Biodegradation of olive washing wastewater pollutants by highly efficient phenol-degrading strains selected from adapted bacterial community

The bacterial community of an olive washing water (OWW) storage basin was characterized, by both cultivation and cultivation-independent methods. PCR-TGGE fingerprints analysis of different samples, taken along the olive harvesting season, revealed important variations of the bacterial community structure showing rapid establishment of prevalent bacterial populations. Several bacteria, isolated from OWW, were cultivated, in media containing increasing amounts of polyphenols, in order to select high phenol-degrading strains for the effluent pollutants reduction. Strains PM3 and PM15, affiliated to Raoultella terrigena and Pantoea agglomerans by 16S rRNA gene sequencing, were selected and used for OWW biological treatment under batch conditions in shake flasks cultures. The OWW content of phenols, BOD₅, COD and colour, was reduced by 93, 91, 89 and 62%, respectively, permitting effluent disposal and/or reuse with no additional treatments.     

Microbiological and physicochemical characterization of olive mill wastewaters from a continuous olive mill in Northeastern Portugal

The microbiological and physicochemical characterization of samples from the different wastewaters generated during oil extraction in a continuous olive mill was performed. The main aim was to determine which of the physicochemical parameters were the best fitted to correctly characterize these residual waters. High correlations were obtained for COD, DOC, K, P and N contents with the sampling points, allowing the distinction of olive washing waters (OWW) from olive centrifuge waters (OCW) and olive mill wastewaters (OMW). These parameters were sufficient for a rapid and less costly chemical characterization of these waters. Phenols and oil and grease contents, together with low pH and dissolved oxygen contents, and high organic loads, were the most toxic for microbial populations. Microbial characterization showed that fungi were well adapted to these stressing environmental characteristics and the reuse of OMW after aerobic treatment with microbial species isolated from the effluent is considered.     

Biomass growth monitoring using pressure drop in a cocurrent biofilter

The possibility of following the biomass growth by pressure drop measurement was investigated in an aerated cocurrent upflow fixed-bed bioreactor continuously fed with wastewater containing industrial organic pollutants. The experiments were carried out in a biological filtration oxygenated reactor (Biofor) pilot plant packed with expanded clay balls (Biolite) of 2.7-mm diameter, which served as biomass carriers. The column was equipped for on-line pressure drop measurements. Correlation between pressure drop measurements and Reynolds numbers of air and water were determined in experiments carried out without biomass. Under operating conditions with biomass, it was demonstrated that column clogging and the operating time between washing cycles can be predicted depending on the volumetric organic load for a given total organic carbon inlet concentration. The biological activity of the fixed biomass was estimated from the oxygen consumption rate per unit time and carrier area. The oxygen consumption rate measurements demonstrated that the biological activity depends on the inlet substrate concentration, and that the Biofor column was most efficient between 75 and 100 g m-3 of total organic carbon inlet concentration. In the course of the wastewater treatment, using pressure drop measurements, the equivalent diameter of the Biolite particles, the reduced column macroporosity, and the biofilm thickness were calculated. An expression correlating biofilm density and biofilm thickness, as determined from the pressure drop measurements, was proposed. Good agreement was found between the fixed biomass in the reactor, determined as volatile suspended solids, and the biologically active biomass, estimated by respirometry. Copyright 1998 John Wiley & Sons, Inc.     

TGGE analysis of the diversity of ammonia-oxidizing and denitrifying bacteria in submerged filter biofilms for the treatment of urban wastewater

The spatial and temporal diversity of the bacterial community-forming biofilms in a pilot-scale submerged biofilter used for the treatment of urban wastewater was analyzed by a temperature-gradient gel electrophoresis (TGGE) approach. TGGE profiles based on partial sequence of the 16S rRNA gene showed that the community composition of the biofilms remained fairly stable along the column system and during the whole time of operation of the biofilter (more than 1 year). Community-profiling based on the amplification and separation of partial ammonia monooxygenase (amoA) and nitrous oxide reductase (nosZ) genes demonstrated that ammonia-oxidizing and denitrifying bacteria coexisted in both the anoxic and the aerated parts of the system. Several amoA and nosZ bands separated by TGGE were reamplified and sequenced, in order to further analyze the composition of these microbial communities in the biofilm. Phylogeny inferred from amoA/AmoA revealed the prevalence of Nitrosomonas species with five sequences affiliated to Nitrosomonas oligotropha, six sequences affiliated to Nitrosomonas europaea, and three sequences that showed only 75.7–76.1% identity of the DNA sequence with the closest described species (Nitrosomonas nitrosa). According to literature, this low identity value is indicative of previously undiscovered species. Eighteen new partial nosZ sequences were obtained which were mostly related to nosZ of gamma-proteobacteria (Pseudomonas) or clustered in the periphery of previously known denitrifying alpha-proteobacteria (Bradyrhizobium and Azospirillum).     

Evaluation of kinetic parameters of biochemical reaction in three-phase fluidized bed biofilm reactor for wastewater treatment

This study evaluates the kinetic parameters of biochemical reaction in three-phase fluidized bed biofilm reactor from the steady state values of the response of the system to step changes in inlet concentration. It was observed from the outlet biological oxygen demand (BOD(5)) plot of the response of the system that as the inlet BOD(5) was increased, the outlet BOD(5) also increased, reached a peak value and then decreased until it leveled to a new steady state value corresponding to the new inlet concentration level. The increase in BOD(5) was attributed to the accumulation of substrate within the reactor as well as the decrease in biofilm substrate consumption rate as the microorganisms adjusted to the new environment. Using the substrate balance at steady state and assuming Monod kinetics, an equation relating the substrate consumption rate to substrate concentration (BOD(5)) and total biofilm surface area had been established. Monod kinetic parameters were found to be K=2.20g/m(2)/day, K(m)=17.41g/m(3) and K/K(m)=0.13m/day. The ratio K/K(m) can be taken as the indicator for biofilm substrate degradation effectiveness at low substrate concentrations.     

Edible mushrooms from olive oil mill wastes

The biological remediation of olive oil mill wastes has been attempted several times in the past through the use of different types of microbes. Among them, a relatively large array of fungi were studied for neutralizing the heavy pollutant effects and/or for converting these wastes into new value-added products. The present investigation was aiming at examining whether olive oil mill wastes could be exploited for the cultivation of mushrooms of the genus Pleurotus. At a preliminary stage, two Pleurotus species, i.e. P. eryngii and P. pulmonarius, were tested for their ability to colonize an olive press-cake (OPC) substrate supplemented with various dilutions of raw olive mill wastewater (OWW). Some important cultural characters related to mushroom production (earliness, yield, biological efficiencies and quality of basidiomata) were estimated. The outcome revealed different cultural responses for each Pleurotus species examined; the P. pulmonarius strain showed better earliness values and P. eryngii, although it was a slow growing fungus, produced basidiomata in high yields and of a very good quality. On the other hand, the OPC substrate supplemented with low concentrations of OWW (12.5% v/w) behaved satisfactorily as regards the fungal colonization rates and mushroom yield, but when the addition of higher rates of raw, untreated OWW (75–100% v/w) was attempted then the Pleurotus strains were completely unable to grow. The optimal concentration of OWW for Pleurotus mycelial growth was assessed through measurements of the biomass produced in liquid nutrient media and was found to lie within the 25–50% range, depending on the Pleurotus species and on the properties of the substrates examined. Furthermore, the phytotoxic effects that the spent liquid medium possessed were examined in comparison with the phytotoxicity of the raw liquid waste. The prospects of exploiting olive oil mills wastes for mushroom cultivation is discussed.     

Performance and efficiency of a yeast biofilter for the treatment of a Nigerian fertilizer plant effluent

A biofilter composed of yeasts and cassava peel was used to detoxify fertilizer plant effluent. The biological oxygen demand was reduced on treatment from a range of 1200–1400 mg/l to a range 135–404 mg/l. The ammonia-nitrogen (NH₃–N) and nitrate-nitrogen (NO₃–N) were reduced after treatment from 1000 to 10 mg/l and from 100 to 17.6 mg/l, respectively. The biofilter is simple and easy to handle with high efficiency of 98%.     

Development and evaluation of an experimental protocol for 3-D visualization and characterization of the structure of bacterial biofilms in porous media using laboratory X-ray tomography

The development of a reliable model allowing accurate predictions of biofilm growth in porous media relies on a good knowledge of the temporal evolution of biofilm structure within the porous network. Since little is known about the real 3-D structure of biofilms in porous media, this work was aimed at developing a new experimental protocol to visualize the 3-D microstructure of the inside of a porous medium using laboratory X-ray microtomography. A reliable and reproducible methodology is proposed for (1) growing a biofilm inside a porous medium, and (2) X-ray tomography-based characterization of the temporal development of the biofilm at the inlet of the biofilter. The statistical analysis proposed here also validates the results presented in the literature based on a biofilm structure single measurement.     

Macro-kinetic investigation on phenol uptake from air by biofiltration: Influence of superficial gas flow rate and inlet pollutant concentration

The macro-kinetic behavior of phenol removal from a synthetic exhaust gas was investigated theoretically as well as experimentally by means of two identical continuously operating laboratory-scale biological filter bed columns. A mixture of peat and glass beads was used as filter material. After sterilization it was inoculated with a pure strain of Pseudomonas putida, as employed in previous experimental studies. To determine the influence of the superficial gas flow rate on biofilter performance and to evaluate the phenol concentration profiles along the column, two series of continuous tests were carried out varying either the inlet phenol concentration, up to 1650 mg . m(-3), or the superficial gas flow rate, from 30 to 460 m(3) . m(-2) . h(-1). The elimination capacity of the biofilter is proved by a maximum volumetric phenol removal rate of 0.73 kg . m(-3) . h(-1). The experimental results are consistent with a biofilm model incorporating first-order substrate elimination kinetics. The model may be considered a useful tool in scaling-up a biofiltration system. Furthermore, the deodorization capacity of the biofilter was investigated, at inlet phenol concentrations up to 280 mg . m(-3) and superficial gas flow rates ranging from 30 to 92 m(3) . m(-2) . h(-1). The deodorization of the gas was achieved at a maximum inlet phenol concentration of about 255 mg . m(-3), operating at a superficial gas flow rate of 30 m(3) . m(-2) . h(-1). (c) 1996 John Wiley & Sons, Inc.     

Effect of plant and artificial aeration on solids accumulation and biological activities in constructed wetlands

In constructed wetlands, solids accumulation may have two consequences with opposing effects on treatment efficiency: it decreases the longevity by reducing void space and it enhances biological activity by favoring biofilm development. The goal of our study was to estimate the effect of plants (presence and species) and artificial aeration on solids accumulation (volatile and inorganic). The horizontal and vertical distribution of solids was sampled using solids traps in 12 constructed wetland mesocosms (5 years old). Microbial density and activity were estimated in the biological fraction of the sampled solids. The effect of plant presence reduced accumulated solids by 26% and sulphide content by 50% sulphide content. There was more solids accumulation in Typha angustifolia units than in Phragmites australis. Also, T. angustifolia generated more biological activities at the surface and close to the inlet while conditions were more homogeneous throughout P. australis units. Aeration (1) stimulated biofilm development at the inlet of planted beds, (2) seemed to reduce mineral matter accumulation and (3) generated the same pattern of activities in planted beds enabling to reach a total nitrogen removal rate of up to 0.65 g N m^?2 d^?1.     

A model for treating isopropyl alcohol and acetone mixtures in a trickle-bed air biofilter

A mathematical model that incorporates mass transfer process and biofilm reactions is presented to predict the performance of a trickle-bed air biofilter (TBAB) for treating isopropyl alcohol (IPA) and acetone (ACE) mixtures. The model consists of a set of mass balance equations for IPA, ACE and oxygen in the bulk gas phase and within the biofilm. The effluent gas phase IPA and ACE concentrations predicted by the present model were in good agreement with the measured data available in a previous study. The important parameters were evaluated by sensitivity analysis to determine their respective effects on model performance. Four parameters were identified that strongly influenced model performance: surface area of the biofilm per unit volume of packing material (A_S), empty-bed residence time (EBRT), maximum specific growth rate of microorganism (μ_m), and microbial yield coefficient (Y). Practical applications of the model to derive the performance equation of TBAB for treating different inlet IPA and ACE concentrations were also demonstrated.     

Biofiltration of ethylbenzene vapours: influence of the packing material

In order to investigate suitable packing materials, a soil amendment composed of granular high mineralized peat (35% organic content) locally available has been evaluated as carrier material for biofiltration of volatile organic compounds in air by comparison with a fibrous peat (95% organic content). Both supports were tested to eliminate ethylbenzene from air streams in laboratory-scale reactors inoculated with a two-month conditioned culture. In pseudo-steady state operation, experiments at various ethylbenzene inlet loads (ILs) were carried out. Maximum elimination capacity of about 120 g m(-3) h(-1) for an IL of 135 g m(-3) h(-1) was obtained for the fibrous peat. The soil amendment reactor achieved a maximum elimination capacity of about 45 g m(-3) h(-1) for an inlet load of 55 g m(-3) h(-1). Ottengraf-van den Oever model was applied to the prediction of the performance of both biofilters. The influence of gas flow rate was also studied: the fibrous peat reactor kept near complete removal efficiency for empty bed residence times greater than 1 min. For the soil amendment reactor, an empty bed residence time greater than 2 min was needed to achieve adequate removal efficiency. Concentration profiles along the biofilter were also compared: elimination occurred in the whole fibrous peat biofilter, while in the soil amendment reactor the biodegradation only occurred in the first 65% part of the biofilter. Results indicated that soil amendment material, previously selected to increase the organic content, would have potential application as biofilter carrier to treat moderate VOC inlet loads.     

A study of the effect of isothiazolones on the performance and characteristics of a laboratory-scale rotating biological contactor

AIMS: To study the effect of the isothiazolone biocide (Kathon WT) on the performance of laboratory-scale rotating biological contactors (RBCs) and their component biofilms. METHODS AND RESULTS: Biofilms were established on the RBCs and then exposed to 0.7-15 p.p.m. isothiazolones. Young, 1-week-old, biofilms were found to attain treatment efficiency equal to that of mature, 2-month-old, biofilms. Isothiazolone concentrations at 3 p.p.m. and above caused a progressive decline in treatment efficiency and 15 p.p.m. isothiazolones inhibited all microbial activity and resulted in the death of the biofilms. Bio-oxidation and the biodegradation of isothiazolones within the biofilms ontinued unhindered at concentrations which caused the total inhibition of planktonic bacteria. CONCLUSION: There was at least a 10-fold difference in susceptibility of planktonic and biofilm bacteria to isothiazolones. The chemical oxygen demand (COD) test was shown to be a reliable tool for investigating the efficiency of wastewater treatment units when the influent contains isothiazolones, while the biochemical oxygen demand (BOD) was unreliable due to the inhibition of bio-oxidation by the biocide. SIGNIFICANCE AND IMPACT OF THE STUDY: The results show that RBCs can be used to treat effluents containing isothiazolones at concentrations up to 1.5 p.p.m.     

Effects of biofilm formation on membrane performance in submerged membrane bioreactors

The effects of biofilm formation on membrane performance were evaluated for a submerged membrane bioreactor (sMBR) system with six different types of micro- and ultrafiltration membranes (working volume = 19 l). After operation for 24 h the permeability of the membranes with a larger pore size (microfiltration) decreased to that of the membranes with a much smaller pore size (ultrafiltration). Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) confirmed that biofilms could reduce the influence of the membrane surface properties. The chemical oxygen demand (COD) removal efficiency was 95% for the oily wastewater treatment in the sMBR where the filtration process made an important contribution (47% based on feed COD). Significant enhancement in COD removal occurred at the initial filtration stage because of biofilm formation and the dynamic member role of the biofilm layer. Membranes with various pore sizes had approximately the same permeate quality that was attributed to the biofilm on the membrane surfaces. Nevertheless, the ultrafiltration membranes had 43% more COD removal efficiency than the other applied membranes at the beginning of filtration (before biofilm formation) because of the smaller pore sizes and better sieving.     

Aerobic biological treatment of black table olive washing wastewaters: effect of an ozonation stage

The present work is a study of oxidative degradation of the organic matter present in the washing waters from the black table olive industry. Pollutant organic matter reduction was studied by an aerobic biological process and by the combination of two successive steps: ozonation pretreatment followed by aerobic biological degradation. In the single aerobic biological process, the evolution of biomass and organic matter contents was followed during each experiment. Contaminant removal was followed by means of global parameters directly related to the concentration of organic compounds in those effluents: chemical oxygen demand (COD) and total phenolic content (TP). A kinetic study was performed using the Contois model, which applied to the experimental data, provides the specific kinetic parameters of this model: 4.81×10⁻² h⁻¹ for the kinetic substrate removal rate constant, 0.279 g VSS g COD⁻¹ for the cellular yield coefficient and 1.92×10⁻² h⁻¹ for the kinetic constant for endogenous metabolism. In the combined process, an ozonation pretreatment is conducted with experiments where an important reduction in the phenolic compounds is achieved. The kinetic parameters of the following aerobic degradation stage are also evaluated, being 5.42×10⁻² h⁻¹ for the kinetic substrate removal rate constant, 0.280 g VSS g COD⁻¹ for the cellular yield coefficient and 9.1×10⁻³ h⁻¹ for the kinetic constant for the endogenous metabolism.     

Effects of biofilm formation on membrane performance in submerged membrane bioreactors

The effects of biofilm formation on membrane performance were evaluated for a submerged membrane bioreactor (sMBR) system with six different types of micro- and ultrafiltration membranes (working volume=19 l). After operation for 24 h the permeability of the membranes with a larger pore size (microfiltration) decreased to that of the membranes with a much smaller pore size (ultrafiltration). Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) confirmed that biofilms could reduce the influence of the membrane surface properties. The chemical oxygen demand (COD) removal efficiency was 95% for the oily wastewater treatment in the sMBR where the filtration process made an important contribution (47% based on feed COD). Significant enhancement in COD removal occurred at the initial filtration stage because of biofilm formation and the dynamic member role of the biofilm layer. Membranes with various pore sizes had approximately the same permeate quality that was attributed to the biofilm on the membrane surfaces. Nevertheless, the ultrafiltration membranes had 43% more COD removal efficiency than the other applied membranes at the beginning of filtration (before biofilm formation) because of the smaller pore sizes and better sieving.     

Activity and three-dimensional distribution of toluene-degrading Pseudomonas putida in a multispecies biofilm assessed by quantitative in situ hybridization and scanning confocal laser microscopy.

As a representative member of the toluene-degrading population in a biofilter for waste gas treatment, Pseudomonas putida was investigated with a 16S rRNA targeting probe. The three-dimensional distribution of P. putida was visualized in the biofilm matrix by scanning confocal laser microscopy, demonstrating that P. putida was present throughout the biofilm. Acridine orange staining revealed a very heterogeneous structure of the fully hydrated biofilm, with cell-free channels extending from the surface into the biofilm. This indicated that toluene may penetrate to deeper layers of the biofilm, and consequently P. putida may be actively degrading toluene in all regions of the biofilm. Furthermore, measurements of growth rate-related parameters for P. putida showed reduced rRNA content and cell size (relative to that in a batch culture), indicating that the P. putida population was not degrading toluene at a maximal rate in the biofilm environment. Assuming that the rRNA content reflected the cellular activity, a lower toluene degradation rate for P. putida present in the biofilm could be estimated. This calculation indicated that P. putida was responsible for a significant part (65%) of the toluene degraded by the entire community.     

Reaction kinetics in biofilms

A novel in situ microtechnique allows evaluating parameters of diffusion-controlled reactions in biofilms. A microprobe, 15 mum in diameter, was used to simultaneously measure the dissolved oxygen concentration and the optical density at different depths in a submerged biofilm. Based on the results, the biofilm diffusion coefficient for dissolved oxygen, D(f) the dissolved oxygen flux through the biofilm surface, J(02), and the half velocity coefficient, K(s), have been calculated.     

Continuous deodorization and bacterial community analysis of a biofilter treating nitrogen-containing gases from swine waste storage pits

A biofilter inoculated with Arthrobacter sp. was applied to the simultaneous elimination of trimethylamine (TMA) and ammonia (NH3) from the exhaust air of swine waste storage pits. The results showed that the biofilter achieved average removal efficiencies of 96.8+/-2.5% and 97.2+/-2.3% for TMA and NH3, respectively. A near-neutral pH (7.3-7.4) was maintained due to the accumulation of acid metabolites and the adsorption of alkaline NH3. Low moisture demand, low pressure drop and high biofilm stability in the system were other advantages. After long-term operation, the bacterial community structure showed that at least twenty-five bands were explicitly detected by a denaturing gradient gel electrophoresis (DGGE) method. However, the inoculated Arthrobacter sp. still maintained a dominant population (>50%). Paracoccus denitrificans' presence in the biofilter could play an important role in oxidizing NH3 and reducing nitrite by heterotrophic nitrification and anaerobic denitrification.