minimally renneted milk samples (20 and 35%) formed weak gels with low storage modulus, and the acidification range within which gels could form was narrow (pH ≤5.2). Highly renneted milk samples formed more gels with high storage modulus. The results of this study revealed that acidification determined the structural properties of highly renneted milk gels. As pH increased from 5.0 to 6.2, highly renneted milk gels had lower loss tangent, decreased spontaneous syneresis, and smaller pores. For both the low and high rennetings, divalent calcium bonds contributed less at low pH than at high pH. In conclusion, renneting increased the pH range suitable for gel formation; acidification determined the spontaneous syneresis and microstructure of highly renneted milk gels.

Biosorption of Zn2+ and Pb2+ from aqueous solutions using native and microwave treated Flammulina velutipes stipe

Native stipe (NS) and microwave treated stipe (MTS) of Flammulina velutipes were utilized for the biosorption of Zn2+ and Pb2+ ions from aqueous solution. The effects of pH, contact time, and initial concentration on the biosorption were studied for each metal separately. The desired pH of aqueous solution was found to be 6.0 for the removal of Zn2+ ions and 5.0 for the removal of Pb2+ ions. The percent removal of both metals was found to increase with the increase in contact time; biosorption equilibrium was established in about 60 min. The maximum biosorption of Zn2+ and Pb2+ ions from single component systems can be successfully described by Langmuir and Freundlich models; the biosorption kinetics can be accurately described by a second-order kinetic model. The present data from these studies confirms that the native and microwave treated forms of Flammulina velutipes stipe have the potential to be used for the biosorption of Zn2+ and Pb2+ ions from aqueous solution. The metal biosorption capacities of NS for Zn2+ and Pb2+ were 58.14 and 151.51 mg g−1, respectively, while the biosorption capacities of MTS for the both metals were 95.24 and 172.41 mg g−1, respectively.

PH- and thermo-responsive microcontainers as potential drug delivery systems: Morphological characteristic, release and cytotoxicity studies

Polymeric pH- and thermo-sensitive microcontainers (MCs) were developed as a potential drug delivery system for cancer therapy. It is well known that cancer cells exhibit notable characteristics such as acidic pH due to glycolytic cycle and higher temperature due to their higher proliferation rate. Based on these characteristics, we constructed a dual pH- and thermo-sensitive material for specific drug release on the pathological tissue. The MC's fabrication is based on a two-step procedure, in which, the first step involves the core synthesis and the second one is related to the shell formation. The core consists of poly(methyl methacrylate (PMMA), while the shell consists of PMMA, poly(isopropylacrylamide), poly(acrylic acid) and poly(divinylbenzene). Three different types of MCs were synthesized based on the seed polymerization method. The synthesized MCs were characterized structurally by Fourier transform infrared and morphologically by scanning electron microscopy. Dynamic light scattering was also used to study their behavior in aqueous solution under different pH and temperature conditions. For the loading and release study, the anthracycline drug daunorubicin (DNR) was used as a model drug, and its release properties were evaluated under different pH and thermo-conditions. Cytotoxicity studies were also carried out against MCF-7 breast cancer and 3T3 mouse embryonic fibroblast cells. According to our results, the synthesized microcontainers present desired pH and thermo behavior and can be applied in drug delivery systems. It is worth mentioning that the synthesized microcontainers which incorporated the drug DNR exhibit higher toxicity than the free drug.

Investigation on adsorption capacity of TiO2-P25 nanoparticles in the removal of a mono-azo dye from aqueous solution: A comprehensive isotherm analysis

In this work TiO2-P25 nanoparticles with high surface area have been used as adsorbent for the removal of C.I Acid Red 27 (AR27), as an organic contaminant from aqueous solution. Characteristics of phases and crystallite size of TiO2-P25 nanoparticles were achieved from XRD and the surface area and pore size distribution were obtained from BET and BJH techniques. TiO2-P25 nanoparticles with almost 80% anatase and 20% rutile phases, the average crystallite size of 18 nm, have specific surface area of 56.82 m2 g-1. The effect of various parameters like initial AR27 concentration, pH, contact time and adsorbent dosage has been carried out in order to find desired adsorption conditions. The desired pH for adsorption of AR27 onto TiO2-P25 nanoparticles was 3. The equilibrium data were analyzed with various 2-, 3- and 4-parameter isotherm models. Equilibrium data fitted very well by the 4-parameter Fritz-Schluender model. Results of adsorption kinetics study indicated that the pseudo-second order kinetics provided the best fit with correlation coefficients close to unity.

Controlling Release From pH-Responsive Microcapsules

We report a microfluidic approach to produce monodisperse pH-responsive microcapsules with precisely controlled release behavior. The solid microcapsule shells are composed of a biocompatible pH-responsive polymer and robustly encapsulate an active material; however, when exposed to a trigger pH, the shells degrade and ultimately release the microcapsule contents. We control the trigger pH by using polymers that dissolve at different pH values. We independently control the time at which the microcapsule contents are released by carefully controlling the shell thickness. Moreover, we independently control the rate at which the encapsulated contents are released by making hybrid shells composed of a mixture of a pH-responsive polymer and varying proportions of another, solid, pH-unresponsive polymer. This enables us to achieve monodisperse microcapsules that robustly encapsulate an active material, only releasing it when exposed to a desired pH, after a prescribed time delay, and at a prescribed rate.

New solution IEF device for micropreparative separation of peptides and proteins

The article presents a new concept of preparative solution IEF where time requirements and efficiency are similar to gel-based IEF whereas simple fraction handling as well as quick and complete protein recovery typical for solution-based IEF methods are maintained. The presented method is based on the IEF in separation medium soaked in a segmented strip of nonwoven fabric. The strip is positioned in an open horizontal V-shaped trough. Suggested focusing method combines free solution IEF under continuous evaporation and whole channel dispensing. Separation medium based on ethylene glycol/water mixture enhances viscosity enough to reduce electroosmosis and prevents the medium from completely drying out. Generation of pH gradient and final local pH is visually traced by colored low-molecular pI markers added to input mixture, which enables an optimization of focusing process and collection of individual fractions at desired pH range. The proposed method was tested by fractionation of the proteins and bioactive peptides originating from raw whey. Moreover, subsequent HPLC analysis of the individually collected solution IEF fractions was used for identification of whey components. We confirmed that the method is capable to process directly few tenths of milliliters of raw samples including the salty ones.

A study of pH effects on the bacterial surface physicochemical properties of Acinetobacter baumannii

The first step in the biofilm formation is the bacterial attachment to solid surfaces, which is dependent on the bacteria cell surface physico-chemical properties. The purpose of this work was to analyze the effect of pH on the physicochemical cell surface properties of Acinetobacter baumannii by two different methods. The cell surface properties were evaluated using the microbial adhesion to solvents method (MATS) and contact angle measurements (CAM). MATS technique allowed us to enlighten that A. baumannii was hydrophilic at the different values of pH. It was found that at a desired pH of 6.5, the strain presents a maximum and stable value of electron-donor characteristic, while the electron acceptor character increased as the pH increased. Regardless of the methods employed, the obtained results using MATS and CAM confirmed the influence of the pH on the surface physicochemical properties of A. baumannii. The cell surface electron-donor and electron-acceptor character at pH 6.5 was found to be quite similar using both methods.

Bactericidal efficacy of elevated pH on fish pathogenic and environmental bacteria

Ship ballast water is a recognized medium for transfer and introductions of nonindigenous species. There is a need for new ballast water treatment methods that effectively and safely eliminate or greatly minimize movements of these species. The present study employed laboratory methods to evaluate the bactericidal efficacy of increased pH (pH 10.0–12.0) for exposure durations of up to 72h to kill a variety of Gram-negative and Gram-positive bacteria including fish pathogens (Aeromonas spp., Yersinia ruckeri, Edwardsiella ictaluri, Serratia liquefaciens, Carnobacterium sp.), other common aquatic-inhabitant bacteria (Serratia marcescens, Pseudomonas fluorescens, Staphylococcus sp., Bacillus sp.) and indicators listed in International Maritime Organization D2 Standards; namely, Vibrio cholera (an environmental isolate from fish), Escherichia coli and Enterococcus faecalis. Volumes of 5N NaOH were added to tryptic soy broth to obtain desired pH adjustments. Viable cells were determined after 0, 4, 12, 24, 48, and 72h. Initial (0h) cell numbers ranged from 3.40×104cfu/mL for Bacillus sp. to 2.44×107cfu/mL for E. faecalis. The effective endpoints of pH and treatment duration necessary to realize 100% bactericidal effect varied; however, all bacteria tested were killed within 72h at pH 12.0 or lower. The lowest parameters examined, 4h at pH 10.0, were bactericidal to V. cholera, E. ictaluri, three of four isolates of E. coli, and (three of four) Aeromonas salmonicida subsp. salmonicida. Bactericidal effect was attained at pH 10.0 within 12h for the other A. salmonicida subsp. salmonicida, and within 24h for P. fluorescens, and the remaining E. coli.

Role of the Iron Axial Ligands of Heme Carrier HasA in Heme Uptake and Release

The hemophore protein HasA from Serratia marcescens cycles between two states as follows: the heme-bound holoprotein, which functions as a carrier of the metal cofactor toward the membrane receptor HasR, and the heme-free apoprotein fishing for new porphyrin to be taken up after the heme has been delivered to HasR. Holo- and apo-forms differ for the conformation of the two loops L1 and L2, which provide the axial ligands of the iron through His(32) and Tyr(75), respectively. In the apo-form, loop L1 protrudes toward the solvent far away from loop L2; in the holoprotein, closing of the loops on the heme occurs upon establishment of the two axial coordination bonds. We have established that the two variants obtained via single point mutations of either axial ligand (namely H32A and Y75A) are both in the closed conformation. The presence of the heme and one out of two axial ligands is sufficient to establish a link between L1 and L2, thanks to the presence of coordinating solvent molecules. The latter are stabilized in the iron coordination environment by H-bond interactions with surrounding protein residues. The presence of such a water molecule in both variants is revealed here through a set of different spectroscopic techniques. Previous studies had shown that heme release and uptake processes occur via intermediate states characterized by a Tyr(75)-iron-bound form with open conformation of loop L1. Here, we demonstrate that these states do not naturally occur in the free protein but can only be driven by the interaction with the partner proteins.

Effect of sourdough fermentation on the quality of Chinese Northern-style steamed breads

The effect of spontaneous and lactic acid bacteria (LAB) fermentation (Lactobacillus plantarum (LP), Lactobacillus sanfranciscensis (LS), Lactobacillus brevis (LBr) and Lactobacillus delbrueckii subsp bulgaricus (LB) strains) on the specific volume, crumb texture, volatiles profile and other aspects of steamed bread quality were studied. The addition of sourdough starter significantly decreased dough water absorption, development time and stability while increasing steam bread specific volume. The fermentation with LAB starters reached the desired pH 4 five times faster than the spontaneous starters. Steamed bread leavened with LS and LB starters had the softest crumb texture whereas LP and LBr starters produced the lowest setback viscosity. From the total of 77 compounds identified in sourdough steamed bread, ethanol and 3-methyl-1-butanol were the most abundant. All sourdough steamed breads had 22 compounds in common, which represented about 90% of the total peak area. But unique volatile compounds were present in sourdough steamed bread with different starters.

PH control structure design for a periodically operated membrane separation process

A bioreactor integrated with an electrically driven membrane separation process (Reverse Electro-Enhanced Dialysis – REED) is under investigation as potential technology for intensifying lactic acid bioproduction. In this contribution the pH regulation issue in the periodically operated REED module is studied. A methodology for control structure design is proposed to handle the dynamic system. A sensitivity analysis is used for the conceptual design of the control structure. Dynamic simulations are employed to evaluate the sensitivity index. From the analysis a periodic input-resetting control structure is selected. The system controls pH using the imposed current density and resets the current density manipulating the hydroxide inlet concentration to the dialysate channel. The control structure is satisfactorily achieving a desired pH at the outlet of the feed channel in REED from period to period and resetting the current density. Thus suitable performance is achieved within a large part of the operating window.

Improved Acid Stress Survival of Lactococcus lactis Expressing the Histidine Decarboxylation Pathway of Streptococcus thermophilus CHCC1524

Degradative amino acid decarboxylation pathways in bacteria generate secondary metabolic energy and provide resistance against acid stress. The histidine decarboxylation pathway of Streptococcus thermophilus CHCC1524 was functionally expressed in the heterologous host Lactococcus lactis NZ9000, and the benefits of the newly acquired pathway for the host were analyzed. During growth in M17 medium in the pH range of 5-6.5, a small positive effect was observed on the biomass yield in batch culture, whereas no growth rate enhancement was evident. In contrast, a strong benefit for the engineered L. lactis strain was observed in acid stress survival. In the presence of histidine, the pathway enabled cells to survive at pH values as low as 3 for at least 2 h, conditions under which the host cells were rapidly dying. The flux through the histidine decarboxylation pathway in cells grown at physiological pH was under strict control of the electrochemical proton gradient (pmf) across the membrane. Ionophores that dissipated the membrane potential (ΔΨ) and/or the pH gradient (ΔpH) strongly increased the flux, whereas the presence of glucose almost completely inhibited the flux. Control of the pmf over the flux was exerted by both ΔΨ and ΔpH and was distributed over the transporter HdcP and the decarboxylase HdcA. The control allowed for a synergistic effect between the histidine decarboxylation and glycolytic pathways in acid stress survival. In a narrow pH range around 2.5 the synergism resulted in a 10-fold higher survival rate.

Gd‐Aminoethyl‐DO3A Complexes: A Novel Class of pH‐Sensitive MRI Contrast Agents

AbstractMolecular imaging relies on the availability of imaging probes designed to modify their efficiency in the presence of a specific parameter, among which pH is one of the most investigated. Introduction of an aminoethyl moiety into the well‐known DO3A platform imparts the desired pH sensitivity to the corresponding Gd3+ complex. The amine group is accessible and easy to functionalize; in particular, the possibility of tuning its pKAH through N‐substitution makes it a good pH‐responsive functionality to access a novel class of tailored pH‐sensitive MRI contrast agents. This was demonstrated by a relaxometric study on a functionalized Gd‐DO3A complex bearing a simple primary amine group and comparison with the results of the corresponding N,N‐dimethyl analogue.

The use of CO2 for reversible pH shifting, and the removal of succinic acid in a polymer‐based two‐phase partitioning bioreactor

AbstractBACKGROUND: Succinic acid (SA) is an intermediate in the production of commodity chemicals, but SA bioproduction has not yet been commercialized due to end product inhibition and high product separation costs. Two‐phase partitioning bioreactors (TPPBs) can increase volumetric productivity through in situ product removal, although SA uptake by polymers requires a pH below the pKA2 of SA (4.2). It was proposed to reversibly reduce the pH with CO2 sparging for absorption of SA, followed by nitrogen stripping to allow continued bioproduction after returning to metabolic pH levels.RESULTS: At 1 atm CO2 sparging lowered the pH of RO water to 3.8 but only to 4.75 in medium, requiring acid/base pH adjustment in subsequent experiments. Actinobacillus succinogenes was temporarily exposed to pH 4.2 for between 5 min and 4 h to observe the effect on subsequent growth; cells could grow after up to 4 h of low pH exposure, sufficient time for SA uptake. Because atmospheric CO2 could not adequately lower the pH of medium, a TPPB was operated with the pH being shifted using strong acid/base; SA was recovered in situ, however, the accumulation of salts hindered further cell growth.CONCLUSION: Several key elements of this novel processing strategy were successfully demonstrated, and work is continuing with high pressure CO2 to achieve the desired pH adjustment levels. Copyright © 2011 Society of Chemical Industry

Hepatitis C Virus Is Primed by CD81 Protein for Low pH-dependent Fusion

Hepatitis C virus (HCV) entry into permissive cells is a complex process that involves interactions with at least four co-factors followed by endocytosis and low pH-dependent fusion with endosomes. The precise sequence of receptor engagement and their roles in promoting HCV E1E2 glycoprotein-mediated fusion are poorly characterized. Because cell-free HCV tolerates an acidic environment, we hypothesized that binding to one or more receptors on the cell surface renders E1E2 competent to undergo low pH-induced conformational changes and promote fusion with endosomes. To test this hypothesis, we examined the effects of low pH and of the second extracellular loop (ECL2) of CD81, one of the four entry factors, on HCV infectivity. Pretreatment with an acidic buffer or with ECL2 enhanced infection through changing the E1E2 conformation, as evidenced by the altered reactivity of these proteins with conformation-specific antibodies and stable association with liposomes. However, neither of the two treatments alone permitted direct fusion with the cell plasma membrane. Sequential HCV preincubation with ECL2 and acidic buffer in the absence of target cells resulted in a marked loss of infectivity, implying that the receptor-bound HCV is primed for low pH-dependent conformational changes. Indeed, soluble receptor-pretreated HCV fused with the cell plasma membrane at low pH under conditions blocking an endocytic entry pathway. These findings suggest that CD81 primes HCV for low pH-dependent fusion early in the entry process. The simple triggering paradigm and intermediate conformations of E1E2 identified in this study could help guide future vaccine and therapeutic efforts to block HCV infection.

Anti-CD22 Antibody Targeting of pH-responsive Micelles Enhances Small Interfering RNA Delivery and Gene Silencing in Lymphoma Cells

The application of small interfering RNA (siRNA) for cancer treatment is a promising strategy currently being explored in early phase clinical trials. However, efficient systemic delivery limits clinical implementation. We developed and tested a novel delivery system comprised of (i) an internalizing streptavidin-conjugated monoclonal antibody (mAb-SA) directed against CD22 and (ii) a biotinylated diblock copolymer containing both a positively charged siRNA condensing block and a pH-responsive block to facilitate endosome release. The modular design of the carrier facilitates the exchange of different targeting moieties and siRNAs to permit its usage in a variety of tumor types. The polymer was synthesized using the reversible addition fragmentation chain transfer (RAFT) technique and formed micelles capable of binding siRNA and mAb-SA. A hemolysis assay confirmed the predicted membrane destabilizing activity of the polymer under acidic conditions typical of the endosomal compartment. Enhanced siRNA uptake was demonstrated in DoHH2 lymphoma and transduced HeLa-R cells expressing CD22 but not in CD22 negative HeLa-R cells. Gene knockdown was significantly improved with CD22-targeted vs. nontargeted polymeric micelles. Treatment of DoHH2 cells with CD22-targeted polymeric micelles containing 15nmol/l siRNA produced 70% reduction of gene expression. This CD22-targeted polymer carrier may be useful for siRNA delivery to lymphoma cells.

Bioleaching and recovery of metals from final slag waste of the copper smelting industry

Solid waste from the copper smelting industry may be harmful if disposed of in the environment, but it may be a valuable resource if metals can be recovered. The purpose of this study was to evaluate the acid bioleaching of metals from a sample of final smelter slag and the recovery of metals from the leach liquors. Bioleaching was tested in a continuously stirred tank reactor (CSTR) at 20–25 °C with 5% pulp density (particle size 75% <47 μm). The yields of metal solubilization after 29 days of contact were 41% Fe, 62% Cu, 35% Zn and 44% Ni. Metals were precipitated in a separate CSTR by titrating the leach liquors with sulfide-rich effluent from a sulfate-reducing fluidized-bed reactor (FBR) (25 °C) to desired pH values. Over 98% of the Cu precipitated at pH ⩾ 2.8 and over 99% of the Zn precipitated at pH ⩾ 3.9. The precipitation of Ni and Fe required higher pH values and was less efficient than Cu and Zn recovery. In addition, bulk precipitation of metals was also tested by feeding the leach liquor directly to another sulfate-reducing FBR. In order to reduce its toxicity and maintain stable sulfate reduction performance in the FBR, the leach liquor had to be diluted ten-fold and the pH adjusted from 0.6 to approximately 4.

Formulation and Evaluation of Extended Release Asymmetric Membrane Capsules of Atenolol

The objective of this study was to demonstrate that the asymmetric membrane capsule can be used to deliver a poorly water soluble drug with a pH dependent solubility such as atenolol for extended periods of time by modulating solubility with organic acid. In osmotic systems, the release rate of an excipient relative to the release rate of the drug is an important factor that determines the duration of drug release. Consequently, for maintaining the desired pH over the entire period of drug dissolution a suitable thickening and suspending agent can be incorporated. By optimizing the concentration of thickening agent, it is possible to extend the availability of pH modifier in the core to provide an osmotic driving force or solubilization over the entire delivery period, so that the desired profile can be achieved for an active agent that has lower solubility characteristics. Finally, it was observed that the release rate of atenolol was influenced by the concentration of citric acid, mannitol and hydroxypropyl methylcellulose (HPMC). Results of scanning electron microscopy studies showed the formation of pores in the membrane from where the drug release occurred. The optimal formulation was found to be able to deliver atenolol at the rate of approximate zero-order up to 24 h, independent of pH of release media and agitation rate.

Utilisation of steel slags as neutralising agents in biooxidation of a refractory gold concentrate and their influence on the subsequent cyanidation

A study on the possibilities to utilise steel slag as neutralising agent in biooxidation of a refractory gold concentrate has been done with reference to commercial grade slaked lime. The idea has been to reduce the operating costs for neutralisation in the biooxidation plant, which is known to be the second largest operating cost. Other benefits would be savings in cost for landfilling of slag, possibilities to recycle elements present in the slag and savings of virgin limestone deposits. The slags used were an EAF slag and a slag from ladle refining; both originating from Swedish scrap based steel-making. Continuous biooxidation of the refractory gold concentrate was conducted in a single-stage 5 L reactor at a retention time of 56 h. The neutralisation capacity was determined by comparing the amount needed, per ton of feed concentrate added, to maintain the desired pH of 1.5 during steady state operation. Slaked lime had the highest neutralisation capacity with 110 kg/ton feed followed by ladle slag and EAF slag with values of 152 and 267 kg/ton feed, respectively. Sulphide mineral oxidation was similar and high in all cases although the ladle slag results were slightly better. Gold recoveries after cyanide leaching on the residues obtained were also similar and were in the range of 86–89%. However, the cyanide consumption expressed as kilogram cyanide per ton of concentrate fed to biooxidation, was double in the case of ladle slag and three times as much for the EAF slag compared to the slaked lime experiment. The increased cyanide consumption could not be explained only by the increased amount of elemental sulphur obtained in the slag experiments. The elemental sulphur formed had different reactivities as seen from the thiocyanate formation and cyanide losses due to thiocyanate formation were 16%, 32% and 40% for EAF slag, slaked lime and ladle slag, respectively. It is concluded that the ladle slag could be a possible replacement for limestone if they are mixed in proper proportions so that the microbial carbon dioxide demand is met whereas the EAF slag is less suitable due to the very fine reaction products obtained which gave operational problems with filtration and washing. To come further, experiments with the normal multi-stage biooxidation set-up with total retention time of 120 h should be performed which would increase the sulphur oxidation and eventually also reduce the cyanide consumption.