Question

4. You are charged to quantitatively detect a specific microorganism involved in anaerobic ammonium oxidation (ANAMMOX) in a wastewater treatment plant. This organism is very difficult to isolate and grow. Please answer these.

1) What kind of molecular biological method for cell biomass quantification will be most suitable? Why?

2) Please describe procedure of the method briefly.

info is all..

Answer 1

Here i have discribed best two methods.

METHOD 1:-

Isotope pairing technique

IPT is a well-established 15N method used in estimation of denitrification (Nielsen 1992) on many environmental samples, such as marine sediments, lake sediments, rivers, and wetland (Rysgaard et al. 1993; Stepanauskas et al. 1996; Davidsson et al. 1997; Pind et al. 1997; Dong and Tollner 2003; Trimmer et al. 2003). In 2002, IPT was used to estimate the relative contribution of anammox process to the overall total N2 production through coupling with nitrate reduction in marine sediment (Thamdrup and Dalsgaard 2002). Risgaard-Petersen et al. (2003) described the detail methodology for application of IPT in sediments where anammox and denitrification coexist. Experimental samples can be treated for a short-term incubation (e.g., 24 h) in parallel with different inorganic 15N species labeled substrates: (1) 15NH4+ alone, (2) a mixture of 15NH4+ and 14NO2−, and (3) 15NO2− alone. After incubation, N2 production from each treatment can be collected and measured on an isotope mass spectrometer for 15N concentrations. The first incubation is used as a control to detect any oxidation of ammonium without the addition of nitrite, while the second treatment is used to measure the anammox activity, where the production of 29N2 stoichiometrically is a confirmation on the oxidation of ammonium (15NH4+) with reduction of nitrite (14NO2−) through the anammox process. The third incubation is to estimate the relative contribution of anammox and denitrification collectively, where the production of 29N2 and 30N2 indicate the activities of anammox and denitrification, respectively (Risgaard-Petersen 2003; Dalsgaard et al. 2003, 2005). Experimental details of the IPT procedures for the measurement of anammox and denitrification were described by Risgaard-Petersen et al. (2003) and further modifications had been made by Ward et al. (2009). Up to now, IPT has been used as the widely accepted method to substantiate the contribution of anammox process in various environmental samples. However, new evidences had pointed out that anammox bacteria could also reduce 15NO3− to 15NO2− and then to 15NH4+ under certain conditions and as a result, portions of the measured denitrification may be partitioned to the anammox reaction (Kartal et al. 2007a). In addition, IPT provides indisputable information on the specific process involved, but neither abundance nor the organisms responsible for the anammox process is available (Schmid et al. 2005).

Another isotope-labeling technique for detection the presence and activity of anammox bacteria is stable isotope probing (SIP) with 13CO2 and/or 15N labeled inorganic N species. SIP is a powerful technique when combined with current available molecular methods in revealing the anammox community composition and the associated microbial activity in environmental samples (Murrell and Whiteley 2011). This method relies on the incorporation of a substrate that is preferably enriched in a heavier stable isotope, such as 13C or/and 15N which allows the identification of an active population of microorganisms through selective recovery and analysis of isotope-enriched cellular biomolecules, such as DNA, RNA, proteins, and phosphorus lipid fatty acids (Bull et al. 2000; Murrell and Whiteley 2011). Currently, SIP has been successfully applied in the study of microbial nitrogen cycle, e.g., ammonia-oxidizing archaea and bacteria in the soils (Tourna et al. 2010; Zhang et al. 2011; Pratscher et al. 2011). In these studies, 13CO2 and 14N/15N were used as substrates for microbial metabolism in the soil samples, and then total DNA, RNA, or mRNA were extracted after a period of incubation for analysis of the relevant microbial communities and microbial abundance using relevant molecular techniques could be obtained for direct evidence on the active microbial groups and their abundance (Tourna et al. 2010; Zhang et al. 2011). In research on anammox bacteria, 14C labeled substrates were also used for fluorescence in situ hybridization (FISH)–microautoradiography analysis to confirm the chemolithoautotrophic biochemical pathway carried out by anammox bacteria (Strous et al. 1998; Jetten et al. 2002), and no other related reports are available. Therefore, SIP coupling with an array of molecular techniques (see below) is a promising approach for detection and quantification of anammox bacteria in the ecosystem.

METHOD 2:-

Lipid measurement

In contrast to all other known prokaryotes, anammox bacteria have special lipids in cellular membrane surrounding the anammoxosome within the anammox bacterial cells (Sinninghe Damste et al. 2002). These membrane ladderane lipids contain cyclobutane/cyclohexane ring systems, which make the anammoxosome membrane very impermeable comparing to other known non-annamox bacterial membranes (Sinninghe Damste et al. 2002; van Niftrik et al. 2004). Since the unique lipids are only found in anammox bacteria so far, the ladderane lipid can be used as an indicator and a biomarker for the presence of anammox bacteria in environmental samples (Sinninghe Damste et al. 2002, 2005; Kuypers et al. 2003). Furthermore, ladderane lipids are predominately enumerated as the core lipid derivatives (Kuypers et al. 2003, 2005; Jaeschke et al. 2007), but occurring as intact ladderane glycerophospholipids (ladderane IGPs) within cells at high abundances (Boumann et al. 2006), thus the ladderane IGPs, such as C20-[3]-ladderane monoalkylether–phosphocholine, may reflect living biomass more accurately than ladderane core lipids. Because of this, they are more specific biomarkers for viable anammox bacteria (Jaeschke et al. 2009). Up to now, the detection and quantification of ladderane lipids are used not only to infer the presence of anammox bacteria in environmental samples (Strous et al. 1999; Sinninghe Damste et al. 2002, 2005), but also to assess the presence of anammox bacteria associated with some of the geological events (Jaeschke et al. 2007).

In order to analyze ladderane lipids, biomass containing anammox bacteria are firstly extracted with methanol, methanol/dichloromethane, and dichloromethane substantially. The extracts are methylated with B3/methanol after removing the solvent by a rotary evaporator, and then fractionated by a small silica column using ethyl acetate as the eluent. After that, the obtained fraction is silylated with BSTFA in pyridine convert alcohols in TMS ethers for further analysis by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS)(Sinninghe Damste et al. 2002). Various chemicals contained in the ladderane lipids, such as fatty acids, glycerol diethers, glycerol ether-esters, and sn-2 glycerol monoether, would be analyzed through these procedures (Sinninghe Damste et al. 2002, 2005). Recently, a high performance liquid chromatography/atmospheric pressure chemical ionization-MS/MS method allowing determination of low levels of ladderane lipids in complex matrixes (e.g., sediments) had been developed with success, and this technique could detect as low as about 35 pg ladderane lipids, which improves the method resolution compared to the GC/MS method greatly (Hopmans et al. 2006). Although the lipids analysis provides a powerful tool to detect anammox bacteria, the following deficiencies still exist when using this technique on environmental samples: (1) extraction procedures are complicated; (2) high volume of samples is required, such as the seawaters and sediment due to the low abundance of anammox bacteria; (3) difficulties are encountered in purification when dealing with sediment materials because of other contaminants, such as humic acid and fulvic acid; and (4) these lipids may also be present in nonliving organic matter and thus may not necessarily indicate the presence of metabolically active anammox bacteria without error (Schmid et al. 2005), which limits the quantification of active anammox bacteria population with high confidence and precision