Anouk F. Duque

Changes in the bacterial community structure in two-stage constructed wetlands with different plants for industrial wastewater treatment

This study focused on the diversity of bacterial communities from two series of two-stage constructed wetlands (CWs) treating tannery wastewater, under different hydraulic conditions. Series were separately planted with Typha latifolia and Phragmites australis in expanded clay aggregates and operated for 31 months. The effect of plant species, hydraulic loading and unit stage on bacterial communities was addressed through bacterial enumeration and denaturating gradient gel electrophoresis (DGGE). Diverse and distinct bacterial communities were found in each system unit, which was related in part to the type of plant and stage position (first or second unit in the series). Numerical analysis of DGGE profiles showed high diversity in each unit with an even distribution of species. No clear relation was established between the sample collection time, hydraulic loading applied and the bacterial diversity. Isolates retrieved from plant roots and substrates of CWs were affiliated with gamma-Proteobacteria, Firmicutes, alpha-Proteobacteria, Sphingobacteria, Actinobacteria and Bacteroidetes. Both series were effective in removing organic matter from the inlet wastewater, however, based on batch degradation experiments it seems that biodegradation was limited by the recalcitrant properties of the wastewater.

Bacterial community dynamics in horizontal flow constructed wetlands with different plants for high salinity industrial wastewater polishing

This study is focused on the diversity of bacterial communities from two series of horizontal subsurface flow constructed wetlands (CW) polishing high salinity tannery wastewater. Each series was planted with Arundo donax or Sarcocornia sp. in a substrate composed by expanded clay and sand. Chemical and biochemical oxygen demand removal efficiencies were similar in each series, varying between 58 and 67% (inlet COD 218 ± 28 mg L(-1)) and 60 and 77% (inlet BOD(5) 37 ± 6 mg L(-1)), respectively. High numbers of culturable bacteria were obtained from substrate and root samples - 5.75 × 10(6)-3.95 × 10(8) CFU g(-1) recovered on marine agar and 1.72 × 10(7)-8.46 × 10(8) CFU g(-1) on nutrient agar. Fifty bacterial isolates were retrieved from the CW, related phylogenetically to Firmicutes, Actinobacteria, Bacteroidetes, α-, β-, and γ-Proteobacteria. Changes in the bacterial communities, from roots and substrate of each series, related to the plant species, hydraulic loading rates and along CW operation were examined using denaturating gradient gel electrophoresis (DGGE). The clustering analysis suggested that a diverse and distinct bacterial community inhabits each series, which was related to the type of plant present in each CW.

2-Fluorophenol degradation by aerobic granular sludge in a sequencing batch reactor

Aerobic granular sludge is extremely promising for the treatment of effluents containing toxic compounds, and it can economically compete with conventional activated sludge systems. A laboratory scale granular sequencing batch reactor (SBR) was established and operated during 444 days for the treatment of an aqueous stream containing a toxic compound, 2-fluorophenol (2-FP), in successive phases. Initially during ca. 3 months, the SBR was intermittently fed with 0.22 mM of 2-FP added to an acetate containing medium. No biodegradation of the target compound was observed. Bioaugmentation with a specialized bacterial strain able to degrade 2-FP was subsequently performed. The reactor was thereafter continuously fed with 0.22 and 0.44 mM of 2-FP and with 5.9 mM of acetate (used as co-substrate), for 15 months. Full degradation of the compound was reached with a stoichiometric fluoride release. The 2-FP degrading strain was successfully retained by aerobic granules, as shown through the recovering of the strain from the granular sludge at the end of the experiment. Overall, the granular SBR has shown to be robust, exhibiting a high performance after bioaugmentation with the 2-FP degrading strain. This study corroborates the fact that bioaugmentation is often needed in cases where biodegradation of highly recalcitrant compounds is targeted.

Response of a three-stage process for PHA production by mixed microbial cultures to feedstock shift: impact on polymer composition.

Polyhydroxyalkanoates (PHA) can be produced by mixed microbial cultures (MMC) using a three-stage process. An attractive feature of MMC for PHA production is the ability to use waste/surplus feedstocks. In this study, the effect of a feedstock shift, mimicking a seasonal feedstock scenario and/or as a strategy for controlling polymer composition, on a MMC PHA production process was assessed using cheese whey (CW) and sugar cane molasses (SCM) as model feedstocks. The acidogenic stage responded immediately to the feedstock shift by changing the fermented products profile, with acetate and butyrate being the main acids produced from CW, while for SCM propionate and valerate were the dominant products. The fermentation process was then quite stable during long term operation. The PHA culture selection stage also responded quickly to the fermented feestocks shift, generating a polymer whose composition was linearly dependent on the concentration of HV and HB precursors produced in the acidogenic stage. The selected culture reached a maximum PHA content of 56% and 65% with fermented SCM and CW, respectively. Mixing fermented CW and SCM, in equal volume proportions, demonstrated the possibility of using different fermented feedstocks for tailoring polymer composition.

Bioaugmentation of a rotating biological contactor for degradation of 2-fluorophenol.

The performance of a laboratory scale rotating biological contactor (RBC) towards shock loadings of 2-fluorophenol (2-FP) was investigated. During a period of ca. 2 months organic shock loadings of 25 mg L⁻¹ of 2-FP were applied to the RBC. As no biodegradation of 2-FP was observed, bioaugmentation of the RBC with a 2-FP degrading strain was carried out and, along ca. 6 months, organic shock loadings within a range of 25-200 mg L⁻¹ of 2-FP were applied. Complete biodegradation of 50 mg L⁻¹ of 2-FP was observed during operation of the reactor. The RBC showed to be robust towards starvation periods, as after ca. 1month of non-supply of the target compound, the reactor resumed 2-FP degradation. The inoculated strain was retained within the biofilm in the disks, as the 2-FP degrading strain was recovered from the biofilm by the end of the experiment, thus bioaugmentation was successfully achieved.

Labrys portucalensis sp. nov., a fluorobenzene-degrading bacterium isolated from an industrially contaminated sediment in northern Portugal

A detailed classification of a novel bacterial strain, designated F11(T), capable of degrading fluorobenzene as a sole carbon and energy source, was performed by using a polyphasic approach. This Gram-negative, rod-shaped, non-motile, non-spore-forming, aerobic bacterium was isolated from a sediment sample collected from an industrially contaminated site in northern Portugal. The predominant whole-cell fatty acids were C(19 : 0) cyclo omega8c, C(16 : 0), C(18 : 1)omega7c, C(18 : 0), C(18 : 0) 3-OH and C(16 : 0) 3-OH. The G+C content of the DNA was 62.9 mol% and the major respiratory quinone was ubiquinone 10 (UQ-10). 16S rRNA gene sequence analysis revealed that strain F11(T) was a member of the class Alphaproteobacteria and was phylogenetically related to the genus Labrys, having sequence similarities of 95.6 and 93.1 % to the type strains of Labrys monachus and Labrys methylaminiphilus, respectively. DNA-DNA hybridization experiments revealed levels of relatedness of <70 % between strain F11(T) and the type strains of L. monachus and L. methylaminiphilus (38.6 and 34.1 %, respectively), justifying the classification of strain F11(T) as representing a novel species of the genus Labrys. The name Labrys portucalensis sp. nov. is proposed for this organism. The type strain is F11(T) (=LMG 23412(T)=DSM 17916(T)).

Bioaugmentation for treating transient 4-fluorocinnamic acid shock loads in a rotating biological contactor

A rotating biological contactor (RBC) was used to treat shock loadings of 4-fluorocinnamic acid (4-FCA). Intermittent 4-FCA shocks of 35 mg L(-1) were applied (ca. 3 months) with only limited mineralization occurring and accumulation of 4-fluorobenzoate (4-FBA) as an intermediate. After bioaugmentation with a degrading bacterium the RBC was able to deal with 4-FCA intermittent loading of 80 mg L(-1) however, a gradual decline in RBC performance occurred, leading to 4-FBA accumulation. The degrading strain was recovered from the biofilm during 2 months but intermittent feeding may have led to diminishing strain numbers. Distinct bacterial communities in the 1st and the 5th and 10th stages of the RBC were revealed by denaturating gradient gel electrophoresis. Several isolates retrieved from the RBC transformed 4-FCA into 4-FBA but only two strains mineralized the compound. Bioaugmentation allowed removal of the fluorinated compound however intermittent feeding may have compromised the bioreactor efficiency.

Biological treatment of a contaminated gaseous emission from a leather industry in a suspended-growth bioreactor.

A suspended-growth bioreactor (SGB) was operated for the treatment of a gaseous stream mimicking emissions generated at a leather industrial company. The main volatile organic compounds (VOCs) present in the gaseous stream consisted of 1-methoxy-2-propanol, 2,6-dimethyl-4-heptanone, 2-butoxyethanol, toluene and butylacetate. A microbial consortium able to degrade these VOCs was successfully enriched. A laboratory-scale SGB was established and operated for 210-d with an 8h cycle period and with shutdowns at weekends. Along this period, the SGB was exposed to organic loads (OL) between 6.5 and 2.3 x 10(2) g h(-1) m(-3). Most of the compounds were not detected at the outlet of the SGB. The highest total VOC removal efficiency (RE) (ca 99%) was observed when an OL of 1.6 x 10(2) g h(-1) m(-3) was fed to the SGB. The maximum total VOC elimination capacity (1.8 x 10(2) g h(-1) m(-3)) was achieved when the OL applied to the SGB was 2.3 x 10(2) g h(-1) m(-3). For all the operating conditions, the SGB showed high levels of degradation of toluene and butylacetate (RE approximately equal to 100%). This study also revealed that recirculation of the gaseous effluent improved the performance of the SGB. Overall, the SGB was shown to be robust, showing high performance after night and weekend shutdown periods.

Characterization of the bacterial communities of aerobic granules in a 2-fluorophenol degrading process

Aerobic granular sludge constitutes a novel technology for wastewater treatment. This study focused on the effect of 2-fluorophenol (2-FP) shock loadings on the microbial community diversity present in aerobic granules before and after inoculation with a bacterial strain able to degrade 2-FP, Rhodococcus sp. strain FP1. After bioaugmentation, apart from strain FP1, five culturable bacteria were isolated from the 2-FP degrading granules, belonging to the following genera: Serratia, Chryseobacterium, Xanthomonas, Pimelobacter and Rhodococcus. The latter two isolates are able to degrade 2-FP. Changes in the aerobic granules’ bacterial communities related to 2-FP shock loadings were examined using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene pool. Numerical analysis of the DGGE profiles showed high diversity with an even distribution of species. Based on cluster analysis of the DGGE profiles, the bacterial communities present in the aerobic granules changes were related to the sampling time and the 2-FP concentration fed.

Development of flow injection potentiometric methods for the off-line and on-line determination of fluoride to monitor the biodegradation of a monofluorophenol in two bioreactors.

Water treatment has become a source of concern as new pollutants and higher volumes of waste water must be treated. Emerging biological approaches, namely the use of bioreactors, for cleaning processes have been introduced. The use of bioreactors requires the development of efficient monitoring tools, preferably with real-time measurements. In this work, a couple of flow injection systems were developed and optimized for the potentiometric determination of fluoride to monitor a rotating biological contactor (RBC) bioreactor and a sequencing batch reactor (SBR) with off-line and on-line sampling. Both the RBC and the SBR bioreactors were set up for the biodegradation of the halogenated organic compound 2-fluorophenol and, as fluoride was a degradation byproduct, the process was monitored by following up its concentration. The described flow injection potentiometric methods enabled the fluoride determination within the required quantification range 0.10-100mM. The possible interferences from the growth medium were minimized in-line. The determination rate was 78 h(-1) for the off-line monitoring of RBC and 50(-1)h for the on-line monitoring of the SBR, with a sample consumption of 0.500 mL and 0.133 mL per determination, respectively. Furthermore, the overall reagent consumption was quite low. The accuracy of the system was evaluated by comparison with a batch procedure. The SBR efficiency was monitored both on-line by the flow system and off-line by HPLC, for comparison purposes.