J. González-López

Influence of carbon source on nitrate removal of contaminated groundwater in a denitrifying submerged filter.

The effectiveness of three selected carbon sources (sucrose, ethanol and methanol) on submerged filters for the removal of nitrate from contaminated groundwater (100mg NO3(-)/litre), was studied. Process yields, nitrite accumulation, biomass production and growth of denitrifying bacteria were compared. Process yields, represented as ratio C/N were 2.5, 1.08 and 1.1 for sucrose, ethanol and methanol assays, respectively, making sucrose the least efficient carbon source. Nitrite accumulation in treated groundwater was more notable for sucrose assays, reaching values of 5mg NO2(-)/l. However, when ethanol or methanol were used as carbon sources, nitrite accumulation in treated water was practically zero during the experiments. On the other hand, a greater biomass production was observed in these assays with sucrose, causing clogging of the filter. Higher density of denitrifying bacteria in the biofilm, observed when ethanol and methanol were amended to the influent, suggested that these carbon sources increased the denitrification activity compared with the experiments performed with sucrose. Since methanol is toxic, ethanol is considered the most suitable carbon source out of the three tested, under the experimental conditions.

Application of bioemulsifiers in soil oil bioremediation processes. Future prospects

Biodegradation is one of the primary mechanisms for elimination of petroleum and other hydrocarbon pollutants from the environment. It is considered an environmentally acceptable way of eliminating oils and fuel because the majority of hydrocarbons in crude oils and refined products are biodegradable. Petroleum hydrocarbon compounds bind to soil components and are difficult to remove and degrade. Bioemulsifiers can emulsify hydrocarbons enhancing their water solubility and increasing the displacement of oily substances from soil particles. For these reasons, inclusion of bioemulsifiers in a bioremediation treatment of a hydrocarbon polluted environment could be really advantageous. There is a useful diversity of bioemulsifiers due to the wide variety of producer microorganisms. Also their chemical compositions and functional properties can be strongly influenced by environmental conditions. The effectiveness of the bioemulsifiers as biostimulating agent in oil bioremediation processes has been demonstrated by several authors in different experimental assays. For example, they have shown to be really efficient in combination with other products frequently used in oil bioremediation such as they are inorganic fertilizer (NPK) and oleophilic fertilizer (i.e. S200C). On the other hand, the bioemulsifiers have shown to be more efficient in the treatment of soil with high percentage of clay. Finally, it has been proved their efficacy in other biotechnological processes such as in situ treatment and biopiles. This paper reviews literature concerning the application of bioemulsifiers in the bioremediation of soil polluted with hydrocarbons, and summarizes aspects of the current knowledge about their industrial application in bioremediation processes.

Effect of dissolved oxygen concentration on nitrate removal from groundwater using a denitrifying submerged filter.

A unidirectional submerged filter system was employed to purify groundwater contaminated with nitrate by biological denitrification. The influence of the concentration of dissolved oxygen (DO) in the process was tested using ethanol, methanol and sucrose as carbon sources. Inorganic-nitrogen removal, growth of the biofilm, platable denitrifying bacteria and nitrate reducing bacteria in biofilm were studied. With regard to the type of electron donor used, the presence of oxygen decreased the removal efficiency of inorganic nitrogen and caused an increase of nitrite concentration in the treated water. These negative effects depended on utilised carbon source. Biological denitrification with alcohols such as ethanol and methanol was less affected by DO than with sucrose. The development of the biofilm was also influenced by the DO concentration as excess O(2) caused reduced biofilm growth. These biofilms developed in oxygen presence had a smaller bacterial density and a lower denitrifying bacteria versus nitrate reducing bacteria ratio, which led to an unfavorable inorganic nitrogen removal and presence of nitrite in the treated water. All these effects are more pronounced when sucrose is used as carbon source.

Analysis of microbial communities developed on the fouling layers of a membrane-coupled anaerobic bioreactor applied to wastewater treatment.

The structure of the biofouling layers formed on a pilot-scale membrane-coupled upflow anaerobic sludge blanket bioreactor (UASB) used to treat urban wastewater was analyzed by scanning electron microscopy and electron-dispersive X-ray microanalysis. For comparison, control samples of the membranes were fed either UASB effluent or raw wastewater in a laboratory-scale experiment. Microbial diversity in the fouling materials was analyzed by temperature gradient gel electrophoresis (TGGE) combined with sequence analysis of partial 16S rRNA. Significant differences in structure of the Bacteria communities were observed amongst the different fouling layers analyzed in the UASB membranes, particularly following a chemical cleaning step (NaClO), while the Archaea communities retained more similarity in all samples. The main Bacteria populations identified were evolutively close to Firmicutes (42.3%) and Alphaproteobacteria (30.8%), while Archaea were mostly affiliated to the Methanosarcinales and Methanospirillaceae. Sphingomonadaceae-related bacteria and methanogenic Archaea were persistently found as components of biofouling, regardless of chemical cleaning.

Influence of hydraulic loading and air flowrate on urban wastewater nitrogen removal with a submerged fixed-film reactor.

Nutrient disposal to sensitive areas, particularly nitrogen and phosphorus from wastewater treatment plants, provokes eutrophication reducing water quality. Fixed film technology is widely used for the removal of organic matter and nitrogen by the biological process of nitrification-denitrification. This paper studies a nitrification and post-denitrification lab-scale plant with a downflow aerobic submerged filter for removal of organic matter and nitrification, followed by an anoxic upflow biofilter for denitrification. Recycled construction material (clay shists) was employed as support material and methanol was used as carbon source. After 2 weeks of acclimation in which nitrification reached steady-state conditions, different hydraulic loadings (0.35-1.59 m(3)/m(2)h) and air flowrates (7.78-43.5 m(3)/m(2)h) were applied for 1 year. The highest hydraulic loading which complied with the EU regulation on nitrogen disposal was 0.71 m(3)/m(2)h (1.6 h). Hydraulic retention time (HRT), which corresponded to a nitrogen removal of 0.64 kg N/m(3) per day operating at an air flowrate of 25.6 m(3)/m(2)h. Concerning to organic matter removal efficiency, the aerobic reactor accepted a maximum chemical oxygen demand (COD) volumetric loading of 16.0 kg COD/m(3) per day with a 75% COD removal efficiency. For all the tests carried out, suspended solids (SS) concentration in the outlet water was less than 35 mg/l.

Isolation and characterization of Azotobacter and Azospirillum strains from the sugarcane rhizosphere

Bacteria with the ability to grow on nitrogen-free media and with nitrogenase activity under aerobic or microaerobic conditions were isolated from sugarcane roots collected from four different agricultural locations in Granada (Spain). Isolates were Gram negative rods and were identified as Azotobacter chroococcum and Azospirillum brasilense. Our results suggest that Azotobacter isolates do not have a particular affinity for sugarcane rhizospheres and that, on the contrary, Azospirillum isolates show specific association and perhaps endophytic colonization of sugarcane. However, obligate endophytes (Gluconacetobacter diazotrophicus) were not found in the apoplastic fluid of the stems and macerates extracts of sugarcane tissues with the procedure applied. Population of this microorganism might be in low number in the Spanish sugarcane varieties studied which is also discussed.

Effects of culture conditions on the production of polyhydroxyalkanoates by Azotobacter chroococcum H23 in media containing a high concentration of alpechín (wastewater from olive oil mills) as primary carbon source.

Large amounts of homopolymers containing beta-hydroxybutyrate (PHB) and copolymers containing beta-hydroxyvalerate (P[HB-co-HV]) are produced by Azotobacter chroococcum strain H23 when growing in culture media amended with alpechín (wastewater from olive oil mills) as the sole carbon source. Copolymer was formed when valerate (pentanoate) was added as a precursor to the alpechín medium, but it was not formed with the addition of propionate as a precursor. A. chroococcum formed homo- and copolymers of polyhydroxyalkanoates (PHAs) up to 80% of the cell dry weight, when grown on NH(4)(+)-medium supplemented with 60% (v/v) alpechín, after 48 h of incubation at 100 rev min(-1) and 30 degrees C. Production of PHAs by strain H23 using alpechín looks promising, as the use of a cheap substrate for the production of these materials is essential if bioplastics are to become competitive products.

Comparative analysis of the bacterial diversity in a lab-scale moving bed biofilm reactor (MBBR) applied to treat urban wastewater under different operational conditions

Different types of carriers were tested as support material in a lab-scale moving bed biofilm reactor (MBBR) used to treat urban wastewater under three different conditions of hydraulic retention time (HRT) and carrier filling ratios (FR). The bacterial diversity developed on the biofilms responsible of the treatment was studied using a cultivation-independent approach based on the polymerase chain reaction-temperature gradient gel electrophoresis technique (PCR-TGGE). Cluster analysis of TGGE fingerprints showed significant differences of community structure dependent upon the different operational conditions applied. Redundancy analysis (RDA) was used to determine the relationship between the operational conditions (type of carrier, HRT, FR) and bacterial biofilm diversity, demonstrating a significant effect of FR=50%. Phylogenetic analysis of PCR-reamplified and sequenced TGGE bands revealed that the prevalent Bacteria populations in the biofilm were related to Betaproteobacteria (46%), Firmicutes (34%),Alphaproteobacteria (14%) and Gammaproteobacteria (9%).

Microbial community structure and dynamics in a pilot-scale submerged membrane bioreactor aerobically treating domestic wastewater under real operation conditions.

A pilot scale submerged ultra-filtration membrane bioreactor (MBR) was used for the aerobic treatment of domestic wastewater over 9 months of year 2006 (28th March to 21st December). The MBR was installed at a municipal wastewater facility (EMASAGRA, Granada, Spain) and was fed with real wastewater. The experimental work was divided in 4 stages run under different sets of operation conditions. Operation parameters (total and volatile suspended solids, dissolved oxygen concentration) and environmental variables (temperature, pH, COD and BOD(5) of influent water) were daily monitored. In all the experiments conducted, the MBR generated an effluent of optimal quality complying with the requirements of the European Law (91/271/CEE 1991). A cultivation-independent approach (polymerase chain reaction-temperature gradient gel electrophoresis, PCR-TGGE) was used to analyze changes in the structure of the bacterial communities in the sludge. Cluster analysis of TGGE profiles demonstrated significant differences in community structure related to variations of the operation parameters and environmental factors. Canonical correspondence analysis (CCA) suggested that temperature, hydraulic retention time and concentration of volatile suspended solids were the factors mostly influencing community structure. 23 prominent TGGE bands were successfully reamplified and sequenced, allowing gaining insight into the identities of predominantly present bacterial populations in the sludge. Retrieved partial 16S-rRNA gene sequences were mostly related to the alpha-Proteobacteria, beta-Proteobacteria and gamma-Proteobacteria classes. The community established in the MBR in each of the four stages of operation significantly differed in species composition and the sludge generated displayed dissimilar rates of mineralization, but these differences did not influence the performance of the bioreactor (quality of the permeate). These data indicate that the flexibility of the bacterial community in the sludge and its ability to get adapted to environmental changes play an important role for the stable performance of MBRs.

Production of B-group vitamins by two Azotobacter strains with phenolic compounds as sole carbon source under diazotrophic and adiazotrophic conditions

Azotobacter vinelandii strain ATCC 12837 and A. chroococcum strain H23 (CECT 4435) were able to grow on N‐free or NH4Cl‐amended chemically‐defined (Burks) media, with protocatechuic acid (1–2 mmol l−1) or sodium p‐hydroxybenzoate (1–10 mmol l−1) as sole carbon (C) sources. At a concentration of 2 mmol l−1, both substrates supported nitrogen fixation (acetylene reduction assay) at similar or higher rates than bacteria grown in control media amended with 2 mmol l−1 sodium succinate as C source. The two strains produced the B‐group vitamins niacin, pantothenic acid, thiamine, riboflavin and biotin after 72 h of growth in chemically‐defined media with 2 mmol l−1 protocatechuic acid, sodium p‐hydroxybenzoate or sodium succinate as sole C source, either in N‐free media or in media amended with 0·1% NH4Cl. Quantitative production of all vitamins was affected by the use of the different C and N substrates.