The document describes a study that used laboratory-scale bioreactors to investigate the biodegradation of two-phase olive mill waste (TPOMW) by its indigenous microbiota under different conditions. The effects of nutrient addition and aeration on bioremediation and microbial community changes were evaluated. Analysis found that nutrient addition and aeration led to greater decreases in polyphenolic content and increases in the fungal to bacterial ratio. Molecular identification of bacteria and fungi in the bioreactors identified several genera present, with fungi like Penicillium and Candida dominant.
2019 - Profiling of filamentous bacteria in activated sludge by 16s RNA ampli...WALEBUBLÉ
Abstract: In this study, filamentous bacteria in the activated sludge of a WWTP were investigated throughout a one-year period using high-throughput short-read (Illumina) and full-length (PacBio) 16S rRNA gene amplicon sequencing. The results showed that a total of 28 filamentous bacteria genera
were identified using Illumina sequencing. Also, we found 25 species using PacBio sequencing, belonging to Curvibacter, Mycobacterium, Haliscomenobacter, Defluvicoccus, Sphaerotilus, Thiothrix, Leptothrix, Gordonia and Tetrasphaera genera. Active Volatile Suspended Solids (AVSS) were
calculated from ATP data contained in living microorganisms, this parameter represents the living biomass concentration, and the food/microorganisms ratio (F/M ratio) was calculated using AVSS instead of MLVSS. To assess the contribution of the F/M ratio to the variability observed in the filamentous bacteria structure we carried out distance-based linear models (DISTLM) and distancebased redundancy analysis (dbRDA).
2017 - Environmental Ordination of Filamentous Bacteria in Activated SludgeWALEBUBLÉ
Reference:
Zornoza, A., Serrano, S. and Alonso, J.L. (2017) Environmental Ordination of Filamentous Bacteria in Activated Sludge. In: Abstracts of the 7th congress of European microbiologists FEMS 2017, Valencia, Spain, 9-13 July 2017.
2017 - Comparison of nitrifying microbial communities of two full-scale membr...WALEBUBLÉ
Barbarroja, P., Moreno-Mesonero, L., Zornoza, A., Fernández-Navarro, J., Alonso, J.L., Muñagorri, F., García, C., Álvarez, C. (2017) Comparison of nitrifying microbial communities of two full-scale membrane bioreactors treating wastewaters from municipal solid wastes using 16S rDNA gene amplicon sequencing. 7th congress of European microbiologists FEMS 2017, Valencia, Spain, 9-13 July 2017.
2017 - Environmental ordination of nitrifying bacterial community dynamics in...WALEBUBLÉ
Biological nitrification-denitrification is commonly used for nitrogen removal in Wastewater Treatment Plants (WWTPs). Nitrification, is the sequential oxidation of ammonia via nitrite to nitrate. This process is catalysed by ammonia-oxidizing bacteria and archaea (AOB and AOA) and nitrite-oxidizing bacteria (NOB), whose cooperation is needed to achieve complete nitrification. They are a phylogenetically diverse guild with pronounced ecological niche specialization and they differ from each other in fundamental physiological and molecular traits. Although the nitrification process in WWTPs has been investigated in depth, the response of microbial
communities are still a focus of considerable interest due to their high sensitivity to inhibitory compounds and environmental factors, that results in repeated breakdowns of nitrification performance. Most of studies have been mainly descriptive and/or exploratory and environmental interpretation has not been addressed. In this study, we focus on the environmental ordination of the relationships between biological variables (nitrifying bacterial community) and physicochemical variables (nitrogen compounds and environmental conditions), to propose new strategies to improve the performance of the nitrogen removal process in WWTPs.
2017 - Effect of ozone addition to control Gordonia foaming on the nitrifying...WALEBUBLÉ
The ozonation of activated sludge has been used as a technical measure for bulking control in a high number of full-scale wastewater treatment plants (WWTP), despite a lack of precise
predictions on the level of reduction in filament growth or the lack of knowledge of impact on microbial community from this technique. Ozone is a strong oxidant reacting rapidly with
suspended solids. Various studies have suggested that ozone attacks the bacterial cell surface, alters the permeability of the cell membrane and ultimately results in the leakage of cell
contents. However, the microbes in the sludge form a complex matrix, and ozone may affect bacterial populations at different rates different depending on their locations in the floc or their
capacity for adaptation. Nitrification, a key step of the nitrogen cycle, is the sequential oxidation of ammonia via nitrite to nitrate. This process is catalysed by ammonia-oxidizing bacteria
(AOB) and nitrite-oxidizing bacteria (NOB), whose cooperation is needed to achieve complete nitrification. Although the nitrification process in WWTPs has been investigated in depth, the response of microbial communities are still a focus of considerable interest due to their high sensitivity to inhibitory compounds and environmental factors that results in repeated
breakdowns of nitrification performance. In this study, we focus on two aspects that have not been thoroughly considered in previous studies; the use of ozone for Gordonia foaming
elimination on dynamic population of a nitrifying bacterial community, and the nitrification performance of activated sludge system.
2010 - A new species of genus Metacystis from a Wastewater Treatment PlantWALEBUBLÉ
ABSTRACT. Unusual prostomatid specimens were found in the biological reactor of a wastewater treatment plant in a health resort in Valencia, Spain. These ciliates were attached to flocs unlike other free-swimming prostomatid ciliates described to date in the mixed liquor of activated sludge plants. The morphological study of this species led to a typically different combination of characteristics: elongated
cell shape, 20–30 somatic kineties, 2 perioral kineties, and 1 circumoral kinety, 1 large vacuole protruding at the terminal end, a lorica tapered toward the aperture with a smooth neck, and 11–16 annular ridges. These characteristics place this representative as a new species of the genus Metacystis—Metacystis galiani n. sp. This species became the dominant population within the biological reactor when high values of conductivity (4,244 mS/cm) and temperature (26.8 1C) were recorded.
2010 - Assessment of plausible bioindicators for plant performance in advance...WALEBUBLÉ
Reference
Pérez-Uz, B., Arregui, L., Calvo P., Salvadó H., Fernandez N., Rodríguez E., Zornoza, A., Serrano, S. (2010) Assessment of plausible bioindicators for plant performance in advanced wastewater treatment. Water Research 44: 5059-5069.
2019 - Profiling of filamentous bacteria in activated sludge by 16s RNA ampli...WALEBUBLÉ
Abstract: In this study, filamentous bacteria in the activated sludge of a WWTP were investigated throughout a one-year period using high-throughput short-read (Illumina) and full-length (PacBio) 16S rRNA gene amplicon sequencing. The results showed that a total of 28 filamentous bacteria genera
were identified using Illumina sequencing. Also, we found 25 species using PacBio sequencing, belonging to Curvibacter, Mycobacterium, Haliscomenobacter, Defluvicoccus, Sphaerotilus, Thiothrix, Leptothrix, Gordonia and Tetrasphaera genera. Active Volatile Suspended Solids (AVSS) were
calculated from ATP data contained in living microorganisms, this parameter represents the living biomass concentration, and the food/microorganisms ratio (F/M ratio) was calculated using AVSS instead of MLVSS. To assess the contribution of the F/M ratio to the variability observed in the filamentous bacteria structure we carried out distance-based linear models (DISTLM) and distancebased redundancy analysis (dbRDA).
2017 - Environmental Ordination of Filamentous Bacteria in Activated SludgeWALEBUBLÉ
Reference:
Zornoza, A., Serrano, S. and Alonso, J.L. (2017) Environmental Ordination of Filamentous Bacteria in Activated Sludge. In: Abstracts of the 7th congress of European microbiologists FEMS 2017, Valencia, Spain, 9-13 July 2017.
2017 - Comparison of nitrifying microbial communities of two full-scale membr...WALEBUBLÉ
Barbarroja, P., Moreno-Mesonero, L., Zornoza, A., Fernández-Navarro, J., Alonso, J.L., Muñagorri, F., García, C., Álvarez, C. (2017) Comparison of nitrifying microbial communities of two full-scale membrane bioreactors treating wastewaters from municipal solid wastes using 16S rDNA gene amplicon sequencing. 7th congress of European microbiologists FEMS 2017, Valencia, Spain, 9-13 July 2017.
2017 - Environmental ordination of nitrifying bacterial community dynamics in...WALEBUBLÉ
Biological nitrification-denitrification is commonly used for nitrogen removal in Wastewater Treatment Plants (WWTPs). Nitrification, is the sequential oxidation of ammonia via nitrite to nitrate. This process is catalysed by ammonia-oxidizing bacteria and archaea (AOB and AOA) and nitrite-oxidizing bacteria (NOB), whose cooperation is needed to achieve complete nitrification. They are a phylogenetically diverse guild with pronounced ecological niche specialization and they differ from each other in fundamental physiological and molecular traits. Although the nitrification process in WWTPs has been investigated in depth, the response of microbial
communities are still a focus of considerable interest due to their high sensitivity to inhibitory compounds and environmental factors, that results in repeated breakdowns of nitrification performance. Most of studies have been mainly descriptive and/or exploratory and environmental interpretation has not been addressed. In this study, we focus on the environmental ordination of the relationships between biological variables (nitrifying bacterial community) and physicochemical variables (nitrogen compounds and environmental conditions), to propose new strategies to improve the performance of the nitrogen removal process in WWTPs.
2017 - Effect of ozone addition to control Gordonia foaming on the nitrifying...WALEBUBLÉ
The ozonation of activated sludge has been used as a technical measure for bulking control in a high number of full-scale wastewater treatment plants (WWTP), despite a lack of precise
predictions on the level of reduction in filament growth or the lack of knowledge of impact on microbial community from this technique. Ozone is a strong oxidant reacting rapidly with
suspended solids. Various studies have suggested that ozone attacks the bacterial cell surface, alters the permeability of the cell membrane and ultimately results in the leakage of cell
contents. However, the microbes in the sludge form a complex matrix, and ozone may affect bacterial populations at different rates different depending on their locations in the floc or their
capacity for adaptation. Nitrification, a key step of the nitrogen cycle, is the sequential oxidation of ammonia via nitrite to nitrate. This process is catalysed by ammonia-oxidizing bacteria
(AOB) and nitrite-oxidizing bacteria (NOB), whose cooperation is needed to achieve complete nitrification. Although the nitrification process in WWTPs has been investigated in depth, the response of microbial communities are still a focus of considerable interest due to their high sensitivity to inhibitory compounds and environmental factors that results in repeated
breakdowns of nitrification performance. In this study, we focus on two aspects that have not been thoroughly considered in previous studies; the use of ozone for Gordonia foaming
elimination on dynamic population of a nitrifying bacterial community, and the nitrification performance of activated sludge system.
2010 - A new species of genus Metacystis from a Wastewater Treatment PlantWALEBUBLÉ
ABSTRACT. Unusual prostomatid specimens were found in the biological reactor of a wastewater treatment plant in a health resort in Valencia, Spain. These ciliates were attached to flocs unlike other free-swimming prostomatid ciliates described to date in the mixed liquor of activated sludge plants. The morphological study of this species led to a typically different combination of characteristics: elongated
cell shape, 20–30 somatic kineties, 2 perioral kineties, and 1 circumoral kinety, 1 large vacuole protruding at the terminal end, a lorica tapered toward the aperture with a smooth neck, and 11–16 annular ridges. These characteristics place this representative as a new species of the genus Metacystis—Metacystis galiani n. sp. This species became the dominant population within the biological reactor when high values of conductivity (4,244 mS/cm) and temperature (26.8 1C) were recorded.
2010 - Assessment of plausible bioindicators for plant performance in advance...WALEBUBLÉ
Reference
Pérez-Uz, B., Arregui, L., Calvo P., Salvadó H., Fernandez N., Rodríguez E., Zornoza, A., Serrano, S. (2010) Assessment of plausible bioindicators for plant performance in advanced wastewater treatment. Water Research 44: 5059-5069.
2017 - Analysis of nitrifying microbial communities by FISH and 16S rRNA ampl...WALEBUBLÉ
Nitrification, the sequential oxidation of ammonia via nitrite to nitrate, is an important process for nitrogen removal from municipal wastewater. This process is catalysed by ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB), two different groups of slow-growing microorganisms whose cooperation is needed to achieve complete nitrification. High efficiency and stability of this process is required for wastewater treatment plants (WWTPs) operational optimization due to
nitrification is often subjected to recurring collapse in many WWTPs. Therefore, a better understanding of the microbial ecology of nitrifying bacteria in WWTPs could
potentially improve the nitrification stability. Novel high-throughput molecular methods, as next generation sequencing (NGS), are nowadays providing detailed knowledge on the microorganisms governing wastewater treatment systems. This
methods in conjunction with the environmental ordination of the relationships between biological variables (nitrifying bacterial community) and physicochemical variables (nitrogen compounds and environmental conditions) provide a powerful
tool to elucidate how selection pressures imposed by operational and environmental conditions affect community diversity and dynamics within activated sludge systems.
2017 - Plausible Bioindicators of Biological Nitrogen Removal Process in WWTPsWALEBUBLÉ
Reference:
Zornoza, A., Alonso, J.L. and Serrano, S. (2017) Plausible Bioindicators of Biological Nitrogen Removal Process in WWTPs. In: Abstracts of the 7th congress of European microbiologists FEMS 2017, Valencia, Spain, 9-13 July 2017.
2009 - Efficiency of nitrogen removal and protist communities the potential f...WALEBUBLÉ
Article published in the International Workshop on Integrated vision of urban and agro-industrial wastewater treatment, monitoring and reclamation: key role played by the Waste Water Treatment Plant. 2-3 Julio, 2009. ISRIM / LIFE (CEE n. 1973/92 EU Financial Instrument for the Environment) , Terni, Italy.
abstract
Extracts of the medicinal plant Palicourea rigida Kunth, popularly known as douradinha, are
widely used for treating urinary tract disorders. Unfortunately, nowadays this is one of the
species endemic to Brazilian Cerrado that is at greatest risk of extinction.
The aim of the this work was to use AFLP molecular markers to determine the genetic
structure and diversity of eight natural populations of P. rigida and to associate their genetic
characteristics with loganin production in order to obtain provide relevant information
to promote programs for the conservation of this valuable medicinal plant.
A total of 120 polymorphic bands were scored and higher proportion of genetic diversity
was found in inter-populations (64%) rather than in intra-populations (36%). Fst value was
found to be significantly greater than zero (0.3601), demonstrating the complex genetic
structure of P. rigida populations. Accessions collected from Cristalina, GO, showed higher
percentage of polymorphic loci (65.5%) and the highest genetic diversity. Analysis of
Molecular variance (AMOVA) demonstrated 63.9% of intra-population genetic variation.
The lowest genetic variability was detected among accessions from the population found
in Sacramento, MG. No spatial standard was observed for P. rigida population, suggesting a
partially isolated island model. It was observed a minor but significant positive correlation
(r ¼ 0.22) between chemical and genetic matrices. The association between chemical and
genetic data indicated that environmental factors promoted the loganin production in
populations growing in Luziânia, GO, and therefore accessions from those populations
should be considered as prime material for initiating the conservation process of P. rigida.
2013 Elsevier Ltd. All rights reserved.
In order to clean up soils contaminated with hydrocarbons, the bioremediation activity of Pseudomonas putida was studied. Pseudomonas putida is a bacterium that can withstand the harshest environmental conditions. It is able to metabolize a wide range of petroleum hydrocarbons which is used as a source of carbon and energy. Given the potential of this microorganism, an experiment wasconducted on this strain.
For the isolation of this microorganism, a sample ofsoil from the Vakinankaratra region in the urban commune of Antsirabe II, Madagascar was microbiologically analysed. The bacterial identification was based on a study of the morphological, physicochemical and sequential analysis of the 16S rDNA gene.
Characterization of organic compounds from biosolids of Buenos Aires City, Silvana Torri
Como citar este trabajo
Torri S.I., C. Alberti. 2012. Characterization of organic compounds from biosolids of Buenos Aires City, Journal of Soil Science and Plant Nutrition, 12 (1), 143-152
The International Journal of Engineering & Science is aimed at providing a platform for researchers, engineers, scientists, or educators to publish their original research results, to exchange new ideas, to disseminate information in innovative designs, engineering experiences and technological skills. It is also the Journal's objective to promote engineering and technology education. All papers submitted to the Journal will be blind peer-reviewed. Only original articles will be published.
his study investigated the microbial community in a full scale anaerobic baffled reactor and sequencing batch reactor system for oil-produced water treatment in summer and winter. The community structures of fungi and bacteria were analyzed through polymerase chain reaction–denaturing gradient gel electrophoresis and Illumina high-throughput sequencing, respectively. Chemical oxygen demand effluent concentration achieved lower than 50 mg/L level after the system in both summer and winter, however, chemical oxygen demand removal rates after anaerobic baffled reactor treatment system were significant higher in summer than that in winter, which conformed to the microbial community diversity. Saccharomycotina, Fusarium, and Aspergillus were detected in both anaerobic baffled reactor and sequencing batch reactor during summer and winter. The fungal communities in anaerobic baffled reactor and sequencing batch reactor were shaped by seasons and treatment units, while there was no correlation between abundance of fungi and chemical oxygen demand removal rates. Compared to summer, the total amount of the dominant hydrocarbon degrading bacteria decreased by 10.2% in anaerobic baffled reactor, resulting in only around 23% of chemical oxygen demand was removed in winter. Although microbial community significantly varied in the three parallel sulfide reducing bacteria, the performance of these bioreactors had no significant difference between summer and winter.
Abstract— Biofuel production from microalgae biomass appears as a promising long term alternative. Dunaliella tertiolecta is a microalgae with high tolerance to salinity, temperature, and light, making it relatively easy to grow. The aim of this study was to establish a pilot-scale culture to evaluate the biomass yield and bioethanol production. The cell culture of D. tertiolecta was started in 20 ml tubes and escalated to 20 L containers. The biomass yield was 0.153 g L-1 of dry basis (db) and its characterization showed protein (37% db) as major component followed by carbohydrates (35.6), lipids (13% db) and ash (6.5%). The carbohydrate fraction was composed of starch (27.1% db) and fiber (8.5 %) and its neutral sugar characterization yield glucose (91% molar). The main components of the lipid fraction were linolenic and palmitic acids. The biomass was subjected to an acid pre-treatment for the saccharification of complex carbohydrates, and the hydrolyzed biomass was fermented by Saccharomyces cerevisiae. It was possible to produce 0.615 ml g-1 of ethanol. In conclusion, D. tertiolecta has the potential for bioethanol production, making it a promising option for the biofuels future.
Welcome to International Journal of Engineering Research and Development (IJERD)IJERD Editor
call for paper 2012, hard copy of journal, research paper publishing, where to publish research paper,
journal publishing, how to publish research paper, Call For research paper, international journal, publishing a paper, IJERD, journal of science and technology, how to get a research paper published, publishing a paper, publishing of journal, publishing of research paper, reserach and review articles, IJERD Journal, How to publish your research paper, publish research paper, open access engineering journal, Engineering journal, Mathemetics journal, Physics journal, Chemistry journal, Computer Engineering, Computer Science journal, how to submit your paper, peer reviw journal, indexed journal, reserach and review articles, engineering journal, www.ijerd.com, research journals
2017 - Analysis of nitrifying microbial communities by FISH and 16S rRNA ampl...WALEBUBLÉ
Nitrification, the sequential oxidation of ammonia via nitrite to nitrate, is an important process for nitrogen removal from municipal wastewater. This process is catalysed by ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB), two different groups of slow-growing microorganisms whose cooperation is needed to achieve complete nitrification. High efficiency and stability of this process is required for wastewater treatment plants (WWTPs) operational optimization due to
nitrification is often subjected to recurring collapse in many WWTPs. Therefore, a better understanding of the microbial ecology of nitrifying bacteria in WWTPs could
potentially improve the nitrification stability. Novel high-throughput molecular methods, as next generation sequencing (NGS), are nowadays providing detailed knowledge on the microorganisms governing wastewater treatment systems. This
methods in conjunction with the environmental ordination of the relationships between biological variables (nitrifying bacterial community) and physicochemical variables (nitrogen compounds and environmental conditions) provide a powerful
tool to elucidate how selection pressures imposed by operational and environmental conditions affect community diversity and dynamics within activated sludge systems.
2017 - Plausible Bioindicators of Biological Nitrogen Removal Process in WWTPsWALEBUBLÉ
Reference:
Zornoza, A., Alonso, J.L. and Serrano, S. (2017) Plausible Bioindicators of Biological Nitrogen Removal Process in WWTPs. In: Abstracts of the 7th congress of European microbiologists FEMS 2017, Valencia, Spain, 9-13 July 2017.
2009 - Efficiency of nitrogen removal and protist communities the potential f...WALEBUBLÉ
Article published in the International Workshop on Integrated vision of urban and agro-industrial wastewater treatment, monitoring and reclamation: key role played by the Waste Water Treatment Plant. 2-3 Julio, 2009. ISRIM / LIFE (CEE n. 1973/92 EU Financial Instrument for the Environment) , Terni, Italy.
abstract
Extracts of the medicinal plant Palicourea rigida Kunth, popularly known as douradinha, are
widely used for treating urinary tract disorders. Unfortunately, nowadays this is one of the
species endemic to Brazilian Cerrado that is at greatest risk of extinction.
The aim of the this work was to use AFLP molecular markers to determine the genetic
structure and diversity of eight natural populations of P. rigida and to associate their genetic
characteristics with loganin production in order to obtain provide relevant information
to promote programs for the conservation of this valuable medicinal plant.
A total of 120 polymorphic bands were scored and higher proportion of genetic diversity
was found in inter-populations (64%) rather than in intra-populations (36%). Fst value was
found to be significantly greater than zero (0.3601), demonstrating the complex genetic
structure of P. rigida populations. Accessions collected from Cristalina, GO, showed higher
percentage of polymorphic loci (65.5%) and the highest genetic diversity. Analysis of
Molecular variance (AMOVA) demonstrated 63.9% of intra-population genetic variation.
The lowest genetic variability was detected among accessions from the population found
in Sacramento, MG. No spatial standard was observed for P. rigida population, suggesting a
partially isolated island model. It was observed a minor but significant positive correlation
(r ¼ 0.22) between chemical and genetic matrices. The association between chemical and
genetic data indicated that environmental factors promoted the loganin production in
populations growing in Luziânia, GO, and therefore accessions from those populations
should be considered as prime material for initiating the conservation process of P. rigida.
2013 Elsevier Ltd. All rights reserved.
In order to clean up soils contaminated with hydrocarbons, the bioremediation activity of Pseudomonas putida was studied. Pseudomonas putida is a bacterium that can withstand the harshest environmental conditions. It is able to metabolize a wide range of petroleum hydrocarbons which is used as a source of carbon and energy. Given the potential of this microorganism, an experiment wasconducted on this strain.
For the isolation of this microorganism, a sample ofsoil from the Vakinankaratra region in the urban commune of Antsirabe II, Madagascar was microbiologically analysed. The bacterial identification was based on a study of the morphological, physicochemical and sequential analysis of the 16S rDNA gene.
Characterization of organic compounds from biosolids of Buenos Aires City, Silvana Torri
Como citar este trabajo
Torri S.I., C. Alberti. 2012. Characterization of organic compounds from biosolids of Buenos Aires City, Journal of Soil Science and Plant Nutrition, 12 (1), 143-152
The International Journal of Engineering & Science is aimed at providing a platform for researchers, engineers, scientists, or educators to publish their original research results, to exchange new ideas, to disseminate information in innovative designs, engineering experiences and technological skills. It is also the Journal's objective to promote engineering and technology education. All papers submitted to the Journal will be blind peer-reviewed. Only original articles will be published.
his study investigated the microbial community in a full scale anaerobic baffled reactor and sequencing batch reactor system for oil-produced water treatment in summer and winter. The community structures of fungi and bacteria were analyzed through polymerase chain reaction–denaturing gradient gel electrophoresis and Illumina high-throughput sequencing, respectively. Chemical oxygen demand effluent concentration achieved lower than 50 mg/L level after the system in both summer and winter, however, chemical oxygen demand removal rates after anaerobic baffled reactor treatment system were significant higher in summer than that in winter, which conformed to the microbial community diversity. Saccharomycotina, Fusarium, and Aspergillus were detected in both anaerobic baffled reactor and sequencing batch reactor during summer and winter. The fungal communities in anaerobic baffled reactor and sequencing batch reactor were shaped by seasons and treatment units, while there was no correlation between abundance of fungi and chemical oxygen demand removal rates. Compared to summer, the total amount of the dominant hydrocarbon degrading bacteria decreased by 10.2% in anaerobic baffled reactor, resulting in only around 23% of chemical oxygen demand was removed in winter. Although microbial community significantly varied in the three parallel sulfide reducing bacteria, the performance of these bioreactors had no significant difference between summer and winter.
Developing tools to attenuate emerging contaminants in onsite wastewater trea...
Similar to 2008 - Molecular microbial and chemical investigation of the bioremediation of two-phase olive mill waste using laboratory-scale bioreactors
Abstract— Biofuel production from microalgae biomass appears as a promising long term alternative. Dunaliella tertiolecta is a microalgae with high tolerance to salinity, temperature, and light, making it relatively easy to grow. The aim of this study was to establish a pilot-scale culture to evaluate the biomass yield and bioethanol production. The cell culture of D. tertiolecta was started in 20 ml tubes and escalated to 20 L containers. The biomass yield was 0.153 g L-1 of dry basis (db) and its characterization showed protein (37% db) as major component followed by carbohydrates (35.6), lipids (13% db) and ash (6.5%). The carbohydrate fraction was composed of starch (27.1% db) and fiber (8.5 %) and its neutral sugar characterization yield glucose (91% molar). The main components of the lipid fraction were linolenic and palmitic acids. The biomass was subjected to an acid pre-treatment for the saccharification of complex carbohydrates, and the hydrolyzed biomass was fermented by Saccharomyces cerevisiae. It was possible to produce 0.615 ml g-1 of ethanol. In conclusion, D. tertiolecta has the potential for bioethanol production, making it a promising option for the biofuels future.
Welcome to International Journal of Engineering Research and Development (IJERD)IJERD Editor
call for paper 2012, hard copy of journal, research paper publishing, where to publish research paper,
journal publishing, how to publish research paper, Call For research paper, international journal, publishing a paper, IJERD, journal of science and technology, how to get a research paper published, publishing a paper, publishing of journal, publishing of research paper, reserach and review articles, IJERD Journal, How to publish your research paper, publish research paper, open access engineering journal, Engineering journal, Mathemetics journal, Physics journal, Chemistry journal, Computer Engineering, Computer Science journal, how to submit your paper, peer reviw journal, indexed journal, reserach and review articles, engineering journal, www.ijerd.com, research journals
Evaluation of the Effectiveness of Fungi (Candida Tropicalis and Aspergillus ...IJEABJ
Used engine oil is a petroleum or synthetic oil that has been used and as a result of such use, is contaminated by physical and chemical pollutants. These pollutants are harmful to humans, animals and plants following exposure. Evaluation of the effectiveness of fungi in bioremediation of used engine oil (UEO) contaminated soil was investigated. Fungi were isolated from soil samples obtained from automobile workshops in Mgbuka-Nkpor, Nigeria. The isolates were screened for UEO biodegradation potentials in mineral salt broth. They were identified using the cultural and microscopic characteristics and confirmed using the 18SrRNA gene sequence. The effectiveness of the isolates in bioremediation of UEO contaminated soil was also investigated using bioaugmentation technique. A total of 8 fungal isolates were obtained from this study. Two that showed the highest extent of biodegradation of UEO in the screen flasks were identified and confirmed as Candida tropicalis and Aspergillus clavatus. At the end of the experimental period, oil contaminated soil inoculated with the mixed culture of the isolates (C. tropicalis and A. clavatus) showed the highest reduction in concentration of UEO (95.42%). Higher biodegradation rate and shorter half-life of total petroleum hydrocarbon (TPH) was observed in soil microcosm containing the isolates, when compared to the uninoculated control. Therefore fungi such as C. tropicalis and A. clavatus isolated from automobile workshops can facilitate the bioremediation of UEO contaminated soil.
Effect of nitrogen and phosphorus amendment on the yield of a Chlorella sp. s...Agriculture Journal IJOEAR
Abstract— A strain of microalgae was isolated from phytoplankton samples collected from the sea coast of Amsheet, North Lebanon. Molecular diagnosis based on ribosomal RNA genes showed it to be most closely related to Chlorella sp. (GenBank accession KC188335.1) with over 90 % nucleotide identity. It was then evaluated whether N and P amendments of seawater fertilized with Guillard’s f/2 medium would improve algal growth and production. Addition of nitrogen (30 ppm) and/or phosphorus (2 ppm) to microalgae grown under laboratory conditions in 3L bioreactors resulted in improved biomass yield (mg dry matter/ L) by approximately 48%, and increased protein yield by approximately 56%, from 19.5% to 30.6% of DM content. Total protein yield/L of culture medium was therefore increased by approximately 83%. Total lipid content and carotenoid levels of the microalgal culture were not affected by the N+P amendement, whereas chlorophyll content was almost doubled. When lower levels of N+P supplementations, 10 and 20 ppm N, were tried, the biomass yield was also improved. The experiment was repeated in 20 L bioreactors in a plastic greenhouse, under normal environmental conditions, with an average temperature of 28°C and a maximum temperature of 36°C. At these relatively high temperatures, the growth rate was slowed down, but N supplementations at 10 and 20 ppm resulted in improved dry matter yield by 25 and 45% respectively, and protein content by 17 and 35%, respectively. Knowledge of the optimal culturing conditions of this local Chlorella strain is essential for its efficient production and is expected to serve future environmental and biotechnological purposes.
Optimization of Experimental Biomethanation Applied to Poultry Droppings for ...IJEAB
The fight against climate change is first and foremost passed by the reduction of greenhouse gases (GHG). Mainly in the form of methane CH4, the GHGs emitted by the waste originate from the decomposition of organic matter which is more commonly known as Anaerobic Digestion (AD) or Biomethanation. Livestock manure is one of the major hazards to the environment and human health due to the nuisances and pollution generated. The present study consists of optimizing the methane fermentation applied to poultry droppings. This optimization focuses on the daily monitoring of experimental digesters, on the physico-chemical characterization of the inputs used and on the study of the effect of temperature and inoculum changes on the daily production of biogas and its composition (CH4, CO2 and H2S). The main results show, on the one hand, that the stability of the DA process after initial filling depends on experimental conditions, the general characteristics of the anaerobic digester, the initial biomass activity and the nature of the introduced inoculum. On the other hand, the production of biogas is better at a temperature of 35°C than at a temperature of 55°C and the addition of the inoculum has improved the production of biogas and the CH4 content, especially the use Of liquid poultry digestate.
International Journal of Engineering and Science Invention (IJESI) inventionjournals
International Journal of Engineering and Science Invention (IJESI) is an international journal intended for professionals and researchers in all fields of computer science and electronics. IJESI publishes research articles and reviews within the whole field Engineering Science and Technology, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online
Development of a Bioactive Food Additive for Controlling of Fungal GrowthIJEAB
Fresh foods have a great importance in human nutrition. However, they are marketed with greatly reduced shelf life mainly due to fungal spoilage. In this work, cell wall degrading enzymes produced by Trichoderma asperellum T00 (TCWDE) were immobilized onto cashew gum polysaccharide (CGP) in order to evaluate the potential use of this material as food additive aiming to increase the shelf life by inhibiting fungal growth. Results from factorial design (32) evidenced that the best conditions for TCWDE immobilization was achieved with 20 min of reaction using 1 mmol L-1 of NaIO4. On these conditions it was observed 91% of retention yield for NAGase (30.1 ± 0.38 U mL-1), 41% for chitinase (0.67 ± 0.05 U mL-1), and 24% for β-1,3-glucanase (0.017± 0.001 U mL-1). CGP/TCWDE was effective for growth inhibition of Aspergillus fumigatus and Penicillium sp. and the inhibition mechanism seems to involve changes in the cell wall of those microorganisms. Finally, the CGP/TCWDE presented high stability after drying, maintaining enzymatic and biological activity after 200 days of storage at room temperature (25 ºC).
Enhancing the Nutritive Values of Agrowastes for Animal Feed Production Using...iosrjce
IOSR Journal of Environmental Science, Toxicology and Food Technology (IOSR-JESTFT) multidisciplinary peer-reviewed Journal with reputable academics and experts as board member. IOSR-JESTFT is designed for the prompt publication of peer-reviewed articles in all areas of subject. The journal articles will be accessed freely online.
Fruit and Vegetable Waste Hydrolysates as Growth Medium for Higher Biomass an...Premier Publishers
Fruit and vegetable wastes include peels, pulp and seeds that constitute about 40% of the total mass and constitute huge environmental problems. Cultivation of microalgae that utilizes fruit and vegetable wastes as feedstock to produce value added products such as biomass and lipids is a unique approach. Different concentrations of fruit waste hydrolysate (FWH) and vegetable waste hydrolysate (VWH) were used for heterotropic cultivation of Chlorella vulgaris thereby optimizing the suitable hydrolysate concentration for higher biomass and lipid production. FWH in the ratio of 8:2 has produced maximum specific growth rate of 1.92 µ d-1. Higher biomass was recorded in growth medium supplemented with FWH (0.16 mg L-1) than VWH medium. Highest chlorophyll content of 7.2 mg L-1 was observed in 8:2 ratio of FWH whereas it was 4.3 mg L-1 in VWH at the same concentration. Carotenoid content was highest in VWH than FWH media with a maximum content of 0.52 and 0.42 mg L-1 respectively. Fruit waste hydrolysates significantly increased the total lipid content than the vegetable waste hydrolysate medium. Highest lipid content of 6.63 mg L-1 was recorded in 8:2 ratio of FWH. This work demonstrates the feasibility of fruit waste hydrolysate as a nutrient source for algal cultivation and a cost reduction of growth medium in algal biomass and lipid production.
Similar to 2008 - Molecular microbial and chemical investigation of the bioremediation of two-phase olive mill waste using laboratory-scale bioreactors (20)
2013 - Estudio de las relaciones de las bacterias filamentosas no ramificadas...WALEBUBLÉ
Tena, S. (2013) Estudio de las relaciones de las bacterias filamentosas no ramificadas (Microthrix y tipo 0581) formadoras de espumas con los parámetros Operacionales y Físico- Químicos en una EDAR de la Comunidad Valenciana. Trabajo final de Máster en Ingeniería Ambiental. Valencia: Universitat Politècnica de València.
www.abgc
2016 - Estudio de la dinámica de protistas y metazoos en un reactor biológico...WALEBUBLÉ
Martínez. I. (2016) Estudio de la dinámica de protistas y metazoos en un reactor biológico de aireación prolongada con macrófitas en flotación y su relación con las variables fisicoquímicas. Trabajo final de Máster en Ingeniería Ambiental. Valencia: Universitat Politècnica de València.
2014 - Estudio de las relaciones del morfotipo Nosotocoida limicola con los p...WALEBUBLÉ
Calvo, S. (2014) Estudio de las relaciones del morfotipo Nosotocoida limicola con los parámetros operacionales y fisico-químicos en EDAR de la Comunidad Valenciana. Trabajo final de Máster en Ingeniería Ambiental. Valencia: Universitat Politècnica de València.
www.abgc.es
2014 - Identificación y cuantificación del morfotipo Haliscomenobacter hydros...WALEBUBLÉ
Ferrer, M. (2014) Identificación y cuantificación del morfotipo Haliscomenobacter hydrossis formador de bulking mediante la técnica FISH y estudio de su relación con los parámetros operacionales y físico-químicos en EDAR de la Comunidad Valenciana. Trabajo final de Máster en Ingeniería Ambiental. Valencia: Universitat Politècnica de València.
www.abgc.es
2012 - Microscopía convencional versus FISH en la identificación, abundancia ...WALEBUBLÉ
Andújar, A. B. (2012) Microscopía convencional versus FISH en la identificación, abundancia y ecofisiología de los morfotipos filamentosos 0803, 0914 y 0092 en fangos activos. Trabajo final de máster en Ingeniería Ambiental. Valencia: Universitat Politècnica de València.
www.abgc.es
2017 -Study of enzymatic activities and bacterial communities in two full-sca...WALEBUBLÉ
Reference:
Fernández-Navarro, J., Moreno-Mesonero, L., Zornoza, A., Amorós, I., Alonso-Molina, J.L., Muñagorri, F., Álvarez, C. (2017) Study of enzymatic activities and bacterial communities in two full-scale MBR plants treating wastewaters from municipal solid wastes. In: Abstracts of the 7th congress of European microbiologists FEMS 2017, Valencia, Spain, 9-13 July 2017.
www.abgc.es
2013 - Estudio de las relaciones de Gordonia con parámetros operacionales y f...WALEBUBLÉ
Nuñez, J.M. (2013) Estudio de las relaciones de Gordonia con parámetros operacionales y físico-químicos en EDAR de la Comunidad Valenciana. Trabajo final de Máster. Valencia: Universitat Politècnica de València.
Lynx ASM1 (v2.5) es un software de simulación de tratamiento de aguas residuales en estaciones depuradoras de aguas residuales (EDAR), basado en el modelo matemático ASM1 (Activated Sludge Model no 1).
Más información en: www.abgc.es
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
Deep Behavioral Phenotyping in Systems Neuroscience for Functional Atlasing a...Ana Luísa Pinho
Functional Magnetic Resonance Imaging (fMRI) provides means to characterize brain activations in response to behavior. However, cognitive neuroscience has been limited to group-level effects referring to the performance of specific tasks. To obtain the functional profile of elementary cognitive mechanisms, the combination of brain responses to many tasks is required. Yet, to date, both structural atlases and parcellation-based activations do not fully account for cognitive function and still present several limitations. Further, they do not adapt overall to individual characteristics. In this talk, I will give an account of deep-behavioral phenotyping strategies, namely data-driven methods in large task-fMRI datasets, to optimize functional brain-data collection and improve inference of effects-of-interest related to mental processes. Key to this approach is the employment of fast multi-functional paradigms rich on features that can be well parametrized and, consequently, facilitate the creation of psycho-physiological constructs to be modelled with imaging data. Particular emphasis will be given to music stimuli when studying high-order cognitive mechanisms, due to their ecological nature and quality to enable complex behavior compounded by discrete entities. I will also discuss how deep-behavioral phenotyping and individualized models applied to neuroimaging data can better account for the subject-specific organization of domain-general cognitive systems in the human brain. Finally, the accumulation of functional brain signatures brings the possibility to clarify relationships among tasks and create a univocal link between brain systems and mental functions through: (1) the development of ontologies proposing an organization of cognitive processes; and (2) brain-network taxonomies describing functional specialization. To this end, tools to improve commensurability in cognitive science are necessary, such as public repositories, ontology-based platforms and automated meta-analysis tools. I will thus discuss some brain-atlasing resources currently under development, and their applicability in cognitive as well as clinical neuroscience.
What is greenhouse gasses and how many gasses are there to affect the Earth.moosaasad1975
What are greenhouse gasses how they affect the earth and its environment what is the future of the environment and earth how the weather and the climate effects.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
2008 - Molecular microbial and chemical investigation of the bioremediation of two-phase olive mill waste using laboratory-scale bioreactors
1. ENVIRONMENTAL BIOTECHNOLOGY
Molecular microbial and chemical investigation
of the bioremediation of two-phase olive mill waste
using laboratory-scale bioreactors
J. A. Morillo & M. Aguilera & B. Antízar-Ladislao &
S. Fuentes & A. Ramos-Cormenzana & N. J. Russell &
M. Monteoliva-Sánchez
Received: 31 December 2007 /Revised: 14 February 2008 /Accepted: 15 February 2008 /Published online: 18 March 2008
# Springer-Verlag 2008
Abstract Two-phase olive mill waste (TPOMW) is a semi-
solid effluent that is rich in contaminating polyphenols and
is produced in large amounts by the industry of olive oil
production. Laboratory-scale bioreactors were used to
investigate the biodegradation of TPOMW by its indige-
nous microbiota. The effect of nutrient addition (inorganic
N and P) and aeration of the bioreactors was studied.
Microbial changes were investigated by PCR-temperature
time gradient electrophoresis (TTGE) and following the
dynamics of polar lipid fatty acids (PLFA). The greatest
decrease in the polyphenolic and organic matter contents of
bioreactors was concomitant with an increase in the PLFA
fungal/bacterial ratio. Amplicon sequences of nuclear
ribosomal internal transcribed spacer region (ITS) and16S
rDNA allowed identification of fungal and bacterial types,
respectively, by comparative DNA sequence analyses.
Predominant fungi identified included members of the
genera Penicillium, Candida, Geotrichum, Pichia, Clado-
sporium, and Aschochyta. A total of 14 bacterial genera
were detected, with a dominance of organisms that have
previously been associated with plant material. Overall, this
work highlights that indigenous microbiota within the
bioreactors through stimulation of the fungal fraction, is
able to degrade the polyphenolic content without the
inoculation of specific microorganisms.
Keywords Bioremediation . Microbial diversity.
Olive mill waste
Introduction
The olive oil industry generates large amounts of byproducts
that are harmful to the environment. Olive mill wastes
(OMWs) contain phytotoxic components capable of inhibiting
microbial growth (Capasso et al. 1995; Ramos-Cormenzana
et al. 1996) and the germination and vegetative growth of
plants (Linares et al. 2003). Traditionally, the disposal of
OMWs has become a great problem in Mediterranean
countries because of their polluting effects on soil and water
(Sierra et al. 2001; Piotrowska et al. 2006). The production of
olive oil is increasing worldwide, with a growth rate that is
expected to be between 3.5 and 4% per year, according to the
International Olive Oil Council (Anonymous 2004). More-
over, this production is no longer restricted to the Mediter-
ranean Basin, and new producers such as Australia, USA,
and South American countries will have to face the
environmental problems posed by OMWs.
At present, three systems are used worldwide for the
industrial-scale extraction of oil from olives, viz. the
traditional press-cake system, the three-phase decanter
system and the modern two-phase centrifugation system.
The present paper focuses on the two-phase centrifugation
Appl Microbiol Biotechnol (2008) 79:309–317
DOI 10.1007/s00253-008-1422-5
J. A. Morillo (*) :M. Aguilera :S. Fuentes :
A. Ramos-Cormenzana :M. Monteoliva-Sánchez
Departamento de Microbiología, Facultad de Farmacia,
Universidad de Granada,
Campus de Cartuja, s/n,
18071 Granada, Spain
e-mail: jamorillo@yahoo.com
B. Antízar-Ladislao :N. J. Russell
Imperial College London, Wye campus,
Wye, Ashford, Kent TN25 5AH, UK
B. Antízar-Ladislao
School of Engineering and Electronics, University of Edinburgh,
The King’s Buildings, Mayfield Road,
Edinburgh EH9 3JL, UK
2. system, which was introduced in the 1990s as an ecological
approach for olive oil production, since it drastically
reduces water consumption during the process. The waste
stream of this system consists of a semi-solid waste: the
two-phase olive mill waste (TPOMW) or alpeorujo. The
resulting waste comprises about 800 kg per 1,000 kg of
the processed olives, and its production may exceed 4
million tons annually in Spain alone (Aragón and Palancar
2001; Alburquerque et al. 2004). The TPOMW consists of
a thick sludge that contains water and pieces of pit plus the
pulp of the olive fruit. This semi-solid effluent has a water
content of about 65% (Arjona et al. 1999), a slightly acidic
pH, and a very high content of organic matter, mainly
composed of lignin, hemicellulose and cellulose; it also has
a considerable proportion of fats, proteins, water-soluble
carbohydrates, and a small but active fraction of hydro-
soluble (poly)phenolic substances (Alburquerque et al.
2004).
The polyphenol content of olive residues consists of a
complex mixture of compounds and is considered to be the
most problematic fraction of OMWs, being associated with
the well-known antimicrobial and phytotoxic effects of
these residues (Ramos-Cormenzana et al. 1996). Bioreme-
diation is a valuable tool for the detoxification of TPOMW
by breaking down these phenolic compounds. Recent research
on TPOMW bioremediation has included different ap-
proaches, such as anaerobic digestion (Borja et al. 2005;
Rincón et al. 2006), natural biodegradation in evaporation
ponds (Buyer et al. 1999), removal of phenols by sapro-
phytic fungi (Sampedro et al. 2004), biodegradation by co-
composting with agricultural wastes (Paredes et al. 2002),
vermicomposting (Benitez et al. 2005), and the production of
metal-binding microbial exopolysaccharides (Morillo et al.
2006). Despite the importance of bioremediation as an
alternative to TPOMW detoxification and recycling, surpris-
ingly little information is available on the indigenous
microbiota of the residue and their potential for carrying
out biodegradation of the waste (Jones et al. 2000;
Giannoutsou et al. 2004; Ntougias et al. 2006). To our
knowledge, the only detailed molecular characterization of
the microbial communities of TPOMW is that of Rincón et al.
(2006), where the molecular identification of the microbial
species involved in a process of anaerobic treatment of
diluted TPOMW was performed by PCR-DGGE. The
Firmicutes (mainly represented by Clostridiales) were the
most abundant phylotype, followed by the Chloroflexi and
the Gammaproteobacteria (Pseudomonas species; Rincón
et al. 2006).
Thus, the aim of the present investigation was twofold:
first, to evaluate at laboratory scale the effect of nutrient
amendment and aeration on the bioremediation of
TPOMW, and second, to characterize the indigenous
microbial communities present in TPOMW (i.e., bacteria
and fungi) and to elucidate how they change in response to
nutrient amendment and aeration. The results of this study
establish the foundations of a systematic study to identify
optimal bioremediation conditions.
Materials and methods
Two-phase olive mill waste The TPOMW was obtained
from the factory “Aceites Jimena S.A.” (Granada, Spain),
collected in sterile plastic containers and stored at −20°C.
The sample was dark brown in color and had a smooth
dough-like consistency, with a high content of water (56%)
and organic matter (41.14%), slightly acidic pH (5.53) and
a high C/N ratio (40.77 mol/mol).
Experimental design and sampling Four experimental con-
ditions were tested in triplicate using 63 in-vessel glass
bioreactors (each 200 ml) at 25°C. For each bioreactor,
TPOMW, water, and wheat straw at a ratio of 15:18:2.4, by
weight basis was thoroughly mixed in a glass beaker
(500 ml), and then the mixture introduced into the reactor.
Initial moisture content (MC) of the mixture was measured,
and double distilled water (DDW) was added to reach 65%
MC (Antizar-Ladislao et al. 2006). Compost moisture
content was measured at intervals to ensure that it was
maintained at the required level and amended with DDW
when needed. The logistic approach to pinpoint the effect
of nutrient amendment and aeration on the bioremediation
of TPOMW and microbial community changes was to
investigate four different scenarios: non-aerated (OA), non-
aerated + nutrients (OAN), aerated (AA), and aerated +
nutrients (AAN). For the aerated treatments (AA and
AAN), the reactor units stood vertically with air flowing
continuously up through the mixture to avoid oxygen
limitation. The air was vented outdoors to avoid accumu-
lation of volatile compounds in the bioreactors. Airflow
through the mixture was achieved by means of a stainless
steel air-delivery tube inserted into the bottom of the
bioreactors attached by silicone tubing to a 100% oil-free
diaphragm pump (Model PXW-600-DIOV, VP1, 5 l min-1
,
Fisher Scientific). The air inlet was bubbled through a
DDW reservoir to avoid excessive water evaporation during
aeration at 12 h intervals on and off. For the non-aerated
bioreactors (OA and OAN), the set-up was exactly the
same, except that the bioreactors were not aerated. For the
nutrient-amended bioreactors (AAN and OAN), the mixture
in each bioreactor was amended at the beginning of the
experiment with 1 g of nitrogen and 50 mg of phosphorus,
using NH4NO3 and KH2PO4, respectively. Destructive
sampling, in triplicate, for each bioreactor mixture was
done after 0, 7, 14, 21, 35, 55, and 77 days. The contents of
each bioreactor were thoroughly mixed in a 500-ml beaker,
310 Appl Microbiol Biotechnol (2008) 79:309–317
3. and different subsamples were collected. Moisture content,
ash content, and pH were analyzed on the same day of the
sampling. Subsamples for polyphenol content determina-
tions and microbial community analysis were kept frozen
(−20°C) in sterile containers.
Determination of moisture content and total organic
matter Total organic matter (TOM) was determined by
ashing using a loss-on-ignition procedure. Triplicate 2 g
samples were dried for 24 h at 110°C and reweighed to give
the moisture content. The dried samples were transferred to
a muffle furnace held at 550°C for 12 h. Ash content was
calculated from the ratio of pre-ignition and post-ignition
sample weights. The TOM losses were calculated as
described previously (Alburquerque et al. 2006).
Polyphenol analysis Extraction of polyphenolic substances
from the mixture was performed as follows. Approximately
1 g of sample was weighed and extracted with 20 ml of
methanol/water (20:80, v/v) using an orbital shaker
(150 rpm) for 2 h at 25°C. A modification of the Folin–
Ciocalteu reagent assay was used to determine the total
phenolic content (Maestro-Durán et al. 1991). All the
assays were performed in triplicate. Total amount of
phenolic compounds was calculated and expressed as
caffeic acid-equivalent, CAE (mg g−1
).
Polar lipid fatty acid analysis of the microbial community Po-
lar lipids were extracted from mixture samples (1 g) using a
modified Bligh and Dyer method and their fatty acyl chains
converted to fatty acid methyl esters (FAMEs) by trans-
methylation using 2.5% (v/v) sulfuric acid in dry methanol
(Kates 1985). The FAMEs were analyzed by GC-MS using
a Hewlett Packard 6890 series gas chromatograph and a
7673 series auto-sampler and a 5973 series mass selective
detector, as previously described (Antizar-Ladislao et al.
2007). The sum of the following fatty acids was used to
represent total bacteria: i15:0, a15:0, i16:0, i17:0, cy17:0,
18:157c, and cy19:0 (Frostegård and Bååth 1996). Gram-
positive bacteria were represented by i15:0, a15:0, and
i17:0 (Buyer et al. 1999) and Gram-negative bacteria by
cy17:0, 18:157c, and cy19:0 (Klamer and Bååth 1998).
Thermophilic bacteria (largely thermotolerant bacilli) were
represented by i15:0 and i17:0 (Carpenter-Boggs et al.
1998). Fungi were represented by 18:256,9 (Frostegård and
Bååth 1996).
Molecular characterization of the microbial community
1. DNA isolation and 16S rRNA gene (bacterial) and ITS2
(fungal) PCR amplification. Total DNA was isolated from
the samples employing the PowerSoilTM DNA isolation kit
(MoBio Laboratories) following the manufacturer’s instruc-
tions. Following electrophoresis on a 1% agarose gel
containing ethidium bromide, the integrity of bands was
assessed visually and the concentration of nucleic acids
measured spectrophotometrically. Primers U968-GC (5′-
CGC CCG GGG CGC GGC CCG GGC GGG GCG GGG
GCA CGG GGG GAA CGC GAA GAA CCT TAC) and
L1401 (5′-GCG TGT GTA CAA GAC CC) were used to
amplify the V6–V8 region of the bacterial 16S rRNA gene,
giving a product of 450 bp. The ITS2 region of fungal
genomic DNA was amplified with the primer pair ITS3-GC
(5′-CGC CCG GGG CGC GGC CCG GGC GGG GCG
GGG GCA CGG GGC ATC GAT GAA GAA CGC AGC)
and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC), resulting
in 370 bp product (Nikolcheva et al. 2005; White et al.
1990). PCR was performed using Hot Star Taq DNA
polymerase (QIAGEN, Courtaboeuf, France). PCR mix-
tures (25 μl) contained the following: 1×PCR buffer,
1.5 mM MgCl2, 0.1 mM of each dNTP, 0.5 μM of primers,
2.5 U of Hot Star Taq polymerase, and approximately 1 ng
of DNA. The DNA fragments were amplified using a
GeneAmp PCR System 2700 (Applied Biosystem, Singa-
pore). The following program was used with the primers
U968-GC and L-1401: 95°C for 15 min; 30 cycles of 94°C
for 1 min, 56°C for 1 min, and 72°C for 1 min 30 s, and
finally 72°C for 15 min. Amplification of genomic DNA
with the primers ITS3-GC and ITS4 was performed with
the following program: 94°C for 15 min; 35 cycles of 94°C for
30 s, 47°C for 1 min, and 72°C for 1 min, and finally 72°C for
10 min. Negative (without DNA) controls were used in every
series of reaction. Amplicons were analyzed by electrophore-
sis on a 1.5% agarose gel containing ethidium bromide to
check the correct size and concentration.
2. TTGE analysis of PCR amplicons. The Dcode Universal
Mutation Detection System (Bio-Rad, Paris, France) was used
for sequence-specific separation of PCR products. Electro-
phoresis was performed through a 1-mm-thick, 16×16 cm
polyacrylamide gel [8% (w/v) acrylamide–bisacrylamide,
7 M urea, 1.25×Tris–acetate–EDTA (TAE), 55 and 550 μl
of Temed, and 10% ammonium persulfate] using 7 l of
1.25×TAE as electrophoresis buffer. Electrophoresis was
performed at a fixed voltage of 65 V for 969 min with
an initial temperature of 66°C and a ramp rate of 0.2°C
per hour. For better resolution, the voltage was fixed at
20 V for 5 min at the beginning of electrophoresis.
Each well was loaded with 100 to 200 ng of amplified
DNA plus an equal volume of 2×gel loading dye
(0.05% bromophenol blue, 0.05% xylene cyanol, and
70% glycerol). After completion of electrophoresis, the
gel was stained in a SYBR Green I solution (Sigma-
Aldrich, St. Quentin Fallavier, France), and destained in
1.25×TAE. Gels were visualized under UV illumination
using a gel image system (Kodak Gel Logic 100).
Predominant bands were excised and treated for further
analysis.
Appl Microbiol Biotechnol (2008) 79:309–317 311
4. 3. Comparison of TTGE patterns. The digital images of the
acrylamide gels were analyzed as previously described (Marie
et al. 2006) using the Cross-Checker software (available from
http://www.dpw.wau.nl/pv/pub/CrossCheck/).
4. Sequence analysis. To perform sequence-based phyloge-
netic identification, specific bands were cut from the
polyacrylamide gel. Gel fragments were washed once in
200 μl of PCR water and kept in 100 μl of PCR water
overnight at 4°C for diffusion. Both bacterial 16S rRNA
and fungal ITS2 gene fragments were then amplified from
the dialysate. The PCRs were performed in the same
conditions as described above. The amplicons were purified
using the GFX PCR DNA Purification Kit (Amershan
Biosciences), and the size and concentration of the ampli-
cons were evaluated on 1.5% agarose gels containing
ethidium bromide. PCR products were sequenced with an
Applied Biosystems 373A DNA sequencer by automated
Sanger method. Similarity searches with sequences in the
GenBank database were performed by using Blast algorithm.
5. Nucleotide sequence accession numbers. The ITS-2
region gene (fungal) and 16S region rRNA gene (bacterial)
amplicon sequences identified in each treatment have been
submitted to GenBank and the accession numbers are
shown in Tables 1 and 2.
Statistical analysis The effect of different treatments on
polyphenols biodegradation was investigated using a post
hoc Tukey test. All the statistical tests were performed with
StatistiXL Version 1.8.
Results
Chemical changes The changes in concentration of poly-
phenols are shown in Fig. 1a. Generally, there was greater
biodegradation of polyphenols during the first few days of
the experiment (depending on the treatment), and thereafter,
their loss was slow. The greatest amount of biodegradation
was in aerated bioreactors nutrient amended, in which there
was 36% and 54% losses after 7 and 55 days, respectively.
The biodegradation in aerated and not aerated bioreactors that
were not amended with nutrients was not significant (p>0.1).
The addition of nutrients stimulated the biodegradation of
polyphenols during the first 7 days of treatment under both
aerated and non-aerated conditions (p<0.005; Fig. 1a);
however, after 55 days, this stimulation was only apparent
in the aerated bioreactors, and after 77 days, the biodegra-
dation curves reached a plateau (data not shown). Following
55 days of continuous treatment in non-aerated bioreactors,
approximately 22% of polyphenols were lost, irrespective of
whether they were amended with nutrients.
The bulk (about 95%) of the bioreactor material was
organic matter, measured as total organic matter (TOM),
and the losses over a 55-day incubation period are shown in
Fig. 1b. Both aeration and nutrient addition influenced the
rate of TOM losses. The addition of nutrient stimulated
TOM loss in aerated bioreactors but decreased TOM loss in
non-aerated bioreactors. In all bioreactors, the rate of loss of
TOM was faster during the initial 14 days of incubation.
Thereafter, the rate of loss was slow. Following 55 days of
Table 1 Bacteria identified by PCR-TTGE analysis in TPOMW bioreactors according to their similarity to 16S rRNA gene sequences in the
GenBank nucleotide sequence database
Band no. Treatment Closest match (accession no.) Percent similarity Accession no.
Gamma proteobacteria
OAN15 Non-aerated + nutrients Stenotrophomonas maltophilia (AM261992) 94 EU450807
OAN18 Non-aerated + nutrients Erwinia persicina (AJ937837) 97 EU540808
AA1 Aerated Luteibacter sp. (DQ986460) 99 EU450809
AA3 Aerated Stenotrophomonas maltophilia (AM262002) 99 EU4508010
Actinobacteria
OA7 Non-aerated Rhodococcus fascians (AY730713) 98 EU4508011
OAN8 Non-aerated + nutrients Clavibacter michiganensis (L43096) 97 EU4508012
OAN10 Non-aerated + nutrients Frigoribacteirum sp.(AY439250) 97 EU4508013
OAN9 Non-aerated + nutrients Curtobacterium albidum (AM042692) 95 EU4508014
Firmicutes
AA6 Aerated Clostridium saccharolyticum (AY604565) 98 EU4508015
AA7 Aerated Clostridium xylanolyticum (X71855) 98 EU4508016
AA8 Aerated Clostridium sp.(AB114227) 97 EU4508017
OA3 Non-aerated Clostridium tyrobutyricum (L08062) 93 EU4508018
OA6 Non-aerated Uncultured Clostridium sp. (DQ168846) 98 EU4508019
OA2 Non-aerated Lactobacillus vaccinostercus (AB218801) 99 EU4508020
OA4 Non-aerated Leuconostoc mesenteroides (EF068254) 98 EU4508021
OA5 Non-aerated Leuconostoc mesenteroides (EF068254) 99 EU4508022
D10 Initial mixture Bacillus aquimaris (DQ923229) 97 EU4508023
OA1 Non-aerated Sporolactobacillus inulinus (D16283) 98 EU4508024
312 Appl Microbiol Biotechnol (2008) 79:309–317
5. incubation, the loss of TOM was in the range of 10–25% in
all bioreactors, and greatest losses were observed in nutrient
amended aerated bioreactors (Fig. 1b).
Microbial community structure
1. Phospholipid fatty acid (PLFA) analysis. The changes
in relative amounts of fungal and bacterial biomass in
the bioreactors during 55 days incubation are shown in
Fig. 2. The addition of nutrients increased the fungal/
bacterial PLFA index (F/B index) in both aerated and
non-aerated bioreactors, the greatest changes occurring
during the initial 14 days of incubation, and generally
being slow or static thereafter. The increase in F/B
index was greater in the nutrient amended aerated
bioreactors, in which the presence of fungal biomass
was sufficient to be seen as white mycelium by simple
visual observation: this was not seen in any other
bioreactor conditions, even in the nutrient amended
non-aerated bioreactors, which also displayed a steady
rise in the F/B index throughout the 55 days incubation
period (Fig. 2). In contrast, in both aerated and non-
aerated bioreactors without nutrient amendment, there
was a decrease in the F/B index. However, the kinetics
of the decrease differed: in the aerated bioreactors, the
decrease occurred during the initial 7 days of incuba-
tion, in comparison with the non-aerated bioreactors in
which it occurred during the latter half of the
incubation period (Fig. 2).
0
1
2
3
4
5
6
7
0
5
10
15
20
25
30
0 10 20 30 40 50 60
Time (days)
Polyphenols
(mg
g
dry
matter
-1
)
TOM-losses
(%
of
initial
TOM
content)
AA
AAN
OA
OAN
0
1
2
3
4
5
6
7
0
1
2
3
4
5
6
7
0
5
10
15
20
25
30
0
5
10
15
20
25
30
0 10 20 30 40 50 60
0 10 20 30 40 50 60
Time (days)
Polyphenols
(mg
dry
matter
-1
)
TOM-losses
(%
of
initial
TOM
con
ent)
AA
AAN
OA
OAN
a
b
Fig. 1 Polyphenol biodegradation (a) and organic matter losses (b).
The data points are mean values of samplings from triplicate reactors
(bars correspond to ± standard deviation) on the days shown. Symbols
refer to the different treatments: aerated (AA), aerated + nutrients
(AAN), non-aerated (OA) and non-aerated + nutrients (OAN)
Table 2 Fungi identified by PCR-TTGE analysis in TPOMW bioreactors according to their similarity to ITS2 region sequences in the GenBank
nucleotide sequence database
Band no. Treatment Closest match (accession no.) Percent similarity Accession no.
AAN1 Aerated + nutrients Penicillium roquefortii (DQ148947) 99 EU450825
AAN4 Aerated + nutrients Penicillium roquefortii (AY373929) 99 EU450826
AAN6 Aerated + nutrients Penicillium roquefortii (AY373929) 97 EU450827
AAN7 Aerated + nutrients Candida norvegica (DQ249194) 92 EU450828
AAN10 Aerated + nutrients Geotrichum sp. (AY787702) 100 EU450829
OAN12 Non-aerated + nutrients Pichia membranifaciens (DQ104713) 99 EU450830
OAN15 Non-aerated + nutrients Cladosporium herbarum (AY251078) 100 EU450831
OAN17 Non-aerated + nutrients Ascochyta sp. (AY305377) 100 EU450832
OAN19 Non-aerated + nutrients Geotrichum sp. (AY787702) 99 EU450833
OAN20 Non-aerated + nutrients Geotrichum sp. (AY787702) 99 EU450834
0
5
10
15
20
0 10 20 30 40 50 60
Time (days)
AA
AAN
OA
OAN
Fungal/Bacterial
PLFA
index
0
5
10
15
20
0 10 20 30 40 50 60
Time (days)
AA
AAN
OA
OAN
AA
AAN
OA
OAN
Fungal/Bacterial
PLFA
index
Fig. 2 Time course of the change in fungal/bacterial PLFA index. The
index was calculated as the ratio between fungal (18:2ω6.9) and
bacterial (i15:0, a15:0, i16:0, i17:0, cy17:0, 18:1ω7c and cy19:0)
fingerprints abundances. Each data point corresponds to the mean
value for triplicate reactors (bars correspond to ± standard deviation).
Symbols refer to the different treatments: aerated (AA), aerated +
nutrients (AAN), non-aerated (OA) and non-aerated + nutrients (OAN)
Appl Microbiol Biotechnol (2008) 79:309–317 313
6. 2. TTGE analysis. Overall, the results of the comparative
TTGE analyses revealed significant differences in
bacterial and fungal communities when varying nutrient
addition and aeration to the bioreactors (Figs. 3 and 4).
These differences between the treatments were clear
from the first week of the experiment (day 7), i.e., there
was rapid establishment of the different microbial
communities under all the conditions of nutrient status
and aeration. Thereafter, the communities were stable,
since their individual TTGE profiles did not change
during the remaining 48 days of the experiment.
The greatest bacterial diversity, in terms of number of
discrete bands, was detectable in OAN bioreactors (14–17
bands per sample), followed by AA (6–15 bands), AAN
(6–10 bands) and finally by OAN (4–5 bands). The TTGE
profiles showed that the greatest fungal diversity was
detected in OAN bioreactors (14–17 bands), followed by
AAN (10–13 bands) and AA (7–9 bands). Thus, the
complexity of the microbial communities did not seem to
be linked with the degradation of polyphenols. For
instance, the maximal polyphenol degradation was detected
in AAN bioreactors, but the most complex communities, in
terms of number of TTGE bands, was found in OAN
bioreactors. Therefore, these results show that only a few
specific microorganisms, which were stimulated with
nutrients and oxygen supply, were responsible for the
greatest degradation of polyphenols.
As expected, in both bacterial and fungal TTGE
analyses, samples from the same treatment clustered
together (Fig. 5). No PCR product was obtained from any
of the OA samples using fungi primers. In the cluster
analysis of bacterial TTGE, two major groups could be
observed, one corresponding to the AAN bioreactors and
the other to the rest of the samples. In contrast, in the fungal
TTGE analyses, profiles from the OAN treatment clustered
apart from profiles of aerated bioreactors. Moreover, TTGE
profiles seemed to be independent of the incubation time. It
is important to note that, despite the high similarity of
profiles that belonged to the same treatment (for bacteria
and fungi analysis), each lane of the TTGE gels was
obtained from DNA isolated from a different (independent)
bioreactor.
To perform sequence-based phylogenetic identification,
a sequence analysis of specific bands was performed.
Twenty newly determined sequences were compared
directly with those in GenBank using BlastN. Database
similarity is shown in Table 1. A total of 14 different genera
of bacteria were detected, belonging to Gammaproteobac-
teria (5), Actinobacteria (4), and Firmicultes (5). Only two
sequences were obtained from the samples of the initial
mixture (day 0), both corresponding to the genus Bacillus.
In fungal TTGE gels, a total of 14 sequences were
successfully obtained (Table 2). BLAST analysis confirmed
the specificity of the primers used, as all sequences could
be affiliated to fungal genera in more than 97% similarity.
In total, 6 genera were identified, all of then belonging to
Ascomycota, namely Penicillium, Candida, Geotrichum,
Pichia, Cladosporium, and Aschochyta.
Discussion
The majority of previous studies dealing with the biodeg-
radation of olive wastes have involved sterilization of the
residues before inoculation of a specific microorganism or
consortium. However, not only does autoclaving kill the
indigenous microbiota, but also the high temperature causes
significant physico-chemical modifications such as the
oxidation of aromatic compounds present in the olive
wastes (Aggelis et al. 2003). A simple, unmodified system
was selected in this study since a major aim was to provide
7 14 21 35 55 7 14 21 35 55
AA
7 14 21 35 55 7 14 21 35 55
0
AAN OA OAN
AA1
AA2
AA3
AA6
AA7
AA8
OA2
OA3
OA4
OA5
OA6
OA1
OAN8
OAN9
OAN10
OAN15
OAN18
7 14 21 35 55 7 14 21 35 55
AA
7 14 21 35 55 7 14 21 35 55
0
AAN OA OAN
7 14 21 35 55 7 14 21 35 55
AA
7 14 21 35 55 7 14 21 35 55
0
AAN OA OAN
AA1
AA2
AA3
AA6
AA7
AA8
OA2
OA3
OA4
OA5
OA6
OA1
OAN8
OAN9
OAN10
OAN15
OAN18
Fig. 3 PCR-TTGE profiles of
16S rRNA fragments represent-
ing the bacterial diversity of
species generated from the sam-
ples taken at days 0, 7, 14, 21,
35, and 55. The bands identified
by sequencing are indicated by
the arrowheads. Bioreactor
treatments: aerated (AA),
aerated + nutrients (AAN),
non-aerated (OA) and
non-aerated + nutrients (OAN)
314 Appl Microbiol Biotechnol (2008) 79:309–317
7. fundamental information that is relevant to the development
of a robust biotechnological application for eventual
optimized scale-up to industrial scale. Therefore, in the
work presented here, the TPOMW was not sterilized or
manipulated in any other way before preparing the (bio)
degradation mixture, adding only double distilled water and
wheat straw to improve the porosity of the material and
thus enabling bioremediation to occur. Wheat straw may
have contributed to the mixture with some microbial
activity, but given the organic richness of the TPOMW, it
is most likely that the bulk of the microbiota were derived
from the original olive waste. Therefore, it was assumed
that the genetic potential of the indigenous microbiota of
the waste (olive waste plus wheat straw) is able under
favourable conditions to metabolize polyphenolic com-
pounds present in the mixture. It is here suggested that
the stimulation of this indigenous microbiota is a promising
alternative bioremediation approach. Compared with the
inoculation of a single strain (or consortium) approach,
indigenous microorganisms have a range of different
biodegrading activities that could act simultaneously or
sequentially.
For the purpose of this study, a temperature of 25°C was
selected, rather than a higher value, as being one that could
be readily achieved in an industrial scale development
without the necessity for expensive energy input. Break-
down of polyphenols occurred mainly during the first
7 days of incubation, and organic matter losses occurred
mainly during the first 14 days (Fig. 1). Thus, during the
initial 1–2 weeks incubation period, a microbial community
with the capacity to metabolize polyphenolic compounds
present in TPOMW was established in all bioreactors,
except the aerated non-amended ones. Increases in poly-
phenol concentration (mg g−1
dry matter) during treatment
were occasionally observed in the reactors (Fig. 1a). The
experimental variation of moisture content or flow rate
during the treatment would potentially affect the polyphe-
nol biodegradation extent and rate in the mixtures, although
they would not explain an increase in polyphenol con-
centration. Thus, an occasional increase in polyphenol
concentration might be the consequence of a selective
biodegradation of organic matter within the TPOMW and
straw mixture, changing the ratio of TPOMW to wheat
straw in the mixture and therefore in the calculation of the
concentration of polyphenols in the mixture as previously
observed (Antizar-Ladislao et al. 2005).
Although PLFA analysis detected changes in the relative
proportions of bacteria and fungi, and of Gram-positive and
AAN
OAN
OA
AA
14
55
7
35
21
14
55
21
35
7
7
7
14
14
35
35
55
55
21
21
AAN
OAN
AA
14
55
7
35
21
14
55
21
35
7
7
14
35
55
21
AAN
OAN
OA
AA
14
55
7
35
21
14
55
21
35
7
7
7
14
14
35
35
55
55
21
21
AAN
OAN
OA
AA
14
55
7
35
21
14
55
21
35
7
7
7
14
14
35
35
55
55
21
21
AAN
OAN
AA
14
55
7
35
21
14
55
21
35
7
7
14
35
55
21
AAN
OAN
AA
14
55
7
35
21
14
55
21
35
7
7
14
35
55
21
AAN
OAN
AA
14
55
7
35
21
14
55
21
35
7
7
14
35
55
21
a b
Fig. 5 Cluster analysis of
TTGE band patterns for bacteria
(a), and fungi (b). The numbers
indicate the day of sampling.
Bioreactor treatments: aerated
(AA), aerated + nutrients (AAN),
non-aerated (OA) and non-aer-
ated + nutrients (OAN)
7 14 21 35 55
7 14 21 35 55
0
AA
7 14 21 35 55
AAN OAN
AAN1
AAN5
AAN4
AAN6
AAN7
AAN8
AAN10
AA11
OAN12
OAN15
OAN16
OAN19
OAN17
OAN20
7 14 21 35 55
7 14 21 35 55
0
AA
7 14 21 35 55
AAN OAN
AAN1
AAN5
AAN4
AAN6
AAN7
AAN8
AAN10
AA11
OAN12
OAN15
OAN16
OAN19
OAN17
OAN20
Fig. 4 PCR-TTGE profiles of
ITS region fragments represent-
ing the fungal diversity of spe-
cies generated from samples
taken at days 0, 7, 14, 21, 35,
55. The bands identified by
sequencing are indicated by the
arrowheads. Bioreactor treat-
ments: aerated (AA), aerated +
nutrients (AAN), non-aerated
(OA) and non-aerated +
nutrients (OAN)
Appl Microbiol Biotechnol (2008) 79:309–317 315
8. Gram-negative bacteria, TTGE profiles showed that micro-
bial community structures were established rapidly, within
7 days, and that thereafter, the predominant microbial
species in the bioreactors were stable throughout the
incubation period, i.e., not only during the initial phases
of maximum polyphenol biodegradation and TOM de-
crease, but also during the later stages when less biodeg-
radation occurred. Partial rDNA bacterial and fungal
sequence comparisons with sequences in the database gave
values that ranged from 84 to 99% and from 92 to 100%,
respectively, although most values were higher than 97%.
One should notice that these values could differ if complete
sequences were compared. Most of the bacteria, as
identified by TTGE, which are most likely to comprise
the predominant members of the microbial community in
TPOMW-wheat straw mixtures, are phylogenetically asso-
ciated with bacteria, such as Luteibacter sp., that have
previously been isolated from plant material (Johansen et al.
2005). Additionally, some have been described as plant
pathogens or at least associated with plant diseases, such as
Rhodococcus fascians and Clavibacter michiganensis,
present in the bioreactors and known to be plant pathogens
(Young et al. 1996). Furthermore, Stenotrophomonas
maltophila is another plant pathogen that is able to degrade
polyphenolic compounds (Franco et al. 2005). Actino-
mycetes belonging to the genus Curtobacterium have also
been isolated from plant material (Postma et al. 2005).
Clostridium saccharolyticum, which was isolated from
sewage sludge (Murray et al. 1982), and C. xylanolyticum
(Rogers and Baecker 1991) are both able to biodegrade
polymers that could have a role in the bioremediation of
TPOMW. The finding of anaerobes such as clostridia in
aerated bioreactors could be regarded as an anomaly, but as
the material in the bioreactors is very heterogeneous, it is
possible that there were local anaerobic “pockets” within
the organic material that could harbor clostridia which are
able to tolerate the intermittent presence of oxygen.
The greatest biodegradation of polyphenols occurred in
those bioreactors that were both nutrient amended and
aerated. These reactors presented higher fungal to bacterial
biomass ratios, as indicated by PLFA analyses. Fungi have
previously been studied for their capacity to remove
polyphenols from olive residues by the release of extracel-
lular enzymes, particularly lignin peroxidases, Mn-depen-
dent peroxidases and laccases, which not only biodegrade
polyphenols but also decolorize olive-waste residues (Assas
et al. 2000; Linares et al. 2003). Yet, the rate and extent of
the removal of polyphenols depends on the particular fungi
present and also on the nature of the polyphenols
(Sampedro et al. 2004). This study reports for the first time
a molecular biological investigation of fungal communities
involved in the process of olive mill waste bioremediation.
Strains belonging to Penicillium, Geotrichum, Pichia, and
Candida were the major fungi detected by TTGE. These
genera have previously been isolated from different OMWs
including TPOMW (Assas et al. 2000; Ettayebi et al. 2003;
Millan et al. 2000), demonstrating that they are likely to be
part of the natural microbiota of these residues. Some of
these fungi show a promising range of biotechnological
applications in connection with the treatment of OMWs
(D’Annibale et al. 2005; Ayed et al. 2005).
In conclusion, this study highlights that probably a
relatively small number of strains within the indigenous
microbiota of the TPOMW is able to biodegrade the
polyphenolic content of the waste without the inoculation
of specific microorganisms. The results presented here
indicate that the fungal community should be stimulated by
the addition of nutrients and air for improved polyphenol
biodegradation. However, the contribution of the bacteria
able to grow in this waste should be considered. Further
research is necessary to optimize the conditions for
improved bioremediation of olive waste, and different
biotechnological applications could be coupled to the
process.
Acknowledgement This study was possible thanks to the project
REN 2000-1502 and the FPI fellowship program financed by the
Ministerio de Educación y Ciencia (Spain). We also wish to thank
Katerina Spanova (Imperial College London) for her help with the
PLFA analysis, and Dr Belén Rodelas and Raquel Ferrer (University
of Granada) for their assistance in the PCR-TTGE analysis. We should
also like to mention the contribution of Dr Angus Beck (Imperial
College London), without whom this study could not have been
possible.
References
Aggelis G, Iconomou D, Christou M, Bokas D, Kotzailias S, Christou
G, Tsagou V, Papanikolaou S (2003) Phenolic removal in a
model olive mill wastewater using Pleurotus ostreatus in
biobioreactor cultures and biological evaluation of the process.
Water Res 37:3897–3904
Alburquerque JA, Gonzálvez J, García D, Cegarra J (2004) Agro-
chemical characterisation of “alpeorujo”, a solid by-product of
the two-phase centrifugation method for olive extraction. Biores
Technol 91:195–200
Alburquerque JA, Gonzálvez J, García D, Cegarra J (2006) Compost-
ing of a solid olive-mill by-product (“alperujo”) and the potential
of the resulting compost for cultivating pepper under commercial
conditions. Waste Manag 26:620–626
Anonymous. International Olive Oil Council, http://www.internatio
naloliveoil.org. [last access on 22.11.2004]
Antizar-Ladislao B, Lopez-Real J, Beck AJ (2005) Laboratory studies
of the remediation of polycyclic aromatic hydrocarbon contam-
inated soil by in-vessel composting. Waste Manag 25:281–289
Antizar-Ladislao B, Lopez-Real J, Beck AJ (2006) Degradation of
polycyclic aromatic hydrocarbons (PAHs) in an aged coal-tar
contaminated soil under in-vessel composting conditions. Envi-
ron Pollut 141:459–468
316 Appl Microbiol Biotechnol (2008) 79:309–317
9. Antizar-Ladislao B, Spanova K, Beck AJ, Russell NJ (2007)
Microbial community structure changes during bioremediation
of PAHs in an aged coal-tar contaminated soil by in-vessel
composting. Int Biodet Biodeg (in press) DOI 10.1016/j.
ibiod.2007.10.002
Aragón JM, Palancar MC (2001) Improlive 2000. Present and future
of alperujo. Editorial Complutense Press Madrid, Spain
Arjona R, García A, Ollero P (1999) The drying of alpeorujo, a
waste product of the olive oil mill industry. J Food Eng 41:
229–234
Assas N, Marouani L, Hamdi M (2000) Scale down and optimization
of olive mill wastewaters decolourisation by Geotrichum candi-
dum. Biochem Eng 22:503–507
Ayed L, Assas N, Sayadi S, Hamdi M (2005) Involvement of lignin
peroxidase in the decolourization of black olive mill wastewaters
by Geotrichum candidum. Lett Appl Microbiol 40:7–11
Benitez E, Sainz H, Nogales R (2005) Hydrolytic enzyme activities of
extracted humic substances during the vermicomposting of a
lignocellulosic olive waste. Biores Technol 96:785–790
Borja R, Sánchez E, Rincón B, Raposo F, Martín MA, Martín A
(2005) Study and optimisation of the anaerobic acidogenic
fermentation of two-phase olive pomace. Process Biochem
40:281–291
Buyer JS, Roberts DP, Russek-Cohen E (1999) Microbial community
structure and function in the spermosphere as affected by soil and
seed type. Can J Microbiol 45:138–144
Carpenter-Boggs L, Kennedy AC, Reganold JP (1998) Use of
phospholipid fatty acids and carbon source utilization patterns
to track microbial community succession in developing compost.
Appl Environ Microbiol 64:4062–4064
Capasso R, Evidente A, Schivo L, Orru G, Marcialis MA, Cristinzio G
(1995) Antibacterial polyphenols from olive oil mill waste
waters. J Appl Bacteriol 79:393–398
D’Annibale A, Ricci M, Leonardi V, Quaratino D, Mincione E,
Petruccioli M (2005) Degradation of aromatic hydrocarbons by
white-rot fungi in a historically contaminated soil. Biotechnol
Bioeng 90:723–731
Ettayebi K, Errachidi F, Jamai L, Tahri-Jouti MA, Sendide K, Ettayebi
M (2003) Biodegradation of polyphenols with immobilized
Candida tropicalis under metabolic induction. FEMS Microbiol
Lett 223:215–219
Franco AR, Calheiros CSC, Pacheco CC, De Marco P, Manaia CM,
Castro PML (2005) Isolation and characterization of polymeric
galloyl-ester-degrading bacteria from a tannery discharge place.
Microb Ecol 50:550–556
Frostegård A, Bååth E (1996) The use of phospholipid fatty acid
analysis to estimate bacterial and fungal biomass in soil. Biol
Fertil Soils 22:59–65
Giannoutsou EP, Meintanis C, Karagouni AD (2004) Identification of
yeast strains isolated from a two-phase decanter system olive oil
waste and investigation of their ability for its fermentation.
Biores Technol 93:301–306
Johansen JE, Binnerup SJ, Kroer N, Molbak L (2005) Luteibacter
rhizovicinus gen. nov., sp. nov., a yellow-pigmented gammapro-
teobacterium isolated from the rhizosphere of barley (Hordeum
vulgare L.). Int J Syst Evol Microbiol 55:2285–2291
Jones CE, Murphy PJ, Russell NJ (2000) Diversity and osmoregulatory
responses of bacteria isolated from two-phase olive oil extraction
waste products. World J Microbiol Biotechnol 16:555–561
Kates M (1985) Techniques of lipidology. Amsterdam, Elsevier
Klamer M, Bååth E (1998) Microbial community dynamics during
composting of straw material studied using phospholipid fatty
acid analysis. FEMS Microbiol Lett 27:9–20
Linares A, Caba JM, Ligero F, de la Rubia T, Martínez J (2003)
Detoxification of semisolid olive-mill wastes and pine-chip
mixtures using Phanerochaete flavido-alba. Chemosphere
51:887–891
Maestro-Durán R, Borja R, Martín A, Fiestas JA, Alba J (1991)
Biodegradación de los compuestos fenólicos presentes en el
alpechín. Grasas y Aceites 42:271–276
Marie D, Fei Z, Balagué V, Ras J, Vaulot D (2006) Eukaryotic
picoplankton communities of the Mediterranean Sea in summer
assessed by molecular approaches (DGGE, TTGE, QPCR).
FEMS Microbiol Lett 55:403–415
Millan B, Lucas R, Robles A, Garcia T, De Cienfuegos GA, Galvez A
(2000) A study on the microbiota from olive-mill wastewater
(OMW) disposal lagoons, with emphasis on filamentous fungi
and their biodegradative potential. Microbiol Res 155:143–147
Morillo JA, Aguilera M, Ramos-Cormenzana A, Monteoliva-Sánchez
M (2006) Production of a metal binding exopolysaccharide by
Paenibacillus jamilae using two-phase olive-mill waste as
fermentation substrate. Curr Microbiol 53:189–193
Murray WD, Khan AW, Van den Berg L (1982) Clostridium
saccharolyticum sp. nov., a saccharolytic species from sewage
sludge. Int J Syst Bacteriol 32:132–135
Nikolcheva LG, Bärlocher F (2005) Seasonal and substrate prefer-
ences of fungi colonizing leaves in streams: traditional versus
molecular evidence. Environ Microbiol 7:270–280
Ntougias S, Zervakis GI, Ehaliotis C, Kavroulakis N, Papadopoulou
KK (2006) Ecophysiology and molecular phylogeny of bacteria
isolated from alkaline two-phase olive mill wastes. Res Microbiol
157:376–385
Paredes C, Bernal MP, Cegarra J, Roig A (2002) Bio-degradation of
olive mill wastewater sludge by its co-composting with agricul-
tural wastes. Biores Technol 85:1–8
Piotrowska A, Iamarino G, Rao MA, Gianfreda L (2006) Short-term
effects of olive mill waste water (OMW) on chemical and
biochemical properties of a semiarid Mediterranean soil. Soil
Biol Biochem 38:600–610
Postma J, Geraats BPJ, Pastoor R, Van Elsas JD (2005) Characteriza-
tion of the microbial community involved in the suppression of
Pythium aphanidermatum in cucumber grown on rockwool.
Phytopathology 95:808–818
Ramos-Cormenzana A, Juárez-Jiménez B, García-Pareja MP (1996)
Antimicrobial activity of olive mill waste-waters (Alpechin) and
biotransformed olive oil mill wastewater. Int Biodet Biodeg
38:283–290
Rincón B, Raposo F, Borja R, Gonzalez JM, Portillo MC, Saiz-Jimenez C
(2006) Performance and microbial communities of a continuous
stirred tank anaerobic bioreactor treating two-phases olive mill solid
wastes at low organic loading rates. J Biotechnol 121:534–543
Rogers GM, Baecker AAW (1991) Clostridium xylanolyticum sp.
nov., an anaerobic xylanolytic bacterium from decayed Pinus
patula wood chips. Int J Syst Bacteriol 41:140–143
Sampedro I, Romero C, Ocampo JA, Brenes M, García I (2004)
Removal of monomeric phenols in dry mill olive residue by
saprobic fungi. J Agr Food Chem 52:4487–4492
Sierra J, Marti E, Montserrat G, Cruanas R, Garau MA (2001)
Characterisation and evolution of a soil affected by olive oil mill
wastewater disposal. Sci Total Environ 279:207–214
White TJ, Bruns T, Lee S, Taylor JW (1990) Amplification and direct
sequencing of fungal ribosomal RNA genes for phylogenetics.
PCR Protocols: A Guide to Methods and Applications 315–322
Young JM, Saddler GS, Takikawa Y, de Boer SH, Vauterin L, Gardan
L, Gvozdyak RI, Stead DE (1996) Names of plant pathogenic
bacteria 1864–1995. Rev Plant Pathol 75:721–763
Appl Microbiol Biotechnol (2008) 79:309–317 317