Understanding the influence of free nitrous acid on microalgal-bacterial consortium in wastewater treatment: A critical review.

Microalgal-bacterial consortium (MBC) constitutes a sustainable and efficient alternative to the conventional activated sludge process for wastewater treatment (WWT). Recently, integrating the MBC process with nitritation (i.e., shortcut MBC) has been proposed to achieve added benefits of reduced carbon and aeration requirements. In the shortcut MBC system, nitrite or free nitrous acid (FNA) accumulation exerts antimicrobial influences that disrupt the stable process performance. In this review, the formation and interactions that influence the performance of the MBC were firstly summarized. Then the influence of FNA on microalgal and bacterial monocultures and related mechanisms together with the knowledge gaps of FNA influence on the shortcut MBC were highlighted. Other challenges and future perspectives that impact the scale-up of the shortcut MBC for WWT were illustrated. A potential roadmap is proposed on how to maximize the stable operation of the shortcut MBC system for sustainable WWT and high-value biomass production.

Free nitrous acid-based nitrifying sludge treatment in a two-sludge system obtains high polyhydroxyalkanoates accumulation and satisfied biological nutrients removal.

A novel strategy to achieve substantial polyhydroxyalkanoates (PHA) accumulation in waste activated sludge (WAS) was developed, which was conducted in a two-sludge system consisted of an anaerobic/anoxic/oxic reactor (AAO-SBR) and a nitrifying reactor (N-SBR), where the nitrifying-sludge was treated by free nitrous acid (FNA). Initially, 0.98 ±â€¯0.09 and 1.46 ±â€¯0.10 mmol-c/g VSS of PHA were respectively determined in the control-SBR and AAO-SBR. When 1/16 of nitrifying sludge was daily treated with 1.49 mg N/L FNA for 24 h, ∼46.5% of nitrite was accumulated in the N-SBR, ∼2.43 ±â€¯0.12 mmol-c/g VSS of PHA was accumulated in WAS in AAO-SBR without deteriorating nutrient removal. However, nutrient removal of control-SBR was completely collapsed after implementing the same FNA treatment. Further investigations revealed that the activity and abundance of nitrite oxidizing bacteria (NOB) was decreased significantly after FNA treatment. Finally, sludge with high PHA level to generate more methane was confirmed.

Anesthesia effects on the low frequency blood flow oscillations in mouse skin.

BACKGROUND: When laboratory animals are used one needs to anesthetize them before recording. However, the influence of anesthesia on animal blood flow oscillations has not been studied. The effects of two ways of anesthesia, zoletil-xylazine, and zoletil-nitrous oxide mixtures, on mouse skin perfusion using laser Doppler flowmetry (LDF) technique were studied. METHODS: BALB/c mice were used. LDF probe was placed on the ventral surface of the left hind paw. Spectral analysis of LDF signals was performed with continuous adaptive wavelet transform to identify and describe peripheral blood flow oscillations in mouse skin. RESULTS: Low-frequency oscillation interval boundaries (myogenic, neurogenic, and endothelial) for mice were shown to coincide with the boundaries determined for human and rats, that demonstrate their independence from the body size. Zoletil-xylazine anesthesia significantly decreased neurogenic and endothelial oscillation amplitudes by 29% and 50% respectively and increased the amplitude of cardiac oscillations by 23% compared to zoletyl-nitrous oxide anesthesia. There were no significant changes of the amplitudes of myogenic and respiratory oscillations with zoletil-nitrous oxide anesthesia compared to the zoletil-xylazine mixture. CONCLUSION: We suggest that the different influence of anesthesia modes on the amplitudes of skin blood flow oscillations is associated with sympathetic activity suppressed by zoletil-xylazine anesthesia.

Effect of Free Nitrous Acid on Nitrous Oxide Production and Denitrifying Phosphorus Removal by Polyphosphorus-Accumulating Organisms in Wastewater Treatment.

The inhibition of free nitrous acid (FNA) on denitrifying phosphorus removal has been widely reported for enhanced biological phosphorus removal; however, few studies focus on the nitrous oxide (N2O) production involved in this process. In this study, the effects of FNA on N2O production and anoxic phosphorus metabolism were investigated using phosphorus-accumulating organisms (PAOs) culture highly enriched (91 ± 4%) in Candidatus Accumulibacter phosphatis. Results show that the FNA concentration notably inhibited anoxic phosphorus metabolism and phosphorus uptake. Poly-ß-hydroxyalkanoate (PHA) degradation was completely inhibited when the FNA concentration was approximately 0.0923 mgHNO2-N/L. Higher initial FNA concentrations (0.00035 to 0.0103 mgHNO2-N/L) led to more PHA consumption/TN (0.444 to 0.916 mmol-C/(mmol-N·gVSS)). Moreover, it was found that FNA, rather than nitrite and pH, was likely the true inhibitor of N2O production. The highest proportion of N2O to TN was 78.42% at 0.0031 mgHNO2-N/L (equivalent to 42.44 mgNO2-N/L at pH 7.5), due to the simultaneous effects of FNA on the subsequent conversion of NO2 into N2O and then into N2. The traditional nitrite knee point can only indicate the exhaustion of nitrite, instead of the complete removal of TN.

Effect of free nitrous acid pre-treatment on primary sludge at low exposure times.

The present study was undertaken to investigate the effect of different free nitrous acid (FNA) concentrations at low pre-treatment times (PTs) (1, 2 and 5h) and without pH control with mild agitation on primary sludge (PS) biodegradability and methane production (MP). Increasing PTs resulted in an increase in the solubility of the organic matter (around 25%), but not on cell-mortality (>75% in all the cases with FNA) and neither on methane generation. FNA pre-treatment at low PTs improve MP (around 16% at PT of 1h and 650mg N-NO2-/L). However, a similar improvement was found with mild agitation of PS without FNA at 2 and 5h. Taking into account the potential costs associated with the FNA pre-treatment, a mild agitation without FNA would be preferred to enhance MP in PS.

Antimicrobial Effects of Free Nitrous Acid on Desulfovibrio vulgaris: Implications for Sulfide-Induced Corrosion of Concrete.

Hydrogen sulfide produced by sulfate-reducing bacteria (SRB) in sewers causes odor problems and asset deterioration due to the sulfide-induced concrete corrosion. Free nitrous acid (FNA) was recently demonstrated as a promising antimicrobial agent to alleviate hydrogen sulfide production in sewers. However, details of the antimicrobial mechanisms of FNA are largely unknown. Here, we report the multiple-targeted antimicrobial effects of FNA on the SRB Desulfovibrio vulgaris Hildenborough by determining the growth, physiological, and gene expression responses to FNA exposure. The activities of growth, respiration, and ATP generation were inhibited when exposed to FNA. These changes were reflected in the transcript levels detected during exposure. The removal of FNA was evident by nitrite reduction that likely involved nitrite reductase and the poorly characterized hybrid cluster protein, and the genes coding for these proteins were highly expressed. During FNA exposure, lowered ribosome activity and protein production were detected. Additionally, conditions within the cells were more oxidizing, and there was evidence of oxidative stress. Based on an interpretation of the measured responses, we present a model depicting the antimicrobial effects of FNA on D. vulgaris These findings provide new insight for understanding the responses of D. vulgaris to FNA and will provide a foundation for optimal application of this antimicrobial agent for improved control of sewer corrosion and odor management.IMPORTANCE Hydrogen sulfide produced by SRB in sewers causes odor problems and results in serious deterioration of sewer assets that requires very costly and demanding rehabilitation. Currently, there is successful application of the antimicrobial agent free nitrous acid (FNA), the protonated form of nitrite, for the control of sulfide levels in sewers (G. Jiang et al., Water Res 47:4331-4339, 2013, http://dx.doi.org/10.1016/j.watres.2013.05.024). However, the details of the antimicrobial mechanisms of FNA are largely unknown. In this study, we identified the key responses (decreased anaerobic respiration, reducing FNA, combating oxidative stress, and shutting down protein synthesis) of Desulfovibrio vulgaris Hildenborough, a model sewer corrosion bacterium, to FNA exposure by examining the growth, physiological, and gene expression changes. These findings provide new insight and underpinning knowledge for understanding the responses of D. vulgaris to FNA exposure, thereby benefiting the practical application of FNA for improved control of sewer corrosion and odor.

Towards energy positive wastewater treatment by sludge treatment using free nitrous acid.

Free nitrous acid (FNA i.e. HNO2) was revealed to be effective in enhancing biodegradability of secondary sludge. Also, nitrite-oxidizing bacteria were found to be more susceptible to FNA than ammonium-oxidizing bacteria. Based on these findings, a novel FNA-based sludge treatment technology is proposed to enhance energy recovery from wastewater/sludge. Energy analysis indicated that the FNA-based technology would make wastewater treatment become an energy generating process (yielding energy at 4 kWh/PE/y; kWh/PE/y: kilowatt hours per population equivalent per year), rather than being a large energy consumer that it is today (consuming energy at 24 kWh/PE/y). Importantly, FNA required for the sludge treatment could be produced as a by-product of wastewater treatment. This proposed FNA-based technology is economically and environmentally attractive, and can be easily implemented in any wastewater treatment plants. It only involves the installation of a simple sludge mixing tank. This article presents the concept of the FNA-based technology.

The role of small acid-soluble proteins (SASPs) in protection of spores of Clostridium botulinum against nitrous acid.

Mutant strains of Clostridium botulinum ATCC 3502 were generated using the ClosTron in four genes (CBO1789, CBO1790, CBO3048, CBO3145) identified as encoding α/ß-type SASP homologues. The spores of mutant strains in which CBO1789 or CBO1790 was inactivated demonstrated a significant increase in sensitivity to the damaging agent nitrous acid (P<0.01), a phenotype that was partially restored to wild-type in complementation studies. In contrast to nitrous acid, the spores of the CBO1789 and CBO1790 mutants showed no change in their resistance to formaldehyde and hydrogen peroxide (P>0.05), two other chemicals commonly used as components of disinfection regimes. These data indicate that the SASPs CBO1789 or CBO1790 play a significant role in resistance to nitrous acid, but not in resistance to formaldehyde or hydrogen peroxide.

Upstream and downstream processing of lovastatin by Aspergillus terreus.

The present study describes the enhanced production and purification of lovastatin by Aspergillus terreus in submerged batch fermentation. The enhancement of lovastatin production from A. terreus was attempted by random mutagenesis using ultraviolet radiations and nitrous acid. UV mutants exhibited increased efficiency for lovastatin production as compared with nitrous acid mutants. Among all the mutants developed, A. terreus UV-4 was found to be the hyper producer of lovastatin. This mutant gave 3.5-fold higher lovastatin production than the wild culture of A. terreus NRRL 265. Various cultural conditions were also optimized for hyper-producing mutant strain. 5 % glucose as carbon source, 1.5 % corn steep liquor as nitrogen source, initial pH value of 6, 120 h of incubation period, and 28 °C of incubation temperature were found as best parameters for higher lovastatin production in shake flasks. Production of lovastatin by wild and mutant strains of A. terreus was also scaled up to laboratory scale fermentor. The fermentation process was conducted at 28 °C, 200 rpm agitation, and 1vvm air flow rate without pH control. After the optimization of cultural conditions in 250 ml Erlenmeyer flasks and scaling up to laboratory scale fermentor, the mutant A. terreus UV-4 gave eightfold higher lovastatin production (3249.95 µg/ml) than its production by wild strain in shake flasks. Purification of lovastatin was carried out by solvent extraction method which yielded 977.1 mg/l of lovastatin with 98.99 % chromatographic purity and 26.76 % recovery. The crystal structure of lovastatin was determined using X-ray diffraction analysis which is first ever reported.

Enhanced lipid extraction from algae using free nitrous acid pretreatment.

Lipid extraction has been identified as a major bottleneck for large-scale algal biodiesel production. In this work free nitrous acid (FNA) is presented as an effective and low cost pretreatment to enhance lipid recovery from algae. Two batch tests, with a range of FNA additions, were conducted to disrupt algal cells prior to lipid extraction by organic solvents. Total accessible lipid content was quantified by the Bligh and Dyer method, and was found to increase with pretreatment time (up to 48 h) and FNA concentration (up to 2.19 mg HNO2-N/L). Hexane extraction was used to study industrially accessible lipids. The mass transfer coefficient (k) for lipid extraction using hexane from algae treated with 2.19 mg HNO2-N/L FNA was found to be dramatically higher than for extraction from untreated algae. Consistent with extraction results, cell disruption analysis indicated the disruption of the cell membrane barrier.

Inactivation kinetics of anaerobic wastewater biofilms by free nitrous acid.

Recent studies have shown that free nitrous acid (FNA) is biocidal to a broad range of microorganisms. Microorganisms residing in anaerobic sewer biofilms were found to be inactivated after a short (6-24 h) exposure to FNA. In this study, we investigate the inactivation kinetics of anaerobic sewer biofilms grown in real wastewater. Microbial viability of biofilms was determined using LIVE/DEAD staining. A two-fraction kinetic model was developed to simulate the inactivation of mixed culture in biofilms. The kinetic parameters were estimated by using Bayesian statistics. Model simulation found that a fraction (85 %) of the biofilm community was highly sensitive to FNA with a high inactivation rate, and a fraction (15 %) was tolerant to FNA and persisted after FNA treatment. This different susceptibility to FNA treatment was likely due to the diverse microbial community and biofilm protection. The fact that nearly 85 % microbes were inactivated confirmed that FNA is a strong biocide to mixed-culture biofilms. It was found that the inactivation rate constant was not affected by pH levels. The kinetic model was successfully used to optimize FNA dosage for sulfide control in sewer biofilms. Also, results suggest that a high FNA concentration is preferred than long exposure time to reduce the total chemical consumption.

Sodium dodecyl sulphate, a strong inducer of thermostable glucanhydrolase secretion from a derepressed mutant strain of Bacillus alcalophilus GCBNA-4.

In the present study, we report the optimisation of batch conditions for improved α-1,4-glucan-glucanohydrolase (GGH) secretion by a nitrous acid (NA)-treated Bacillus alcalophilus. The wild (isolate GCB-18) and NA-derivative (mutant GCBNA-4) were grown in a medium containing 10 g/L nutrient broth, 10 g/L starch, 5 g/L lactose, 2 g/L ammonium sulphate, 2 g/L CaCl2 and phosphate buffer (pH 7.6). Sodium dodecyl sulphate (SDS) was used as an enzyme inducer while batch fermentations were carried out at 40 °C. The mutant produced GGH in 40 h which was 15-fold higher than the wild in presence of SDS. Thermodynamic studies revealed that the mutant culture exhibited the capability for improved enzyme activity over a broad range of temperature (35-70 °C). The enzyme was purified by cation-exchange column chromatography with ~80 % recovery. The performance of fuzzy-logic system control was found to be highly promising for the improved substrate conversion rate. The correlation (1.045E + 0025) among variables demonstrated the model terms as highly significant indicating commercial utility of the culture used (P < 0.05).

The effect of free nitrous acid on key anaerobic processes in enhanced biological phosphorus removal systems.

In this study, the effect of nitrite/FNA on the anaerobic metabolism of polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) is investigated. The results clearly show that FNA has a detrimental effect on the acetate uptake rate by both PAOs and GAOs, but this adverse effect is much stronger on PAOs than on GAOs. Also, when FNA was increased, phosphate release to acetate uptake ratio by PAOs increased substantially (250-300% compared to control), which was accompanied by decreases (40-60%) in glycogen degradation and PHA production to VFA uptake. In contrast, these ratios for GAOs remained constant or increased slightly towards the highest FNA concentration applied. These results indicate that the anaerobic metabolism of PAOs is more adversely affected than that of GAOs when FNA is present. This might provide a competitive advantage to GAOs over PAOs in enhanced biological phosphorus removal systems when nitrite is present.

Improving secondary sludge biodegradability using free nitrous acid treatment.

This study presents a novel strategy based on free nitrous acid (FNA) treatment to improve the biodegradability of secondary sludge. Several experiments were conducted to demonstrate the biocidal effect of FNA on activated sludge. The viable fraction as well as the biological activity of the biomass decreased significantly after 8-48 h treatment with FNA. The biodegradability of the FNA treated sludge was compared to that of the same sludge without FNA treatment by aerobically digesting these sludges with a full-scale activated sludge for 14 and 6 days respectively. Ninety percent of the FNA treated biomass was consumed during the 14-day aerobic digestion compared to 41% achieved with the untreated biomass. During the 6-day aerobic digestion, 50% of the FNA-treated sludge was degraded. The results indicate that FNA treatment substantially increases sludge biodegradability.

Response of poly-phosphate accumulating organisms to free nitrous acid inhibition under anoxic and aerobic conditions.

The response of free nitrous acid (FNA)-adapted poly-phosphate accumulating organisms (PAOs) to FNA inhibition under aerobic and anoxic conditions was studied. Anoxic P-uptake was 1-6 times more sensitive to the inhibition compared to aerobic P-uptake. The aerobic nitrite reduction rate increased with FNA concentration, accompanied by an equivalent decrease in the oxygen uptake rate, suggesting under high FNA concentration conditions, electrons were channeled to nitrite reduction from oxygen reduction. In contrast, the nitrite reduction rate decreased with increased FNA concentration under anoxic conditions. Anaerobic metabolism of PAO under both anoxic and aerobic conditions was observed at high FNA concentrations. Growth of PAOs decreased sharply with FNA concentration and stopped completely at FNA concentration of 10 µg HNO(2)-N/L. This study, for the first time, investigated the function of nitrite/FNA in an aerobic denitrifying phosphate removal process by evaluating electron as well as energy balances, and provides explanation for FNA inhibition mechanisms.

The strong biocidal effect of free nitrous acid on anaerobic sewer biofilms.

Several recent studies showed that nitrite dosage to wastewater results in long-lasting reduction of the sulfate-reducing and methanogenic activities of anaerobic sewer biofilms. In this study, we revealed that the quick reduction in these activities is due to the biocidal effect of free nitrous acid (FNA), the protonated form of nitrite, on biofilm microorganisms. The microbial viability was assessed after sewer biofilms being exposed to wastewater containing nitrite at concentrations of 0-120 mg-N/L under pH levels of 5-7 for 6-24 h. The viable fraction of microorganisms was found to decrease substantially from approximately 80% prior to the treatment to 5-15% after 6-24 h treatment at FNA levels above 0.2 mg-N/L. The level of the biocidal effect has a much stronger correlation with the FNA concentration, which is well described by an exponential function, than with the nitrite concentration or with the pH level, suggesting that FNA is the actual biocidal agent. An increase of the treatment from 6 to 12 and 24 h resulted in only slight decreases in microbial viability. Physical disrupted biofilm was more susceptible to FNA in comparison with intact biofilms, indicating that the biocidal effect of FNA on biofilms was somewhat reduced by mass transfer limitations. The inability to achieve 2-log killing even in the case of disrupted biofilms suggests that some microorganisms may be more resistant to FNA than others. The recovery of biofilm activities in anaerobic reactors after being exposed to FNA at 0.18 and 0.36 mg-N/L, respectively, resembled the regrowth of residual sulfate-reducing bacteria and methanogens, further confirming the biocidal effects of FNA on microorganisms in biofilms.

Modeling the decay of ammonium oxidizing bacteria.

A bench-scale sequencing batch reactor was used to study factors affecting the endogenous decay of the ammonium oxidizing biomass (AOB) in different operating conditions. AOB decay was very sensitive to oxygen concentration, and increased up to 0.4 d(-1) for oxygen concentration of 7 mg O(2) L(-1). The decay in anaerobic conditions was shown to be very low (0.03 d(-1)) when compared to literature data. The effect of nitrite and nitrate on AOB decay was also studied. The correlation was quite weak suggesting that both nitrate and nitrite absence had little impact on decay which is contrary to what is typically assumed in some of the existing process models. A simple expression for the decay of AOB was proposed, calibrated and validated using the results of batch kinetic tests and of the continuous sequencing batch reactor monitoring.

Free nitrous acid (FNA) inhibition on denitrifying poly-phosphate accumulating organisms (DPAOs).

Free nitrous acid (FNA) has been identified to be a ubiquitous inhibitor of a wide range of microorganisms, including bacteria involved in wastewater treatment. The FNA-induced inhibition on the anoxic (nitrite as electron acceptor) metabolism of denitrifying poly-phosphate accumulating organisms (DPAOs) was investigated using sludge from a sequencing batch reactor performing carbon, nitrogen, and phosphorus removal from synthetic wastewater. We found that FNA had a much stronger inhibitory effect on phosphorus (P) uptake and glycogen production than on poly-beta-hydroxyalkanoate degradation and nitrite reduction. The intracellular adenosine triphosphate levels decreased sharply during the FNA incubation, and the decreasing rates were positively correlated with increasing FNA concentrations. The electron transport activity of DPAOs when exposed to FNA displayed a similar trend. Further, at FNA concentrations above 0.044 mg HNO(2)-N/L, the anaerobic metabolism of DPAOs was initiated despite of the presence of nitrite, as evidenced by the release of phosphorus and the consumption of glycogen. DPAO metabolism did not recover completely from FNA inhibition in the subsequent FNA-free environment. The recovery rate depended on the concentration of FNA applied in the previous anoxic period. These results suggest that the inhibitory effects are diverse and may be attributable to different mechanisms operating simultaneously.

[The Rdh54 protein role in regulation of DNA repair in yeast Saccharomyces cerevisiae].

In this work, we present the evidences of the involvement of Rdh54 in coordination of DNA repair by several pathways. Previously, we isolated rdh54-29 point mutation demonstrating unique properties different from the full deletion of RDH54 gene. Epistatic interaction between rdh54-29 and apn1delta mutations discloses the function of Rdh54p in the process of base excision repair. However, rdh54-29 mutant exhibits sensitivity to many DNA damaging agents including UV light, methylmethanesulphonate and nitrous acid. Such pleiotrophic effect of rdh54-29 mutation may indicate the role of Rdh54p in the regulation of different DNA repair systems. To check this hypothesis, we estimated the effect of rdh54-29 mutation on recombination and mutagenesis. The data confirm the involvement of Rdh54p in coordination of different DNA repair systems including mutagenic and recombinagenic pathways as well as nucleotide excision repair. Rdh54p presumably operates via chromatin remodulation at the site of damage rendering DNA accessible to the DNA repair enzymes.

The effect of free nitrous acid on the anabolic and catabolic processes of glycogen accumulating organisms.

Nitrite/Free Nitrous Acid (FNA) has previously been shown to inhibit aerobic and anoxic phosphate uptake by polyphosphate accumulating organisms (PAOs). The inhibitory effect of FNA on the aerobic metabolism of Glycogen Accumulating Organisms (GAOs) is investigated. A culture highly enriched (92+/-3%) in Candidatus Competibacter phosphatis (hereafter called Competibacter) was used. The experimental data strongly suggest that FNA likely directly inhibits the growth of Competibacter, with 50% inhibition occurring at 1.5 x 10(-3)mgN-HNO(2)/L (equivalent to approximately 6.3 mgN-NO(2)(-)/L at pH 7.0). The inhibition is well described by an exponential function. The organisms ceased to grow at an FNA concentration of 7.1 x 10(-3) mgN-HNO(2)/L. At this FNA level, glycogen production, another anabolic process performed by GAOs in parallel to growth, decreased by 40%, while the consumption of polyhydroxyalkanoates (PHAs), the intracellular carbon and energy sources for GAOs, decreased by approximately 50%. FNA likely inhibited either or both of the PHA oxidation and glycogen production processes, but to a much less extent in comparison to the inhibition on growth. The comparison of these results with those previously reported on PAOs suggest that FNA has much stronger inhibitory effects on the aerobic metabolism of PAOs than on GAOs, and may thus provide a competitive advantage to GAOs over PAOs in enhanced biological phosphorus removal (EBPR) systems.