Correlation of microbial community with salinity and nitrogen removal in an anammox-based denitrification system.

Anaerobic ammonium oxidation (anammox), a low-energy-consuming technology, can be used to remove nitrogen from industrial saline wastewater. However, high salinity inhibits anammox microbial activity. This study investigated the effect of salinity on nitrogen removal performance and microbial community structure. The experiment used an up-flow anammox reactor fed with synthetic wastewater with salinity increased from 0.5 to 2.5%. Results indicated that 80% nitrogen removal efficiency can be achieved at 2% salinity with a nitrogen loading rate of 2.0 kg-N/m3/d. Anammox performance significantly deteriorated at 2.5% salinity. High-throughput sequencing revealed that Planctomycetes (representative anammox bacteria) increased with salinity, replacing Proteobacteria (representative heterotrophic denitrifying bacteria) in the microbial community. qPCR analysis indicated that relative abundance of "Candidatus Kuenenia" within anammox bacteria increased from 3.96 to 83.41%, corresponding to salinity of 0.5-2.0%, and subsequently decreased to 63.27% at 2.5% salinity, correlating with nitrogen-removal performance. Thus, anammox has potential in nitrogen removal from wastewater with salinity up to 2%.

Anammox biomass carrying efficiency of polyethylene non-woven sheets as a carrier material.

To access the effects of the surface modification and fabric structure of polyethylene (PE) non-woven fabric sheets on retaining the attachment efficiency of anammox biomass, three different non-woven sheets were prepared and inserted in an anammox reactor. The hydrophobic surface modification with 10% KMnO4 and gelatin did not improve the attachment efficiency of the anammox biomass on the surface of the PE non-woven fibers. Densely packed PE-755 having the highest specific surface area to volume ratio (SA/V) (755) retained 221.4 mg biomass per unit sheet, whereas PE-181 having the lowest SA/V (181) retained only 66.4 mg biomass per unit. Accordingly, the volumetric anammox activity of non-woven sheet PE-755 was the highest among the three PE non-woven sheets because of the strong positive relationship between the specific anammox activity and biomass amount (R = 0.835, P < .01). The specific surface area to volume ratio (cm2 cm-3) as well as the bulk density should be considered as important parameters for the selection of non-woven biocarriers for anammox biomass.

Comparison of batch cultivation strategies for cost-effective biomass production of Micractinium inermum NLP-F014 using a blended wastewater medium.

Two competitive strategies, fed-batch and sequencing-batch cultivation, were compared in cost-effective biomass production of a high lipid microalgae, Micractinium inermum NLP-F014 using a blended wastewater medium. For fed-batch cultivations, additional nutrient was supplemented at day 2 (FB1) or consecutively added at day 2 and 4 (FB2). Through inoculum size test, 1.0g-DCWL-1 was selected for the sequencing-batch cultivation (SB) where about 65% of culture was replaced with fresh medium every 2days. Both fed-batch cultivations showed the maximum biomass productivity of 0.95g-DCWL-1d-1, while average biomass productivity in SB was slightly higher as 0.96±0.08g-DCWL-1d-1. Furthermore, remained concentrations of organics (426mg-CODL-1), total nitrogen (15.4mg-NL-1) and phosphorus (0.6mg-PL-1) in SB were much lower than those of fed-batch conditions. The results suggested that SB could be a promising strategy to cultivate M. inermum NLP-F014 with the blended wastewater medium.

Development of a protocol to optimize electric power consumption and life cycle environmental impacts for operation of wastewater treatment plant.

In wastewater treatment plants (WWTPs), the portion of operating costs related to electric power consumption is increasing. If the electric power consumption decreased, however, it would be difficult to comply with the effluent water quality requirements. A protocol was proposed to minimize the environmental impacts as well as to optimize the electric power consumption under the conditions needed to meet the effluent water quality standards in this study. This protocol was comprised of six phases of procedure and was tested using operating data from S-WWTP to prove its applicability. The 11 major operating variables were categorized into three groups using principal component analysis and K-mean cluster analysis. Life cycle assessment (LCA) was conducted for each group to deduce the optimal operating conditions for each operating state. Then, employing mathematical modeling, six improvement plans to reduce electric power consumption were deduced. The electric power consumptions for suggested plans were estimated using an artificial neural network. This was followed by a second round of LCA conducted on the plans. As a result, a set of optimized improvement plans were derived for each group that were able to optimize the electric power consumption and life cycle environmental impact, at the same time. Based on these test results, the WWTP operating management protocol presented in this study is deemed able to suggest optimal operating conditions under which power consumption can be optimized with minimal life cycle environmental impact, while allowing the plant to meet water quality requirements.

Mixotrophic cultivation of oleaginous Chlorella sp. KR-1 mediated by actual coal-fired flue gas for biodiesel production.

Flue gases mainly consist of CO2 that can be utilized to facilitate microalgal culture for bioenergy production. In the present study, to evaluate the feasibility of the utilization of flue gas from a coal-burning power plant, an indigenous and high-CO2-tolerant oleaginous microalga, Chlorella sp. KR-1, was cultivated under mixotrophic conditions, and the results were evaluated. When the culture was mediated by flue gas, highest biomass (0.8 g cells/L·d) and FAME (fatty acid methyl esters) productivity (121 mg/L·d) were achieved in the mixotrophic mode with 5 g/L glucose, 5 mM nitrate, and a flow rate of 0.2 vvm. By contrast, the photoautotrophic cultivation resulted in a lower biomass (0.45 g cells/L·d) and a lower FAME productivity (60.2 mg/L·d). In general, the fatty acid profiles of Chlorella sp. KR-1 revealed meaningful contents (>40 % of saturated and mono-unsaturated fatty acids) under the mixotrophic condition, which enables the obtainment of a better quality of biodiesel than is possible under the autotrophic condition. Conclusively then, it was established that a microalgal culture mediated by flue gas can be improved by adoption of mixotrophic cultivation systems.

Evaluation of thermal, ultrasonic and alkali pretreatments on mixed-microalgal biomass to enhance anaerobic methane production.

Anaerobic digestion was regarded as one of the ways to recover energy from mixed-microalgae biomass in this study. After applying thermal-, ultrasonic-, and alkali-pretreatments to raw microalgae biomass to promote the digestion efficiency, a biochemical methane potential was investigated to evaluate the effectiveness of the pre-treatments for the purpose. As the pretreatment intensity increased, the solubilization of the mixed microalgae increased. However, the increased solubilization was not followed proportionally by the increased methane production. The highest methane productivity was achieved by the thermal-pretreatment at 120 °C (405 mL CH4/g-VS), which was 1.2 times higher than that of the non-pretreatment condition (336 mL CH4/g-VS). The net energy analysis revealed that only the pretreatment adjusted to pH 9 yielded a slightly higher energy gains (12.8 kJ/g-VS) than that of non-pretreatment condition (11.9 kJ/g-VS). These findings recommend direct supply of microalgae biomass for anaerobic digestion.

Microalgae cultivation for bioenergy production using wastewaters from a municipal WWTP as nutritional sources.

In order to reduce input cost for microalgal cultivation, we investigated the feasibility of wastewater taken from a municipal WWTP in Busan, Korea as wastewater nutrients. The wastewaters used in this study were the effluent from a primary settling tank (PS), the effluent from an anaerobic digestion tank (AD), the conflux of wastewaters rejected from sludge-concentrate tanks and dewatering facilities (CR), and two combined wastewaters of AD:PS (10:90, v/v) and AD:CR (10:90, v/v). Chlorella sp. ADE5, which was isolated from the AD, was selected for the feasibility test. The highest biomass production (3.01 g-dry cell weight per liter) of the isolate was obtained with the combined wastewater ADCR, and it was 1.72 times higher than that with BG 11 medium. Interestingly, the cells cultivated with wastewater containing PS wastewater were easily separated from the culture and improved lipid content, especially oleic acid content, in their cells.

Variations of electron flux and microbial community in air-cathode microbial fuel cells fed with different substrates.

Microbial fuel cells (MFCs) can convert chemical energy to electricity using microbes as catalysts and a variety of organic wastewaters as substrates. However, electron loss occurs when fermentable substrates are used because fermentation bacteria and methanogens are involved in electron flow from the substrates to electricity. In this study, MFCs using glucose (G-MFC), propionate (P-MFC), butyrate (B-MFC), acetate (A-MFC), and a mix (M-MFC, glucose:propionate:butyrate:acetate = 1:1:1:1) were operated in batch mode. The metabolites and microbial communities were analyzed. The current was the largest electron sink in M-, G-, B-, and A-MFCs; the initial chemical oxygen demands (COD(ini)) involved in current production were 60.1% for M-MFC, 52.7% for G-MFC, 56.1% for B-MFC, and 68.3% for A-MFC. Most of the glucose was converted to propionate (40.6% of COD(ini)) and acetate (21.4% of COD(ini)) through lactate (80.3% of COD(ini)) and butyrate (6.1% of COD(ini)). However, an unknown source (62.0% of COD(ini)) and the current (34.5% of COD(ini)) were the largest and second-largest electron sinks in P-MFC. Methane gas was only detected at levels of more than 10% in G- and M-MFCs, meaning that electrochemically active bacteria (EAB) could out-compete acetoclastic methanogens. The microbial communities were different for fermentable and non-fermentable substrate-fed MFCs. Probably, bacteria related to Lactococcus spp. found in G-MFCs with fermentable substrates would be involved in both fermentation and electricity generation. Acinetobacter-like species, and Rhodobacter-like species detected in all the MFCs would be involved in oxidation of organic compounds and electricity generation.

Electricity generation and microbial community in a submerged-exchangeable microbial fuel cell system for low-strength domestic wastewater treatment.

A submerged type microbial fuel cell (MFC) system, which consisted of six readily exchangeable air-cathode MFCs, was evaluated for continuous treatment of low-strength domestic wastewater. When supplied with synthetic wastewater (COD 100 mg/L), the system showed increasing maximum power densities from 191 to 754 mW/m2 as COD loading rates increased (0.20-0.40 kg/m3/day). COD removal efficiencies decreased with increased COD loading rates but the effluent COD concentrations met the relevant effluent quality standard (CODMn 20 mg/L) at all conditions. The system was then operated with domestic wastewater (c.a. 100 mg COD/L) at 0.32 and 0.43 kg/m3/day. The system showed much lower power densities (116-149 mW/m2) at both loading rates, compared to synthetic wastewater. Anodic microbial communities were completely different when the wastewater type was changed. These results suggest that the newly developed MFC system could be applied to treat low-strength domestic wastewater without requiring any additional organic removal stage.

Comparison of exoelectrogenic bacteria detected using two different methods: U-tube microbial fuel cell and plating method.

In a microbial fuel cell (MFC), exoelectrogens, which transfer electrons to the electrode, have been regarded as a key factor for electricity generation. In this study, U-tube MFC and plating methods were used to isolate exoelectrogens from the anode of an MFC. Disparate microorganisms were identified depending on isolation methods, despite the use of an identical source. Denaturing gel gradient electrophoresis (DGGE) analysis showed that certain microorganisms became dominant in the U-tube MFC. The predominant bacterium was similar to Ochrobactrum sp., belonging to the Alphaproteobacteria, which was shown to be able to function as an exoelectrogen in a previous study. Three isolates, one affiliated with Bacillus sp. and two with Paenibacillus sp., were identified using the plating method, which belonged to the Gram-positive bacteria, the Firmicutes. The U-tube MFCs were inoculated with the three isolates using the plating method, operated in the batch mode and the current was monitored. All of the U-tube MFCs inoculated with each isolate after isolation from plates produced lower current (peak current density: 3.6-16.3 mA/m(2)) than those in U-tube MFCs with mixed culture (48.3-62.6 mA/m(2)). Although the isolates produced low currents, various bacterial groups were found to be involved in current production.

Effects of carbon and nitrogen sources on fatty acid contents and composition in the green microalga, Chlorella sp. 227.

In order to investigate and generalize the effects of carbon and nitrogen sources on the growth of and lipid production in Chlorella sp. 227, several nutritional combinations consisting of different carbon and nitrogen sources and concentrations were given to the media for cultivation of Chlorella sp. 227, respectively. The growth rate and lipid content were affected largely by concentration rather than by sources. The maximum specific growth was negatively affected by low concentrations of carbon and nitrogen. There is a maximum allowable inorganic carbon concentration (less than 500~1,000 mM bicarbonate) in autotrophic culture, but the maximum lipid content per gram dry cell weight (g DCW) was little affected by the concentration of inorganic carbon within the concentration. The lipid content per g DCW was increased when the microalga was cultured with the addition of glucose and bicarbonate (mixotrophic) at a fixed nitrogen concentration and with the lowest nitrogen concentration (0.2 mM), relatively. Considering that lipid contents per g DCW increased in those conditions, it suggests that a high ratio of carbon to nitrogen in culture media promotes lipid accumulation in the cells. Interestingly, a significant increase of the oleic acid amount to total fatty acids was observed in those conditions. These results showed the possibility to induce lipid production of high quality and content per g DCW by modifying the cultivation conditions.

Microbial community structure and dynamics in a mixotrophic nitrogen removal process using recycled spent caustic under different loading conditions.

A laboratory-scale Bardenpho process was established to investigate the proper nitrogen loading rate (NLR) when modified spent caustic (MSC) is applied as electron donor and alkalinity source for denitrification. MSC injection induced autotrophic nitrogen removal with sulfur as electron donor and heterotrophic denitrification. The nitrogen removal rate (NRR) did not increase proportionally to NLR. Based on the total nitrogen concentration in the effluent observed in the trials with MSC, the NLR in the influent should not exceed 0.15 kg N/m(3)d in order to satisfy water quality regulations. Microbial communities in the anoxic reactors were characterized by pyrosequencing of 16S rRNA gene sequences amplified by the polymerase chain reaction of DNA extracted from sludge samples. Microbial diversity was lower as MSC dosage was increased, and the injection of MSC caused an increase in SOB belonging to the genus Thiobacillus which is responsible for denitrification using sulfur.

Reuse of effluent water from a municipal wastewater treatment plant in microalgae cultivation for biofuel production.

This study assessed the usability of effluent water discharged from a secondary municipal wastewater treatment plant for mass cultivation of microalgae for biofuel production. It was observed that bacteria and protozoa in the effluent water exerted a negative impact on the growth of Chlorella sp. 227. To reduce the effect, filtration or UV-radiation were applied on the effluent water as pre-treatment methods. Of all the pretreatment options tested, the filtration (by 0.2 µm) resulted in the highest biomass and lipid productivity. To be comparable with the growth in the autoclaved effluent water, the filtration with a proper pore size filter (less than 0.45 µm) or UV-B radiation of a proper dose (over 1620 mJ cm(-2)) are proposed. These findings led us to conclude that the utilization can be realized only when bacteria and other microorganisms are greatly reduced or eliminated from the effluent prior to its use.

Development of a simultaneous partial nitrification and anaerobic ammonia oxidation process in a single reactor.

Up-flow oxygen-controlled biofilm reactors equipped with a non-woven fabric support were used as a single reactor system for autotrophic nitrogen removal based on a combined partial nitrification and anaerobic ammonium oxidation (anammox) reaction. The up-flow biofilm reactors were initiated as either a partial nitrifying reactor or an anammox reactor, respectively, and simultaneous partial nitrification and anammox was established by careful control of the aeration rate. The combined partial nitrification and anammox reaction was successfully developed in both biofilm reactors without additional biomass inoculation. The reactor initiated as the anammox reactor gave a slightly higher and more stable mean nitrogen removal rate of 0.35 (±0.19) kg-N m(-3) d(-1) than the reactor initiated as the partial nitrifying reactor (0.23 (±0.16) kg-N m(-3) d(-1)). FISH analysis revealed that the biofilm in the reactor started as the anammox reactor were composed of anammox bacteria located in inner anoxic layers that were surrounded by surface aerobic AOB layers, whereas AOB and anammox bacteria were mixed without a distinguishable niche in the biofilm in the reactor started as the partial nitrifying reactor. However, it was difficult to efficiently maintain the stable partial nitrification owing to inefficient aeration in the reactor, which is a key to development of the combined partial nitrification and anammox reaction in a single biofilm reactor.

Comparison of nitrogen removal and microbial distribution in wastewater treatment process under different electron donor conditions.

The applicability of modified spent caustic (MSC) as an electron donor for denitrification was evaluated in a lab-scale reactor for the Bardenpho process under various electron donor conditions: (A) no electron donor, (B) methanol, (C) thiosulfate and (D) MSC conditions. TN removal efficiency varied in each condition, 23.1%, 87.8%, 83.7% and 71.7%, respectively. The distribution ratio of nitrifying bacteria and DGGE profile including sulfur-reducing or oxidizing bacteria also varied depending on the conditions. These results indicated that the MSC would be used as an efficient electron donor for denitrification by autotrophic denitrifier in wastewater treatment process.

Nitrogen removal performance and microbial community analysis of an anaerobic up-flow granular bed anammox reactor.

We investigated nitrogen removal performance and responsible microbial community in an anaerobic up-flow granular bed anammox reactor. The anammox reactor was operated more than 1 year. Biomass in the reactor formed granules after about 2 months of operation, and a sufficient amount of the granules was retained in the reactor with a metallic net to avoid biomass washout during the entire operation. The average diameter of the granules was 3.6mm at day 310. After 8 months of operation, stable nitrogen removal (60%) was achieved at an average total inorganic nitrogen removal rate of 14 kg-N m(-3)d(-1). The phylogenetic analysis and fluorescence in situ hybridization results revealed that the anammox granules consisted of mono species of anammox bacteria, "Candidatus Brocadia-like species", affiliated with "Candidatus Brocadia anammoxidans" with 16S rRNA gene sequence similarity of 95.7%. The relative abundance of the anammox bacteria in the granules was more than 80% of the total bacteria stained with 4',6-diamidino-2-phenylindole. The anammox bacteria were present throughout the granules whereas the other bacterial groups, Chloroflexi-like filamentous bacteria and betaproteobacterial ammonia-oxidizing bacteria, were mainly present on the surface of the anammox granules and around the anammox bacterial clusters. The in situ anammox activity was detected mainly from near the surface of granules to the upper 800 microm of the granules with microsensors. The granular anammox biomass tolerated higher concentrations of nitrite (400 mg-NL(-1)) than did the homogenized biomass (200 mg-NL(-1)) probably due to substrate diffusion limitation.