Enrichments of methanotrophic–heterotrophic cultures with high poly-β-hydroxybutyrate (PHB) accumulation capacities

Methanotrophic–heterotrophic communities were selectively enriched from sewage sludge to obtain a mixed culture with high levels of poly-β-hydroxybutyrate(PHB)accumulation capacity from methane.Methane was used as the carbon source,N_2as sole nitrogen source,and oxygen and Cu content were varied.Copper proved essential for PHB synthesis.All cultures enriched with Cu could accumulate high content of PHB(43.2%–45.9%),while only small amounts of PHB were accumulated by cultures enriched without Cu(11.9%–17.5%).Batch assays revealed that communities grown with Cu and a higher O_2content synthesized more PHB,which had a wider optimal CH_4:O_2range and produced a high PHB content(48.7%)even though in the presence of N_2.In all methanotrophic–heterotrophic communities,both methanotrophic and heterotrophic populations showed the ability to accumulate PHB.Although methane was added as the sole carbon source,heterotrophs dominated with abundances between 77.2%and 85.6%.All methanotrophs detected belonged to type II genera,which formed stable communities with heterotrophs of different PHB production capacities.     

Screening of flocculant-producing microorganisms and flocculating activity

A strain saccharomycete STSM-I with high flocculanting activity was isolated from activated sludge with conventional methods.The high production rate and the low cost STSM-I medium was obtained by selecting different kinds of media, carbon source, nitrogen source and inorganic salt ion. The best flocculant-producing conditions were found by changing medium initial pH, culture temperature and ventilation flow. The best flocculating effect was obtained by changing positive ion types, density and concentration of flocculant.     

Denitrification and biofilm growth in a pilot-scale biofilter packed with suspended carriers for biological nitrogen removal from secondary effluent

Tertiary denitrification is an effective method for nitrogen removal from wastewater. A pilot-scale biofilter packed with suspended carriers was operated for tertiary denitrification with ethanol as the organic carbon source. Long-term performance, biokinetics of denitrification and biofilm growth were evaluated under filtration velocities of 6, 10 and 14 m/hr. The pilot-scale biofilter removed nitrate from the secondary effluent effectively, and the nitrate nitrogen(NO_3-N) removal percentage was 82%, 78% and 55% at the filtration velocities of 6, 10 and 14 m/hr, respectively. At the filtration velocities of 6 and 10 m/hr, the nitrate removal loading rate increased with increasing influent nitrate loading rates, while at the filtration velocity of 14 m/hr, the removal loading rate and the influent loading rate were uncorrelated.During denitrification, the ratio of consumed chemical oxygen demand to removed NO_3-N was 3.99–4.52 mg/mg. Under the filtration velocities of 6, 10 and 14 m/hr, the maximum denitrification rate was 3.12, 4.86 and 4.42 g N/(m~2·day), the half-saturation constant was 2.61, 1.05 and 1.17 mg/L, and the half-order coefficient was 0.22, 0.32 and 0.24(mg/L)1/2/min,respectively. The biofilm biomass increased with increasing filtration velocity and was 2845,5124 and 7324 mg VSS/m~2 at filtration velocities of 6, 10 and 14 m/hr, respectively. The highest biofilm density was 44 mg/cm~3 at the filtration velocity of 14 m/hr. Due to the low influent loading rate, biofilm biomass and thickness were lowest at the filtration velocity of 6 m/hr.     

Characterization of the e ective cellulose degrading strain CTL-6

An e cient cellulose degrading bacteria exists in the thermophilic wheat straw-degrading community, WDC2. However, this strain cannot be isolated and cultured using conventional separation techniques under strict anaerobic conditions. We successfully isolated a strain of e ective cellulose degrading bacteria CTL-6 using a wash, heat shock, and solid-liquid alternating process. Analysis of its properties revealed that, although the community containing the strain CTL-6 grew under aerobic conditions, the purified strain CTL-6 only grew under anaerobic culture conditions. The strain CTL-6 had a striking capability of degrading cellulose (80.9% weight loss after 9 days of culture). The highest e ciency value of the endocellulase (CMCase activity) was 0.404 mol/(min mL), cellulose degradation e ciency by CTL-6 was remarkably high at 50–65°C with the highest degradation e ciency observed at 60°C. The 16S rRNA gene sequence analysis indicated that the closest relative to strain CTL-6 belonged to the genus Clostridium thermocellum. Strain CTL-6 was capable of utilizing cellulose, cellobiose, and glucose. Strain CTL-6 also grew with Sorbitol as the sole carbon source, whereas C. thermocellum is unable to do so.     

Bulking sludge for PHA production:Energy saving and comparative storage capacity with well-settled sludge

Two acetate-fed sequencing batch reactors (SBR) were operated under an aerobic dynamic feeding (ADF) model (SBR #2) and with anaerobic phase before aerobic phase (SBR #1) to select mixed cultures with a high polyhydroxyalkanoates (PHA) storage response. Although kinetic selection based on storage response should bring about a predominance of floc-formers, a bulking sludge with storage response comparable to well-settled sludge was steadily established. An anaerobic phase was introduced before the aerobic phase in the ADF model to improve the sludge settleability (SBR #1), however, due to the consequent increased feast/famine ratio, the performance of SBR #1, in terms of both the maximum PHB (polyhydroxybutyrate) cell content and ΔPHB, was lower than that of SBR #2. SBR #2 gradually reached a steady state while SBR #1 failed suddenly after 50 days of operation. The maximum specific substrate uptake rate and storage rate for the selected bulking sludge were 0.4 Cmol Ac/(Cmol X.hr) and 0.18 Cmol Ac/(Cmol PHB.hr), respectively, resulting a yield of 0.45 Cmol PHB/(Cmol Ac) in SBR #2 in the culture enrichment phase. A maximum PHB content of 53% of total suspended solids and PHB storage rate of 1.36 Cmol Ac/(Cmol PHB.hr) was achieved at 10.2 hr in batch accumulation tests under nitrogen starvation. The results indicated that it was feasible to utilize filamentous bacteria to accumulate PHA with a rate comparable to well-settled sludge. Furthermore, the lower dissolved oxygen demand of filamentous bacteria would save energy required for aeration in the culture enrichment stage.     

Effects of bacteria on nitrogen and phosphorus release from river sediment

To better understand the mechanisms of eutrophication,we addressed the microbial processes that influence many key aspects of water-sediment systems.In this study,a large column experiment was conducted for 30 d.Along the column,solution samples were collected at different locations at different time.The samples were analyzed for physical,chemical,and biological properties of the sediment and oveflying water.The results showed that the amount of nitrogen transforming bacteria was higher than than that of phosphorous bacteria.The amount of nitrogen transforming bacteria was in the orderammonitier＞denitrifying bacteria＞nitrobacteria and nitrosomonas.Principal component analysis indicared that the three main factors accounted for more than 90%overall contributions for bacterium growth,which represented nutrition,organics and oxygen,and pH and redox potential(Eh)of the environment.Corresponding to the bacteria,the concentrations of nitrogen in the system was in the orderammonia(NH4 -N)＞nitrate(NO3--N)＞nitrite(NO2--N),The fluxes of N and P clearly showed a temporal release and adsorption processes in the water-sediment system.The large magnitude of N fluxes suggested that N might act as an important contamination source for the water quality.However,P exchange between the sediment and overlying water was less intensive during the experiment.     

Screening on oil-decomposing microorganisms and application in organic waste treatment machine

As an oil-decomposable mixture of two bacteria strains ( Bacillus sp. and Pseudomonas sp. ), Y3 was isolated after 50 d domestication under the condition that oil was used as the limited carbon source. The decomposing rate by Y3 was higher than that by each separate individual strain, indicating a synergistic effect of the two bacteria. Under the conditions that T = 25-40℃, pH = 6--8, HRT (Hydraulic retention time) = 36 h and the oil concentration at 0.1%,Y3 yielded the highest decomposing rate of 95.7 %. Y3 was also applied in an organic waste treatment machine and a certain rate of activated bacteria was put into the stuffing. A series of tests including humidity, pH, temperature, C/N rate and oil percentage of the stuffing were carried out to check the efficacy of oil-decomposition. Results showed that the oil content of the stuffing with inoculums was only half of that of the control. Furthermore, the bacteria were also beneficial to maintain the stability of the machine operating. Therefore, the bacteria mixture as well as the machines in this study could be very useful for waste treatment.     

Cd2+ removal from wastewater by sulfate reducing bacteria with an anaerobic fluidized bed reactor

A process of treatment for containing Cd²⁺ wastewater by sulfate reducing bacteria with upflow anaerobic fluidized bed reactor has been studied. When the concentration of COD and Cd²⁺ in the influent were 270 5mg/L and 100mg/L respectively and hydraulic retention time was 4 hours, the removal rate of COD and Cd²⁺ were higher than 73 8% and 99 8% respectively. The reactor can treat as high as 1000mg/L of concentration of Cd²⁺ . The highest removal velocity rate of Cd²⁺ reached 2999 1mg/(L·d). And the possible relationship between sulfate reducing bacteria and methanogenic bacteria was discussed.     

Nitrification characteristics of nitrobacteria immobilized in waterborne polyurethane in wastewater of corn-based ethanol fuel production

A technology to achieve stable and high ammonia nitrogen removal rates for corn distillery wastewater (ethanol fuel production) treatment has been designed. The characteristics of nitrifying bacteria entrapped in a waterborne polyurethane (WPU) gel carrier were evaluated after acclimation. In the acclimation period, nitrification rates of WPU-immobilized nitrobacteria were monitored and polymerase chain reaction (PCR) was also carried out to investigate the change in ammonium-oxidizing bacteria. The results showed that the pellet nitrification rates increased from 21 to 228 mg-N/(L-pellet. hr) and the quantity of the ammonia oxidation bacteria increased substantially during the acclimation. A continuous ammonia removal experiment with the anaerobic pond effluent of a distillery wastewater system was conducted with immobilized nitrifying bacteria for 30 days using an 80 L airlift reactor with pellets at a fill ratio of 15% (V/V). Under the conditions of 75 mg/L influent ammonia, hydraulic retention time (HRT) of 3.7--5.6 hr, and dissolved oxygen (DO) of 4 mg/L, the effluent ammonia concentration was lower than 10 mg/L and the ammonia removal efficiency was 90%. While the highest ammonia removal rate, 162 mg-N/(L-pellet.hr), was observed when the HRT was 1.3 hr.     

Effect of bio-column composed of aged refuse on methane abatement – A novel configuration of biological oxidation in refuse landfill

An experimental bio-column composed of aged refuse was installed around the exhaust pipe as a new way to mitigate methane in refuse landfill.One of the objectives of this work was to assess the effect of aged refuse thickness in bio-column on reducing CH 4 emissions.Over the study period,methane oxidation was observed at various thicknesses,5 cm (small size),10 cm (middle size) and 15 cm (large size),representing one to three times of pipeline diameters.The middle and large size both showed over 90% methane conversion,and the highest methane conversion rate of above 95% occurred in the middle-size column cell.Michaelis-Menten equation addressed the methanotrophs diffusion in different layers of the bio-columns.Maximum methanotrophic activity (V max) measured at the three thicknesses ranged from 6.4 × 10 3 to 15.6 × 10 3 units,and the half-saturation value (K M) ranged from 0.85% to 1.67%.Both the highest V max and K M were observed at the middle-size of the bio-column,as well as the largest methanotrophs population,suggesting a significant efficiency of methane mitigation happened in the optimum zone with greatest affinity and methanotrophic bacteria activities.Therefore,bio-column is a potential style for methane abatement in landfill,and the aged refuse both naturally formed and artificially placed in the column plays a critical role in CH 4 emission.     

Performance and microbial response during the fast reactivation of Anammox system by hydrodynamic stress control

Anaerobic ammonium oxidation （Anammox） has become a promising method for biological nitrogen removal. However, this biotechnology application is always limited due to the low growth rate and biomass yield of Anammox bacteria. This study investigated the process of fast reactivation of an Anammox consortium idled for 2 years uia hydrodynamic stress control. The results showed that the Anammox system was efficiently and quickly reactivated by shortening of the hydraulic retention time （I-IRT） of the reactor from 12 to 6 hr within 68 days of operation. Moreover, at a 4-hr HRT with an influent total nitrogen loading rate of 1.2 kg N/（m3.day）, the reactor maintained high biological performance with an ammonium removal loading rate of 0.52 kg N/（m3.day） and a nitrite removal rate of 0.59 kg N/（m3.day）. In the reactivated Anammox reaction, the stoichiometric coefficients of NH4-N to NOE-N and NH4-N to NO4-N were 1：1.04± 0.08 and 1：0.31 ± 0.03, respectively. The specific Anammox activity and hydrazine oxidoreductase activity, both of which represent the degree of Anammox bacteria present, increased as the hydrodynamic stress increased and were maximally （125.38 ± 3.01 mg N/（g VSS.day） and 339.42 ± 6.83 μmol/（min.g VSS）, respectively） at 4-hr HRT. Microbial response analysis showed that the dominant microbial community was obviously shifted and the dominance of Anammox bacteria was enhanced durinR the hydrodynamic selection.     

Realizing stable operation of anaerobic ammonia oxidation at low temperatures treating low strength synthetic wastewater

The low activity of Anammox bacteria at low temperatures and competition from nitrite oxidation bacteria(NOB) when treating low strength wastewater have been major bottlenecks in implementing Anammox in mainstream wastewater treatment. By intermittent high strength feeding(IHSF) and stepwise temperature reduction, stable operation of a granular Anammox reactor was realized at low temperatures(down to 15°C) for 28 days when treating low strength synthetic wastewater. The nitrogen loading rate reached 1.23–1.34 kgN/m~3/day,and the total nitrogen removal rate reached 0.71–0.98 kgN/m~3/day. The IHSF enriched the Anammox sludge in high strength cycles and compensated for sludge loss in low strength cycles, and the high concentration of ammonium in high strength cycles inhibited NOB. The 16 SrRNA gene sequencing results revealed that Candidatus Kuenenia was predominant in the reactor at low temperatures.     

Characteristics of aerobic granules grown on glucose a sequential batch shaking reactor

Aerobic heterotrophic granular sludge was cultivated in a sequencing batch shaking reactor(SBSR) in which a synthetic wastewater containing glucose as carbon source was fed. The characteristics of the aerobic granules were investigated. Compared with the conventional activated sludge flocs, the aerobic granules exhibit excellent physical characteristics in terms of settleability, size, shape, biomass density, and physical strength.Scanning electron micrographs revealed that in mature granules little filamentous bacteria could be found, rod-shaded and coccoid bacteria were the dominant microorqanisms.     

Effects of C/N ratio on nitrate removal and floc morphology of autohydrogenotrophic bacteria in a nitrate-containing wastewater treatment process

The effects of C/N ratio of a nitrate-containing wastewater on nitrate removal performed by autohydrogenotrophic bacteria as well as on the morphological parameters of floc such as floc morphology, floc number distribution, mean particle size(MPS), aspect ratio and transparency were examined in this study. The results showed that the nitrate reduction rate increased with increasing C/N ratio from 0.5 to 10 and that the nitrogen removal of up to 95% was found at the C/N ratios of higher than 5(between 0.5–10). Besides, high C/N ratio values reflected a corresponding high nitrite accumulation after 12-hr operation, and a fast decreasing rate of nitrite in the rest of operational time. The final p H values increased with the C/N ratio increasing from 0.5 to 2.5, but decreased with the C/N ratio increasing from2.5 to 10. There were no significant changes in floc morphology with the MPSs ranging from35 to 40 μm. Small and medium-sized flocs were dominant in the sludge suspension, and the number of flocs increased with the increasing C/N ratios. Furthermore, the highest apparent frequency of 10% was observed at aspect ratios of 0.5 and 0.6, while the transparency of flocs changed from 0.1 to 0.7.     

Experimental study using the dilution incubation method to assess water biostability

Water biostability is of particular concern to water supply as a major limiting factor for heterotrophic bacterial growth in water distribution systems. This study focused on bacterial growth dynamics in the series dilution of water samples with TOC（total organic carbon） values determined beforehand. The results showed that the specific growth rate of Pseudomonas fluorescens P17 varied dramatically and irregularly with TOC value when TOC concentrations were low enough during the initial periods of incubation under given conditions. According to this relationship between bacterial growth rate and TOC, a dilution incubation method was designed for the study of water biostability. With the method under a given condition, a turning-point TOC value was found at a relatively fixed point in the curve between bacterial growth rate and TOC of water sample, and the variation of growth rate had different characteristics below the turning-point TOC value relative to that over this value. A turning-point TOC value similarly existed in all experiments not only with tap water, but also with acetate and mixed solutions. And in the dilution incubation method study, the affections were analyzed by condition factors such as inoculum amount,incubation time and nature of the organic carbon source. In very low organic carbon water environments, the variation characteristics of bacterial growth rate will be useful to further understand the meaning of water biostability.     

Water quality characteristics along the course of the Huangpu River （China）

Huangpu River is about 114.5 km from upriver Dianfeng to downriver Wusong,near the estuary of the Yangtze River.It plays a key role in supplying water for production,life,shipment and irrigation.With the industrial development,the pollution of the Huangpu River has become serious recently.The biological oxygen demand (BOD),total nitrogen (TN),total phosphorus (TP),oil,phenol and suspended solids (SS) were lower in the upstream sites than in the downstream sites,indicating pollutants being input along its course. Water quality was the worst in the Yangpu site,near the center of Shanghai City.Dissolved oxygen (DO) content was less than 2 mg/L in the site of Yangpu in July.Among relations between thirteen characteristics,relations between BOD,DO,TN,TP,NH_4~ -N, NO_3~--N and the count of total bacteria or Escherichia coli were significant and interdependent.Inner relationships between these main characteristics in the Huangpu River were studied.High nutrient concentration led to growth of microorganisms,including E.coli. Degradation of organic matters and respiration of bacteria made oxygen concentration decreased in the water body,and DO was a key factor for nitrification-denitrification process of nitrogen.In the Yangpu site,DO was decreased to less than 3.0 mg/L with BOD higher than 7.5 mg/L in May and July.Low DO concentration will decrease nitrification rate.Nitrification need at higher DO value than other organic substrate oxidation.Consequently,river water contains low NO_3~--N values with high amounts of TN and NH_4~ -N there.This will block the self-purification of surface water,by decreasing the rate of nitrification-denitrification transformation process in the water body.     

Methanotrophic community structure of aged refuse and its capability for methane bio-oxidation

Aged refuse from waste landfills closed for eight years was examined and found to contain rich methanotrophs capable of biooxidation for methane. Specially, community structure and methane oxidation capability of methanotrophs in the aged refuse were studied. The amount of methanotrophs ranged 61.97 103–632.91 103 cells/g (in dry basis) in aged refuse from Shanghai Laogang Landfill. Type I and II methanotrophs were found in the aged refuse in the presence of sterilized sewage sludge and only Type I methanotrophs were detected in the presence of nitrate minimal salt medium (NMS). The clone sequences of the pmoA gene obtained from the aged refuse were similar to the pmoA gene of Methylobacter, Methylocaldum, and Methylocystis, and two clones were distinct with known genera of Type I methanotrophs according to phylogenetic analysis. Aged refuse enriched with NMS was used for methane biological oxidation and over 93% conversions were obtained.     

Study on the decolorization of dyes by microorganisms

Thirteen strains of bacteria with ability of dye decolonization were isolated from activated sludge and biofilm collected from textile wastewater treatment plants, and identified as Alteromonas, Klebsiella, Pseudomonas, Enterobacter. Alcali-genes and Corynobacterium. These strains D33, D32, D4 of Alteromonas can decolorize 29 kinds of dyes with good decolorizing activities of about 2.6 mg dye/gram wet cells.h-1. The optimum pH was 7-8 and the temperature was 37℃. The growth of bacteria was 3 times better in shaking cultures than in static cultures. But the decolorization rate of dyes was 4-9 times more in the latter than in the former. The results from the examination of the reaction products of Diamond Chrome Red B and Diamond Narry Blue RRN indicated the possibility of biodegradation of these dyes by Enterobacter D17.     

Performance of a completely autotrophic nitrogen removal over nitrite process for treating wastewater with different substrates at ambient temperature

The stability and parameters of a bio-ceramic filter for completely autotrophic nitrogen removal were investigated.The completely autotrophic nitrogen removal over nitrite(CANON) reactor was fed with different concentrations of ammonia(400,300,and 200mg N/L) but constant influent ammonia load.The results showed that the CANON system can achieve good treatment performance at ambient temperature(15-23°C).The average removal rate and removal loading of NH4+-N and TN was 83.90%,1.26kg N/(m3·day),and 70.14%,1.09kg N/(m3·day),respectively.Among the influencing factors like pH,dissolved oxygen and alkalinity,it was indicated that the pH was the key parameter of the performance of the CANON system.Observing the variation of pH would contribute to better control of the CANON system in an intuitive and fast way.Denaturing gradient gel electrophoresis analysis of microorganisms further revealed that there were some significant changes in the community structure of ammonium oxidizing bacteria,which had low diversity in different stages,while the species of anaerobic ammonium oxidizing(anammox) bacteria were fewer and the community composition was relatively stable.These observations showed that anaerobic ammonia oxidation was more stable than the aerobic ammonia oxidation,which could explain that why the CANON system maintained a good removal efficiency under the changing substrate conditions.     

Inhibitive effects of chlortetracycline on performance of the nitritation-anaerobic ammonium oxidation (anammox) process and strategies for recovery

The short-and long-term effects of chlortetracycline(CTC) on the nitritation-anaerobic ammonium oxidation(anammox) process were evaluated. The half maximal inhibitory concentration of CTC in the batch tests of the nitritation-anammox process was 278.91 mg/L at an exposure time of 12 hr. The long-term effects of CTC on the process were examined in a continuous-flow nitritation-anammox reactor. Within 14 days, the nitrogen removal rate significantly decreased from 0.61 to 0.25 kg N/m~3/day with 60 mg/L CTC in the influent.The performance suppressed by CTC barely recovered, even after CTC was removed from the influent. Furthermore, the inhibition of CTC also reduced the relative abundance of ammonium oxidizing bacteria(AOB) and anaerobic ammonium oxidizing bacteria(An AOB)in the reactor, resulting in both a decreased amount of and an imbalance between AOB and An AOB. When fresh anammox sludge was reseeded into the nitritation-anammox reactor,the nitrogen removal rate recovered to 0.09 ± 0.03 kg N/m~3/day.