RESUMEN
The diversity of ammonia-oxidizing bacteria (AOB) within the beta-subclass of Proteobacteria was investigated by genus- and family-specific real-time quantitative PCR (qPCR) assays on samples drawn from wastewater treatment systems. The 16S rRNA gene copy numbers ranged from 7.0 × 103 to 6.8 × 106, 1.1 × 107 to 1.8 × 107, and 2.9 × 105 to 1.5 × 107 copies/mL, respectively. Volumetric ammonium load (VAL) in the wastewater treatment systems calculated using the AOB numbers was in the range of 2.1-12.6 mM/d. Distribution patterns of eutrophic (i.e. Nitrosomonas europaea and Nitrosomonas nitrosa clusters) and oligotrophic (i.e. Nitrosomonas cryotolerans cluster) AOB groups were correlated with the VAL values. A high possibility of potential false-positive detection by family-specific qPCR assays was established by evaluating theoretical specificity in in silico and experimental investigations. The specificities of genus-specific qPCR assays were confirmed by amoA PCR, followed by cloning and sequencing. VAL must be the factor influencing the inclusion of AOB species. However, there was no significant correlation between the volatile suspended solid concentration representing chemical oxygen demand and N. europaea's community population, indicating that the degree of ammonia oxidation influenced the community cluster of Nitrosomonas relatively more.
Asunto(s)
Amoníaco/metabolismo , Nitrosomonas/metabolismo , Aguas Residuales , Oxidación-Reducción , Filogenia , ARN Ribosómico 16SRESUMEN
In this work, a method for simultaneously degrading the toxic pollutant, thiocyanate, and producing microalgal lipids using mixed microbial communities was developed. Aerobic activated sludge was used as the seed culture and thiocyanate was used as the sole nitrogen source. Two cultivation methods were sequentially employed: a lithoautotrophic mode and a photoautotrophic mode. Thiocyanate hydrolysis and a nitrification was found to occur under the first (lithoautotrophic) condition, while the oxidized forms of nitrogen were assimilated by the photoautotrophic consortium and lipids were produced under the second condition. The final culture exhibited good settling efficiency (â¼ 70% settling over 10 min), which can benefit downstream processing. The highest CO2 fixation rate and lipid productivity were observed with 2.5% and 5% CO2, respectively. The proposed integrated algal-bacterial system appears to be a feasible and even beneficial option for thiocyanate treatment and production of microbial lipids.
Asunto(s)
Biocombustibles , Microalgas/metabolismo , Tiocianatos/metabolismo , Dióxido de Carbono/metabolismo , Fermentación , Nitrógeno/metabolismo , Fotobiorreactores , Pigmentos Biológicos/metabolismo , Tiocianatos/toxicidadRESUMEN
Quantitative real-time PCR (qPCR) has been widely used in recent environmental microbial ecology studies as a tool for detecting and quantifying microorganisms of interest, which aids in better understandings of the complexity of wastewater microbial communities. Although qPCR can be used to provide more specific and accurate quantification than other molecular techniques, it does have limitations that must be considered when applying it in practice. This article reviews the principle of qPCR quantification and its applications to microbial ecology studies in various wastewater treatment environments. Here we also address several limitations of qPCR-based approaches that can affect the validity of quantification data: template nucleic acid quality, nucleic acid extraction efficiency, specificity of group-specific primers and probes, amplification of nonviable DNA, gene copy number variation, and limited number of sequences in the database. Even with such limitations, qPCR is reportedly among the best methods for quantitatively investigating environmental microbial communities. The application of qPCR is and will continue to be increasingly common in studies of wastewater treatment systems. To obtain reliable analyses, however, the limitations that have often been overlooked must be carefully considered when interpreting the results.
Asunto(s)
Bacterias , ADN Bacteriano , Consorcios Microbianos/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Aguas Residuales/microbiología , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , ADN Bacteriano/análisis , ADN Bacteriano/genéticaRESUMEN
Despite receiving increasing attention during the last few decades, the production of microalgal biofuels is not yet sufficiently cost-effective to compete with that of petroleum-based conventional fuels. Among the steps required for the production of microalgal biofuels, the harvest of the microalgal biomass and the extraction of lipids from microalgae are two of the most expensive. In this review article, we surveyed a substantial amount of previous work in microalgal harvesting and lipid extraction to highlight recent progress in these areas. We also discuss new developments in the biodiesel conversion technology due to the importance of the connectivity of this step with the lipid extraction process. Furthermore, we propose possible future directions for technological or process improvements that will directly affect the final production costs of microalgal biomass-based biofuels.
Asunto(s)
Biocombustibles , Biotecnología , Lípidos/aislamiento & purificación , Microalgas/metabolismo , Biomasa , Esterificación/genética , Humanos , Lípidos/biosíntesis , Lípidos/química , Microalgas/genéticaRESUMEN
To investigate the effect of hydraulic retention time (HRT) and temperature (T) on bacterial community structure and volatile fatty acids (VFAs) production of an acidogenic process, and VFA production and changes in the bacterial community in three identical automated anaerobic continuously-stirred tank reactors were analyzed using response surface analysis (RSA) and nonmetric multidimensional scaling (NMDS). For RSA, 11 trials were conducted to find the combination of T and HRT under which VFA production was greatest; VFA production was affected more by HRT than by T. To identify the bacterial community structure in each trial, DNA from each experimental point of the RSA was analyzed using denaturating gradient gel electrophoresis (DGGE), and eight bacteria species were detected. NMDS was conducted on band intensities obtained using DGGE, and bacterial community structure was affected more by T than by HRT. Taken together, these results suggest that VFA production during acidogenesis was more dependent on the physicochemical properties of acidogens, such as their specific growth rate or contact time with of substrates, than on changes in the microbial community.
Asunto(s)
Bacterias/crecimiento & desarrollo , Bacterias/metabolismo , Ácidos Grasos Volátiles/biosíntesis , Calor , Porcinos , Aguas Residuales/microbiología , Anaerobiosis/fisiología , AnimalesRESUMEN
Activity of ammonia-oxidizing bacteria (AOB) to simultaneous variation in Zn(2+) concentration (0.01-3.5mg/L), temperature (23-33°C), and AOB concentration (3-30 × 10(6)gene copies/mL) in a steel industry wastewater treatment plant was evaluated. Two equations were developed to describe the lag period (i.e., AOB acclimation) and ammonia oxidation rate (i.e., growth of the AOB) depending on the variables. AOB concentration and temperature both had significant effects on lag period and the ammonia oxidation rate. Zn(2+) concentration only had a significant effect on ammonia oxidation rate at 5% α-level. There was a significant interaction between AOB concentration and temperature for both lag period and ammonia oxidation rate. The effects of the variables were not significant when AOB concentration was higher than 2.0 × 10(7)copies/mL. There was no visible shift or changes in AOB communities based on DGGE analysis with amoA gene primers.
Asunto(s)
Aclimatación/fisiología , Amoníaco/metabolismo , Biota , Nitrosomonadaceae/fisiología , Temperatura , Eliminación de Residuos Líquidos/métodos , Zinc/metabolismo , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Gradiente Desnaturalizante , Espectrometría de Masas , Nitrosomonadaceae/metabolismo , Oxidación-Reducción , Reacción en Cadena de la PolimerasaRESUMEN
Microbial community structures were assessed in a two-stage anaerobic digestion system treating food waste-recycling wastewater. The reactors were operated for 390 d at 10 different hydraulic retention times (HRTs) ranging from 25 to 4 d. Stable operation was achieved with the overall chemical oxygen demand (COD) removal efficiency of 73.0-85.9% at organic loading rate of up to 35.6 g COD/L·d. Performance of the acidogenic reactors, however, changed significantly during operation. This change coincided with transition of the bacterial community from one dominated by Aeriscardovia- and Lactobacillus amylovorus-related species to one dominated by Lactobacillus acetotolerans- and Lactobacillus kefiri-like organisms. In methanogenic reactors, the microbial community structures also changed at this stage along with the shift from Methanoculleus- to Methanosarcina-like organisms. This trend was confirmed by the non-metric multidimensional scaling joint plot of microbial shifts along with performance parameters. These results indicated that the overall process performance was relatively stable compared to the dynamic changes in the microbial structures and the acidogenic performance.
Asunto(s)
Bacterias/metabolismo , Alimentos , Reciclaje/métodos , Eliminación de Residuos Líquidos/métodos , Residuos/análisis , Anaerobiosis , Bacterias/genética , Reactores Biológicos/microbiología , Electroforesis en Gel de Gradiente Desnaturalizante , Filogenia , ARN Ribosómico 16S/genéticaRESUMEN
A quantitative real-time PCR (QPCR) assay with the TaqMan system was used to quantify 16S rRNA genes of beta-proteobacterial ammonia-oxidizing bacteria (AOB) in a batch nitrification bioreactor. Five different sets of primers, together with a TaqMan probe, were used to quantify the 16S rRNA genes of beta-proteobacterial AOB belonging to the Nitrosomonas europaea, Nitrosococcus mobilis, Nitrosomonas nitrosa, and Nitrosomonas cryotolerans clusters, and the genus Nitrosospira. We also used PCR followed by denaturing gradient gel electrophoresis (DGGE), cloning, and sequencing of their 16S rRNA genes to identify the AOB species. Seed sludge from an industrial wastewater treatment process controlling high-strength nitrogen wastewater (500 mg/L NH4+-N) was used as the inoculum for subsequent batch experiment. The Nitrosomonas nitrosa cluster was the predominant AOB (2.3x10(5) copies/mL) in the start-up period of the batch experiment. However, from the exponential growth period, the Nitrosomonas europaea cluster was the most abundant AOB, and its 16S rRNA gene copy number increased to 8.9x10(6) copies/mL. The competitive dominance between the two AOB clusters is consistent with observed differences in ammonia tolerance and substrate affinity. Analysis of the DGGE results indicated the presence of Nitrosomonas europaea ATCC19718 and Nitrosomonas nitrosa Nm90, consistent with the QPCR results.
Asunto(s)
Amoníaco/metabolismo , Biodiversidad , Nitrosomonadaceae/genética , Nitrosomonadaceae/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Reactores Biológicos/microbiología , Cartilla de ADN/genética , ADN Bacteriano/genética , ADN Ribosómico/genética , Nitrosomonadaceae/aislamiento & purificación , ARN Ribosómico 16S/genética , Aguas del Alcantarillado/microbiologíaRESUMEN
For biological nitrification, a set of experiments were carried out to approximate the response of lag period along with ammonia oxidation rate with respect to different concentrations of cyanide (CN-) and ammonia-oxidizing bacteria (AOB), and temperature variation in laboratory-scale batch reactors. The effects of simultaneous changes in these three factors on ammonia oxidation were quantitatively estimated and modeled using response surface analysis. The lag period and the ammonia oxidation rate responded differently to changes in the three factors. The lag period and the ammonia oxidation rate were significantly affected by the CN- and AOB concentrations, while temperature changes only affected the ammonia oxidation rate. The increase of AOB concentration and temperature alleviated the inhibition effect of cyanide on ammonia oxidation. The statistical method used in this study can be extended to estimate the quantitative effects of other environmental factors that can change simultaneously.
Asunto(s)
Amoníaco/metabolismo , Cianuros/metabolismo , Nitrosomonadaceae/metabolismo , Amoníaco/química , Cinética , Nitrosomonadaceae/química , Nitrosomonadaceae/genética , Oxidación-Reducción , TemperaturaRESUMEN
This study was designed to monitor changes in the levels of adenosine 5'-triphosphate (ATP) and deoxyribonucleic acid (DNA) per unit of microbial mass during the autotrophic biodegradation of thiocyanate (SCN(-)). An artificial medium containing trace minerals and 500 mg SCN(-)/L was used as a substrate for bacterial growth. An SCN(-)-degrading bioreactor with a working volume of 6 L, equipped with temperature, pH, and dissolved oxygen controls, was operated in batch mode. During the exponential phase of SCN(-) biodegradation, the ratios of ATP and DNA to microbial dry weight varied from 0.6 to 1.1 microg ATP/mg of volatile suspended solid (VSS), and from 3.5 to 8.8 microg DNA/mg of VSS, respectively. The ATP and DNA concentrations correlated linearly with microbial mass (r (2) > 0.9) within the exponential phase. The linear regression equations were as follows: (1) microbial mass concentration (mg/L) = 0.663 x ATP concentration (microg/L) + 11.1 and (2) microbial concentration (mg/L) = 0.081 x DNA concentration (microg/L) + 10.9. The applicable ranges were 6.8 to 47.4 microg/L for ATP concentration and 41.5 to 395 microg/L for DNA concentration, respectively.
Asunto(s)
Adenosina Trifosfato/metabolismo , ADN Bacteriano/metabolismo , Tiocianatos/metabolismo , Bacterias/metabolismo , Biomasa , Reactores Biológicos/microbiología , Aguas del Alcantarillado/microbiologíaRESUMEN
Use of quantitative real-time PCR (QPCR) with TaqMan probes is increasingly popular in various environmental works to detect and quantify a specific microorganism or a group of target microorganism. Although many aspects of conducting a QPCR assay have become very easy to perform, a proper design of oligonucleotide sequences comprising primers and a probe is still considered as one of the most important aspects of a QPCR application. This work was conducted to design group specific primer and probe sets for the detection of ammonia oxidizing bacteria (AOB) using a real-time PCR with a TaqMan system. The genera Nitrosomonas and Nitrosospira were grouped into five clusters based on similarity of their 16S rRNA gene sequences. Five group-specific AOB primer and probe sets were designed. These sets separately detect four subgroups of Nitrosomonas (Nitrosomonas europaea-, Nitrosococcus mobilis-, Nitrosomonas nitrosa-, and Nitrosomonas cryotolerans-clusters) along with the genus Nitrosospira. Target-group specificity of each primer and probe set was initially investigated by analyzing potential false results in silico, followed by a series of experimental tests for QPCR efficiency and detection limit. In general, each primer and probe set was very specific to the target group and sensitive to detect target DNA as low as two 16S rRNA gene copies per reaction mixture. QPCR efficiency, higher than 93.5%, could be achieved for all primer and probe sets. The primer and probe sets designed in this study can be used to detect and quantify the beta-proteobacterial AOB in biological nitrification processes and various environments.
Asunto(s)
Betaproteobacteria/genética , Betaproteobacteria/aislamiento & purificación , Cartilla de ADN/genética , Sondas de ADN/genética , Nitrosomonas/genética , Nitrosomonas/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Recuento de Colonia Microbiana/métodos , ARN Ribosómico 16S/genética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Especificidad de la EspecieRESUMEN
Biokinetics for autotrophic degradation of thiocyanate using batch culture of Klebsiella sp. were evaluated both analytically and numerically. A sequential approach with an analytical method followed by a numerical approximation was used to evaluate and to ensure the accuracy of the parameter estimation. The nonlinear least-squares method with a 95% confidence interval was employed. The growth conditions were maintained at pH 7 and 38 degrees C for all experiments. With an automated incubation and turbidity reader, a total of 16 different initial thiocyanate concentrations, ranging from 10 to 300 mg L(-1), were used to develop a kinetic expression of specific growth rate as a function of substrate concentration. The biodegradation of thiocyanate with Klebsiella sp. followed a substrate inhibition pattern. Three identical automated bioreactors with working volumes of 1.5 L, equipped with sterilizable sampling ports, were also used for the numerical approximation of the biokinetic parameters in batch mode. A fourth order Runge-Kutta method was used to approximate the substrate inhibition kinetics of the Klebsiella sp. utilizing thiocyanate. Although the kinetic coefficients estimated by analytical and numerical methods were not statistically different at a 0.05 alpha level, model responses of numerical approximation generated a better prediction of changes in thiocyanate and cell mass concentrations. The hypothetical maximum growth rate, micro m, half saturation coefficient, Ks, microbial yield coefficient, Y, cell mass decay rate coefficient, kd, and substrate inhibition coefficient, Ksi, were evaluated as being 0.62 +/- 0.05 d(-1), 85 +/- 8 mg SCN- L(-1), 0.076 +/- 0.011 mg cell mass (mg SCN)(-1), 0.03 +/- 0.002 d(-1), and 131 +/- 22 mg SCN- L(-1), respectively. The calculated maximal substrate concentration, Sm, and apparent maximum specific growth rate, micro'm, were 105.5 +/- 8.7 mg SCN- L(-1) and 0.24 +/- 0.01 d(-1), respectively. Using these estimated parameters, the theoretical performance of the continuous operation was also illustrated, which depicts the residual thiocyanate and Klebsiella sp. concentrations in the non-steady and steady states at different hydraulic retention times (HRTs). Assuming the influent concentration of 250 mg SCN- L(-1), the expected treatment efficiency ranged from 94.9% to 69.4% between 20 and 5 days HRT, respectively. Klebsiella sp. was expected to be washed out at 4.8 days HRT, thus resulting in no treatment of thiocyanate.