Insight into recent advances in microalgae biogranulation in wastewater treatment.

Microalgae-based technology is widely utilized in wastewater treatment and resource recovery. However, the practical implementation of microalgae-based technology is hampered by the difficulty in separating microalgae from treated water due to the low density of microalgae. This review is designed to find the current status of the development and utilization of microalgae biogranulation technology for better and more cost-effective wastewater treatment. This review reveals that the current trend of research is geared toward developing microalgae-bacterial granules. Most previous works were focused on studying the effect of operating conditions to improve the efficiency of wastewater treatment using microalgae-bacterial granules. Limited studies have been directed toward optimizing operating conditions to induce the secretion of extracellular polymeric substances (EPSs), which promotes the development of denser microalgae granules with enhanced settling ability. Likewise, studies on the understanding of the EPS role and the interaction between microalgae cells in forming granules are scarce. Furthermore, the majority of current research has been on the cultivation of microalgae-bacteria granules, which limits their application only in wastewater treatment. Cultivation of microalgae granules without bacteria has greater potential because it does not require additional purification and can be used for border applications.

The most recent development in microalgae biogranulation research is highlighted.Factors affecting microalgae granule development are discussed for the first time.Duration to develop granules is a crucial aspect that needs further research.Cultivation of single-species microalgae for rapid harvesting needs more attention.

Self-flocculation of enriched mixed microalgae culture in a sequencing batch reactor.

Microalgae-based biodiesel has gained widespread interest as an alternative energy source. Low-cost microalgae harvesting technologies are important for economically feasible biodiesel production. This study investigated, for the first time, the impact of adaptation period and height to diameter (H/D) ratio of a reactor on the growth and self-flocculation of microalgae, without the addition of bacteria. Six reactors were grouped into three sets of experiments, and each reactor was operated for 30 days at similar operating conditions (volume exchange ratio = 25% and settling time = 30 min). In set 1, two 8-L reactors, H5a (H/D ratio: 5) and H8a (H/D ratio: 8), were operated under batch operation. In set 2, reactors H5b and H8b were operated as sequential batch reactors (SBRs) without an adaptation period. In set 3, the reactors H5c and H8c were operated as SBRs with an adaptation period. The findings showed a threefold improvement in biomass productivity for the higher H/D ratio (H8c) and a reduction in biomass loss for microalgae. The H8c reactor exhibited 95% settling efficiency within 5 days, in comparison to 30 days for the H5c reactor. This study demonstrated that a higher H/D ratio and the introduction of an adaptation period in SBR operation positively influences growth and self-flocculation of enriched mixed microalgae culture.

In-situ alkaline enhanced two-stage anaerobic digestion system for waste cooking oil and sewage sludge co-digestion.

Anaerobic digestion is a promising way for resource recovery from waste cooking oil (WCO) due to its high bio-methanation potential. In-situ mild alkaline (pH 8) enhanced two-stage continuous stirred tank reactors (ALK-2-CSTRs) were implemented to explore its efficiency in co-digesting WCO and sewage sludge with stepwise increase of WCO in the co-substrates. Results demonstrate that the ALK-2-CSTRs effectively promoted methane yield from the co-substrates via promoting hydrolysis, long chain fatty acids (LCFAs) degradation and protecting methanogens from exposure to high concentration of LCFAs directly. The maximum methane yield of the ALK-2-CSTRs is 39.2% higher than that of a single stage CSTR system at the optimal feed mixture of 45:55 (WCO:SS [VS]). The thermophilic operation applied to the stage-1 of the ALK-2-CSTRs failed to improve the methane yield when the methanogenic performance was stable; while upon WCO overloaded, the elevated temperature mitigated the deterioration of methanogenesis by stimulating the bioconversion of the toxic LCFAs, especially the unsaturated oleic acid. Microbial community analysis reveals the ALK-2-CSTRs stimulated the growth of lipolytic bacteria and hydrogenotrophic methanogens, which suggests the hydrogenotrophic methanogenic pathway was promoted. Cost evaluation demonstrates the economical superiority of the ALK-2-CSTR over the prevailing strategies developed for enhancing methane yield from the co-substrates.

Effect of storage conditions on maintaining anammox cell viability during starvation and recovery.

Anammox bacteria can easily undergo starvation due to fluctuations in feed flowrate and concentration in wastewater treatment plants. In this study, we analyzed the effects of different types of storage conditions (presence of ammonium (Ra), nitrite (Rn), hydrazine (Rh), and no substrate (Rc)) in aiding the viability of anammox bacteria during starvation and recovery. After starvation, the bacteria were subjected to a 15-week recovery period. Anammox bacteria showed better results during starvation and recovery in Rh as compared to other conditions. Decay rate values obtained after starvation in Ra, Rn, Rh, and Rc were 0.032/day, 0.042/day, 0.019/day, and 0.037/day, respectively. Meanwhile, µmax values obtained in Rh, Ra, Rn, and Rc on the 15th week of recovery were 0.092, 0.075, 0.011, and 0.067 d-1, respectively. This indicated that the availability of hydrazine helps to reduce the mortality rate of anammox bacteria during starvation and enhances the recovery of anammox process.

Effect of external hydrazine addition on anammox reactor start-up time.

Hydrazine is an intermediate product of the anaerobic ammonium oxidation (Anammox) process where both ammonium and nitrite in wastewater are converted to nitrogen gas by bacteria. In this study the effect of external hydrazine addition (5, 10, 15, and 20 mg/L) on the start-up period of the Anammox process was studied using sequencing batch reactors (SBRs). The SBR with an addition of 10 mg/L hydrazine took only 7 weeks to stabilize and achieve the maximum removal of ammonium and nitrite, whereas the SBR without the addition of hydrazine took 12 weeks. The amount of Heme C extracted from the biomass indicated that externally added hydrazine accelerated the growth of Anammox bacteria and reduced the release of nitrous oxide gas from the reactors.

Nitrite pre-treatment of dewatered sludge for microbial fuel cell application.

The effect of pre-treatment of dewatered sludge using different nitrite concentrations and pH for microbial fuel cell (MFC) application was investigated. The results show that the addition of nitrite was feasible to increase the solubilization rate of the sludge and may reduce mass transfer limitation at the anode. This helped the MFC to reach higher voltage and to generate more power. The higher free nitrous acid (FNA) concentration under the acidic condition helped to increase sludge solubilization. However, under an alkaline condition, during which the FNA concentration was relatively low, the solubilization of the sludge was higher. The highest voltage and power density produced was 390 mV and 153 mW/m2, respectively, with the addition of nitrite at 100 mg-N/L and pH 9. Furthermore, it was found that elevated levels of FNA could inhibit electrogenic bacteria thus reducing power generation.

The role of nitrite and free nitrous acid (FNA) in wastewater treatment plants.

Nitrite is known to accumulate in wastewater treatment plants (WWTPs) under certain environmental conditions. The protonated form of nitrite, free nitrous acid (FNA), has been found to cause severe inhibition to numerous bioprocesses at WWTPs. However, this inhibitory effect of FNA may possibly be gainfully exploited, such as repressing nitrite oxidizing bacteria (NOB) growth to achieve N removal via the nitrite shortcut. However, the inhibition threshold of FNA to repress NOB (∼0.02 mg HNO2-N/L) may also inhibit other bioprocesses. This paper reviews the inhibitory effects of FNA on nitrifiers, denitrifiers, anammox bacteria, phosphorus accumulating organisms (PAO), methanogens, and other microorganisms in populations used in WWTPs. The possible inhibition mechanisms of FNA on microorganisms are discussed and compared. It is concluded that a single inhibition mechanism is not sufficient to explain the negative impacts of FNA on microbial metabolisms and that multiple inhibitory effects can be generated from FNA. The review would suggest further research is necessary before the FNA inhibition mechanisms can be more effectively used to optimize WWTP bioprocesses. Perspectives on research directions, how the outcomes may be used to manipulate bioprocesses and the overall implications of FNA on WWTPs are also discussed.

Determination of growth rate and yield of nitrifying bacteria by measuring carbon dioxide uptake rate.

Nitrifier growth parameters--the maximum growth rate (microAmax) and yield (YA)--were estimated by measuring the rate of carbon dioxide uptake and additional rates of oxygen uptake and ammonia (or nitrite) use. Batch tests in a combined titrimetric and offgas analyzer with enriched Nitrobacter and Nitrosomonas cultures and an activated sludge sample were performed. The measured microAmax values for the Nitrobacter and Nitrosomonas cultures were 0.67 +/- 0.03 day(-1) and 0.54 +/- 0.09 day(-1), while the YA values were 0.072 +/- 0.01 g volatile suspended solids (VSS) x g nitrogen (N)(-1) and 0.14 +/- 0.02 gVSS x gN(-1), respectively. For the activated sludge sample, microAmax was observed to increase with pH (microAmax = 0.72 x 3.3(pH-7.1)) over the range 6.8 to 7.1. All microAmax and YA values determined by this method were similar to those previously reported. Compared with other microAmax and YA estimation methods, this method allows for unique microAmax and YA estimations for given conditions from a single experiment.

Kinetic characterisation of an enriched Nitrospira culture with comparison to Nitrobacter.

Nitrospira and Nitrobacter are nitrite-oxidising bacteria commonly identified in nitrogen removal wastewater treatment plants. Little is known about the growth parameters of Nitrospira or the effects of environmental conditions or inhibitory compounds on Nitrospira activity. These bacterial properties were investigated using an enriched Nitrospira culture and an enriched Nitrobacter culture or Nitrobacter literature values. Compared to Nitrobacter, Nitrospira was found to have a comparable optimal pH range (8.0-8.3); similar normalised activity-temperature relationship (0.44e(0.055(T-15))) for temperatures between 15 and 30 degrees C and a similar oxygen half-saturation constant, K(O) (0.54+/-0.14 mgL(-1)). The major differences identified were that Nitrospira had a lower nitrite half-saturation constant, K(S) (0.9+/-0.07 mgNO(2)-NL(-1)); lower inhibition threshold concentrations for free ammonia (between 0.04 and 0.08 mg NH(3)-NL(-1)) and free nitrous acid (less than 0.03 mg HNO(2)-NL(-1)) and a higher yield (0.15+/-0.04 g VSS g N(-1)). Therefore, Nitrospira is more likely to dominate nitrite oxidation under conditions with low ammonium and nitrite concentrations, which would provide an advantage to them due to their lower K(S) value while avoiding any free ammonia or free nitrous acid inhibition.

Effect of free ammonia on the respiration and growth processes of an enriched Nitrobacter culture.

The inhibitory effect of free ammonia (FA;NH(3)) on the metabolism of Nitrobacter is investigated using a method that allows decoupling energy generation from growth processes. A lab-scale sequencing batch reactor (SBR) was operated for the enrichment of Nitrobacter. Fluorescent in situ hybridization (FISH) analysis showed that 73% of the bacterial population in the reactor was Nitrobacter, while no Nitrospira was detected. Batch tests were carried out to measure the oxygen uptake rate (OUR) by the culture at various FA levels, in the presence (OUR with CO(2)) or absence (OUR without CO(2)) of inorganic carbon (CO(2), HCO(3)(-) and CO(3)(2-)). The FA inhibition on the respiration initiated at below 1mgNH(3)-NL(-1) in both cases. OUR without CO(2) gradually decreased by 12% when the FA concentration increased from 0 to approximately 4mgNH(3)-NL(-1) and remained at the same level till an FA level of 9mgNH(3)-NL(-1) (the highest FA concentration applied in this study). This indicates that FA has a limited inhibitory effect on the respiratory capability of Nitrobacter. Starting from a level that is 15% higher than OUR without CO(2) when no FA was present, OUR with CO(2)decreased more rapidly than OUR without CO(2) reaching the same level as OUR without CO(2) when FA was between 6-9mgNH(3)-NL(-1). This implies that in this range of FA the presence of inorganic carbon did not cause any increase in the respiration activity of Nitrobacter. The results suggest that, while still oxidizing nitrite at approximately 75% of the non-inhibited rate, Nitrobacter likely ceased to grow at an FA level of above 6mgNH(3)-NL(-1). While the real mechanisms remain to be identified, this study indicates that the FA inhibition on Nitrobacter is likely much more serious than suggested by previous studies where OUR with CO(2) (or the equivalent nitrite oxidation rate) was used as the sole measure of the inhibitory effects.

Effect of free ammonia and free nitrous acid concentration on the anabolic and catabolic processes of an enriched Nitrosomonas culture.

The effects of free ammonia (FA; NH(3)) and free nitrous acid (FNA; HNO(2)) concentrations on the metabolisms of an enriched ammonia oxidizing bacteria (AOB) culture were investigated using a method allowing the decoupling of growth and energy generation processes. A lab-scale sequencing batch reactor (SBR) was operated for the enrichment of an AOB culture. Fluorescent in-situ hybridization (FISH) analysis showed that 82% of the bacterial population in the SBR bound to the NEU probe specifically designed for Nitrosomonas europaea. Batch tests were carried out to measure the oxygen and ammonium consumption rates by the culture at various FA and FNA levels, in the presence or absence of inorganic carbon (CO(2), HCO(3) (-), and CO(3) (2-)). It was revealed that FA of up to 16.0 mgNH(3)-N . L(-1), which was the highest concentration used in this study, did not have any inhibitory effect on either the catabolic or anabolic processes of the Nitrosomonas culture. In contrast, FNA inhibited both the growth and energy production capabilities of the Nitrosomonas culture. The inhibition on growth initiated at approximately 0.10 mgHNO(2)-N . L(-1), and the data suggested that the biosynthesis was completely stopped at an FNA concentration of 0.40 mgHNO(2)-N . L(-1). The inhibition on energy generation initiated at a slightly lower level but the Nitrosomonas culture was still oxidizing ammonia at half of the maximum rate at an FNA concentration of 0.50-0.63 mgHNO(2)-N . L(-1). The affinity constant of the Nitrosomonas culture with respect to ammonia was determined to be 0.36 mgNH(3)-N . L(-1), independent of the presence or absence of inorganic carbon.

The inhibitory effects of free nitrous acid on the energy generation and growth processes of an enriched nitrobacter culture.

The inhibitory effects of nitrite (NO2-)/free nitrous acid (HNO2-FNA) on the metabolism of Nitrobacter were investigated using a method allowing the decoupling of the growth and energy generation processes. A lab-scale sequencing batch reactor was operated forthe enrichment of a Nitrobacter culture. Fluorescent in situ hybridization (FISH) analysis showed that 73% of the bacterial population was Nitrobacter. Batch tests were carried out to assess the oxygen and nitrite consumption rates of the enriched culture at low and high nitrite levels, in the presence or absence of inorganic carbon. It was observed that in the absence of CO2, the Nitrobacter culture was able to oxidize nitrite at a rate that is 76% of that in the presence of CO2, with an oxygen consumption rate that is 85% of that measured in the presence of CO2. This enabled the impacts of nitrite/FNA on the catabolic and anabolic processes of Nitrobacter to be assessed separately. FNA rather than nitrite was likely the actual inhibitor to the Nitrobacter metabolism. It was revealed that FNA of up to 0.05 mg HNO2-N x L(-1) (3.4 microM), which was the highest FNA concentration used in this study, did not have any inhibitory effect on the catabolic processes of Nitrobacter. However, FNA initiated its inhibition to the anabolic processes of Nitrobacterat approximately 0.011 mg HNO2-N x L(-1) (0.8 microM), and completely stopped biomass synthesis at a concentration of approximately 0.023 mg HNO2-N x L(-1) (1.6 microM). The inhibitory effect could be described by an empirical inhibitory model proposed in this paper, but the underlying mechanisms remain to be revealed.

Stoichiometric and kinetic characterisation of Nitrobacter in mixed culture by decoupling the growth and energy generation processes.

The growth, maintenance and lysis processes of Nitrobacter were characterised. A Nitrobacter culture was enriched in a sequencing batch reactor (SBR). Fluorescent in situ hybridisation showed that Nitrobacter constituted 73% of the bacterial population. Batch tests were carried out to measure the oxygen uptake rate and/or nitrite consumption rate when both nitrite and CO2 were in excess, and in the absence of either of these two substrates. The results obtained, along with the SBR performance data, allowed the determination of the maintenance coefficient and in situ cell lysis rate of Nitrobacter. Nitrobacter spends a significant amount of energy for maintenance, which varies considerably with the specific growth rate. At maximum growth, Nitrobacter consume nitrite at a rate of 0.042 mgN/mgCOD(biomass) . h for maintenance purposes, which increases more than threefold to 0.143 mgN/mgCOD(biomass) . h in the absence of growth. In the SBR, where Nitrobacter grew at 40% of its maximum growth rate, a maintenance coefficient of 0.113 mgN/mgCOD . h was found, resulting in 42% of the total amount of nitrite being consumed for maintenance. The above three maintenance coefficient values obtained at different growth rates appear to support the maintenance model proposed in Pirt (1982). The in situ lysis rate of Nitrobacter was determined to be 0.07/day under aerobic conditions at 22 degrees C and pH 7.3. Further, the maximum specific growth rate of Nitrobacter was estimated to be 0.02/h (0.48/day). The affinity constant of Nitrobacter with respect to nitrite was determined to be 1.50 mgNO2(-)-N/L, independent of the presence or absence of CO2.

Stoichiometric and kinetic characterisation of Nitrosomonas sp. in mixed culture by decoupling the growth and energy generation processes.

A novel method that relies on the decoupling of the energy production and biosynthesis processes was used to characterise the maintenance, cell lysis and growth processes of Nitrosomonas sp. A Nitrosomonas culture was enriched in a sequencing batch reactor (SBR) with ammonium as the sole energy source. Fluorescent in situ hybridization (FISH) showed that Nitrosomonas bound to the NEU probe constituted 82% of the bacterial population, while no other known ammonium or nitrite oxidizing bacteria were detected. Batch tests were carried out under conditions that both ammonium and CO2 were in excess, and in the absence of one of these two substrates. The oxygen uptake rate and nitrite production rate were measured during these batch tests. The results obtained from these batch tests, along with the SBR performance data, allowed the determination of the maintenance coefficient and the in situ cell lysis rate, as well as the maximum specific growth rate of the Nitrosomonas culture. It is shown that, during normal growth, the Nitrosomonas culture spends approximately 65% of the energy generated for maintenance. The maintenance coefficient was determined to be 0.14-0.16 mgN mgCOD(biomass)(-1)h(-1), and was shown to be independent of the specific growth rate. The in situ lysis rate and the maximum specific growth rate of the Nitrosomonas culture were determined to be 0.26 and 1.0 day(-1) (0.043 h(-1)), respectively, under aerobic conditions at 30 degrees C and pH 7.