1. Abstract

The study examines the presence of bacterial species that were present in the peat core and their ice nucleating abilities. A peat core was collected from Thrusley, National Nature Reserve (NNR) at a particular point using a GPS navigator. The peat core was labeled and sealed carefully and stored in cold room. The analyzing of Peat samples were done using molecular techniques such as DNA extraction, Agarose gel electrophoresis, PCR, and Ice, Differential scanning calorimetry, Cloning and Sequencing. There were no signs of DNA during the initial DNA extraction from peat. But when the Peat samples were analyzed through ice nucleation process and spread plate method. The analyzing showed various ice nucleating temperatures at which it represents presence of a biological material. We were successful in extracting DNA from the colonies cultured in agar plates inoculated with peat sample. The sequencing results mainly showed Chromobacterium violatium and Lysini bacillus sp when the sequencing results were cited in

1. 1. Introduction

Peat is an undecomposed plant and animal material formed due to water accumulation. Water accumulation enables oxygen to travel slowly than in air. This slow movement oxygen will enable complex anaerobic conditions and plant material will not be decomposed like normal aerobic decomposition. Peat stats accumulating due to slow decomposition of mainly bryophytes (e.g.: Sphagnum species) (Rydin and Jeglum, 2006).

Geographical distribution of Peat lands

Peat lands were distributed through out the world. The total area of peat lands is not known exactly. In Europe and North America the known distribution is approximate one (Gorham, 1991 cited in Charman, 2001). In North America (Canada, USA) peat lands are distributed with a total area of 171127000 ha approximately. In Europe the total area is 179762900 ha with most of the peat lands distributed in former USSR, Finland, Poland and Norway. Peat lands are also distributed in Ice land, Hungary, Norway, and Germany. In United Kingdom they are distributed with a total area of approximately 1640280 ha (Immirizi et al, 1992 cited in charman, 2001).

In England the Most of the peat lands are Fen and bogs. Most of them are open peat lands with plants growing to a maximum height of 50 centimeters. It is very rare to find a plant growing more than 50cm. In England Peat lands are distributed approximately in 384204 ha (Lindsay and Campbell, 1995)

Formation of Peat

Peat land development mainly occurs in two types of pathways. Peat lands development normally occurs in a very small process. The decomposition rate of peat lands depends on the availability of water. Peat development is mainly due to Sphagnum plant species (Rydin and Jeglum, 2006).

1) Paludification

2) Terrestrialisation

Paludification is a process occurs over a less mineral content ground. This type of lands will not have an aquatic condition at the bottom of the peat land. Tree remains such as bark, main stem and leaves are found and this is an indication of past forest existence. Most of the peat lands are expected to have formed by this process (Heinselman, 1963, 1970, 1975 cited in Rydin and Jeglum, 2006).

In terrestralisation formation peat occurs due to flow of water in to a shallow water body with remains of organic and inorganic sources. The peat formation occurs due to over flooding of the lands and water flowing in to side by small ponds and flooding leads death of the plants growing on there and peat starts accumulating.

1.2 Composition of peat

Peat mainly contains remains of dead plant and animal matter. When the plant dies the roots, leaves, stems, and other plant materials stats decomposing, but the decomposition process is very slow in peat lands and this process will lead to peat formation.

Peat is composed of organic matter. The plant material decomposed as slowly as that only a 1mm growth can be seen in one year. Peat contains a variety of complex organic compounds. These organic compounds mainly derived from the vegetation that grows on peat land. These organic compounds mainly contain carbohydrates, nitrogenous compounds, different types of phenols, and lipids. In addition to these other organic compounds such as vitamins, pigments, alkaloids, nucleic acids are present. The ratio of this composition mainly depends on the type of vegetation that grows on these peat lands. The ration even differs in between the species. The composition of peat also depends on the peat acidity, alkalinity, mineral content, redox conditions, and the rate of humification. (Fuchsman, 1983).

Along with the composition of peat degree of humification is also important. Humification is the process at which the peat is decomposed and the plant material decomposed to a level of recognizable or unrecognizable. Different countries use different methods of humification. The von post humification is method is mostly used and it is the simplest (Rydin and Jeglum, 2006)

Importance of peat

Peat lands are used for Fuel purposes agricultural development and other purposes like fertilization. Apart from these factors it has great importance in climatic change study. The CO2 and methane emissions have a great impact on climate as a green house gases. Methane is a potent green house gas, and it has a great effect on climatic change. It has increased sharply from past 200 years as the industrialization began (S.N. Singh et al, 1999).

The concentration of methane is increasing 1.1% per year which is a significant amount that can have a great effect on climatic change. Wet land research has been stimulated due to the emission of green house gases (Blake and Rowland, 1988 cited in S.N. Singh et al, 199).

Emissions of CH4 depend on the peat lands that are man made and naturally formed. Naturally formed peat lands emit high amount of CO2 when compared to man made (S.N. Singh et al, 1999). Therefore study of peat lands has great importance in understanding climatic chance and the factors affecting it.

1.3 Bacterial species in peat

Peat bacterial species have unique characteristics because some of the species take up methane produced in a peat land and some of them releases due to existence of anaerobic conditions. These anaerobic conditions are due to the low availability of oxygen which travels slower in water that in air.

Different bacterial species exists in peat, mainly methanogenic Archaea and methanotrophs. The major part of these contains phylum Actinobacteria, Planctomycetes, verrucomicrobia, and Spirochaetes. (Luka Ausec et al., 2009). The bacterial communities are present through out the whole peat core (Waksman and Stevens, 1929). Peat lands are mainly distributed with Bacillus, Pseudomonas, Achromobacter, Cytophaga, Micrococcus, Chromobacterium, Clostridium, Streptomuces, Actinomyces, Mycobacterium, Micromonospora, and Noardia (Williums and Crawford 1983). There were other bacterial species observed in related to the fen bacterial community analysis. The different bacterial species were Proteobacteria, Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Acidobacteria. Uncultured deltaproteobacterium.(Barbara Kraigher et al., 2006).

The raised bog in mainly contains Bacillus species that were spore forming with an average of (50-60%). Other important bacterial species were Gram-negative non-sporing rods of (30%) and Arthrobacter (5%) (Wheatley et al,1996). The number of bacteria decreases with the increase in depth of the peat (I.P.Martini et al., 2006).Peat land vegetation contains ice nucleating bacterial species such as Pseudomonas (H.E. Ahern et al, 2007). Ice nucleation is discussed in fallowing subtopic

1.4 Ice nucleation

Ice nucleation is naturally found in clouds. The difference in temperatures of cloud water molecules leads to ice nucleation. I has been found that bacteria is responsible to create the difference in temperatures with in the cloud vapor and this process have importance when coming to the study of higher temperature ranges (Vali,1995 and Morris et al.,2005 cited in H.E. Ahern et al, 2007).

Ice nucleation is defrosting of water by bacteria using their surface ice nucleating proteins. These triggered ice nuclei will spread with in the plant cells and damages the cell wall. To avoid this plants will cool down to temperatures up to -14 (Lindow, 1983 cited in H.E. Ahern et al, 2007). Bacterial species that are capable of expressing Ice nucleation are more abundantly found in phyllosphere and these bacterial species are capable of overcoming the strategy of plants and can cause injury in plants and this makes bacterial to use the nutrients from plants (Hirano and Upper, 1995 cited in H.E. Ahern et al, 2007).

Ice nucleation is a process of shifts in between the phases, i.e. from liquid to solid. The shifts in between phases from metastable to a new phase are caused by biological material. (Vali, 1999). The shifts are caused by proteins of ice nucleating bacteria (Elias Anastassopoulous, 2006). Pure water freezes at -35 to -40C.It is believed that certain Bacterial species have specific ice nucleating proteins on its cell wall that cause ice nucleation. Homogeneous and heterogeneous are the two types of ice nucleation, Homogeneous is caused by pure water freezing at -40C and Heterogeneous is caused by accumulation of water molecules on ice nucleating bacteria (Jinkun Li, Tung-Ching Lee, 1995).

The discovery of bacterial species that lives on the leafs of certain plant species and causes damage to leafs by defrosting them. Ice nucleating bacteria can be detected by a Spectrometer (David Sands et al). The spectrometer shows temperatures ranging from -2.0C to -5.0C if a biological material mainly ice nucleating bacteria are present in a sample (Thomas L.Kifet, 1988).The Ice nucleating ability of bacteria is not stable at 30C and more(Naomi Muryoi et al., 2002).Ice nucleation is initiated by bacterial repetitive proteins (Hitoshi Obata et al, 1999). It is very difficult to observe which bacterial colonies caused ice nucleation when grown on agar plates. This is due to transparency of the medium (Elias Anastassopoulos, 2006). There are lots of bacteria strains that will cause ice nucleation e.g. Pseudomonas, Erwinia, Xanthomonas, Chromobacterium (Jinkun Li, Tung-Ching Lee, 1995), (Mariangela Hungria et al., 2004)


Sample collection and analysis

Peat samples were collected from Thrusley, National Nature Reserve (NNR) on1st July 2009 using a GPS navigator, The GPS points were (BNG SU 90245, BNG 41344).The landscape is a raised bog with temperatures. The sample core was collected using Russian peat borer. The sample was sealed and stored carefully in a cold room. The sample core was 40cm in length. The core was divided in to nine different sections of 5cm length. First of all 1gm of sample was extracted at 19cm distance from top to bottom. The weighed 1gm sample was taken in to universal test tube and 2ml of molecular grade water is added and vortex to confirm that the sample was well mixed. 100L of the sample is transferred and centrifuged at 10,000rpm for 10 minutes and pellet is transferred to fresh tube and DNA extraction is continued with phenol chloroform extraction.

Again the same core was used as a sample, but this time the peat core was separated in to nine different sections of each one with a length of 5cm and taken in to 10mL tubes. A sample weighing 1gm was taken from each 5cm sectioned core and mixed with 2mL water. 100L of the sample was taken and centrifuged at 10,000 rpm for 10 minutes. From those samples DNA extraction was performed using phenol chloroform method and heat shock method at 95.

Phenol chloroform extraction

Initially Phenol chloroform DNA extraction was performed to extract DNA from the peat sample. Phenol and chloroform allows DNA to precipitate and forms two layers upper aques layer and bottom organic layer. RNA accumulates in the upper aqueous layer. Phenol, Chloroform and isoamyalchol at a ratio of 25:24:1 was used to extract DNA sample from peat.SDS and proteinase K was used to denature the cell wall of bacteria, and the lipid layer. Absolute Ethanol was used to wash the sample to remove the excess protein and RNA content. The extracted DNA sample was stored at -20C to prevent it from destroying.

A 1gm upper layer of peat was taken and mixed with 2mL of molecular grade water and vortex to make sure that the peat is well dissolved in the water. Centrifugation was performed to separate the two layers supernatant and debris. A 200L sample was taken from the supernatant and DNA extraction was performed on it.

Agarose gel electrophoresis

Agarose gel electrophoresis was performed to observe the bands of DNA under UV light. A multipurpose agar weighing 1 was taken in conical flask and mixed with 100mL of 0.5x TBE buffer(1%gel). Conical flask allows quick dissolving of agar when heated in an oven. The agar was well heated to make sure that it was dissolved. DNA stain with a volume of 10L was added. Then a tank having a cathode and anode was set up and gel was poured in to it before solidifying. Tank should be filled with the buffer until the gel is submerged. The wells were loaded with extracted DNA sample and 1kb ladder (Invitrogen, paisley, UK). The tank was connected to a current supply and the voltage was setup as 120C and it was observed to make sure that bubbles were coming from both the electrodes and the tank should be left undisturbed until the gel sample runs until the end of gel. After running the gel it was observed under UV light to observe the bands. Larger bands will appear at the top and smaller at the bottom. This is due to molecular weight of the bands.

Ice nucleation

Ice nucleation of peat sample was performed in a differential scanning calorimetric (DSC)

Machine and the graphs were analyzed. The fallowing steps show the preparation of a sample for ice nucleation and process of ice nucleation.

Peat samples weighing 1gm was taken from top, middle, bottom of the peat core and 2mL of molecular grade water was added. Vortex was done to make sure that the peat was well dissolved in water. From the solution 200L of the sample was taken and centrifuged at 13000 for 10 minutes to separate supernatant and debris. Separating supernatant and debris makes it to six tubes. From each tube a 10L sample was taken in different tubes. Ice nucleation was performed after treating samples at different temperatures (37C, 37C with lysozyme, 60C, 90C, and an untreated sample) .The 37C, 37CL samples were incubated overnight. A 10L of each sample was taken and placed in to an aluminum crucible that was sealed tightly. The sample was placed in an ice nucleating machine. Graphs were obtained showing the ice nucleating temperature of the respective sample. Lysozyme treatment will shear the cell wall of the bacteria and will allow genomic DNA to be released.

Plating method

The raw samples those were prepared for ice nucleation was used for platting. The platting was done using four different types of agars. In the next step the peat samples were inoculated on four different types of agar and were incubated at room temperature for one week.

1) 15 gm agar+0.25 nutrient broth+ 1 litre water

2) 15 gm agar +1 litre water

3) Malt extract agar

4) 28 gm nutrient broth+1 litre water

The temperatures set will denature the DNA, anneal the primers and elongate the added primers by the action of DNA polymerase. After the PCR the amplified DNA was loaded on to a gel and visualized under the UV light.

Preparation of Agarose gel

Gel was prepared by doing 1/10 dilution. A 1% gel was prepared by adding 1gm of agar in 100mL of 0.5x TBE buffer and heated to make sure that agarose is well dissolved. After cooling 10L of DNA cyber safe stain was added to gel and it was pored in to tank and left undisturbed until it dissolves. A 1kb ladder with a volume of 10L was added to the first well. The sample with a volume of 10L was added to the next well and positive and negative electrodes are connected. Negative electrode should be connected at the top i.e. towards the wells and positive at the bottom. This should be done because DNA is negatively charged and runs towards positive end. Current have been set at 100V and left until the samples run till the bottom and the gel was visualized under UV light.

Agarose gel electrophoresis showed a band out of three samples and this was detected at 6108 and 5090bp. After the sample was visualized under UV light, cloning was performed on the remaining sample.


Cloning was performed to increase the copy number of the sample. This process is performed by cutting the DNA to be inserted and the vector going to carry the DNA fragment are both cut using restriction enzyme to create blunt or sticky ends. Then the fragment is inserted in to vector and the bacterial cell was allowed to multiply in a medium and the multiplication makes a copy of foreign DNA. Thus the foreign DNA will become a large number of copies during overnight. The same process can be performed in a thermocycler. But the advantage of cloning is large fragments of DNA can be cloned.

Before cloning the DNA should be inoculated with E.coli cells. Cloning was performed using TOPO TA Cloning kit (Invitrogen, Paisley, UK). A mixture of 20L DNA+40L binding buffer was taken and vortex was done. The mixture was transferred to the column tube And Centrifuged at 13000 rpm for 30 sec. The collection tube was discarded and the column tube was taken and wash buffer was added to wash out proteins and mRNA. Centrifugation was done at 13000 rpm for 30secs. Again the sample was transferred to a column tube and washed with washing buffer and the collection tube was discarded and centrifugation was performed.TE buffer of volume 10L was added to the column tube and Centrifugation was done at 13000 rpm for 30secs.Column tube was discarded and now the E.coli cells were added and incubated overnight. Three plates were inoculated with sample on it. Petri plates poured with agar on it were taken from the incubator and Xgal of volume 40L was added along with 90L SOC (Super optimal catabolite), 10L of sample from overnight cultures.

On the second plate 20L of SOC and 10L sample were spread using a sterilized glass rod.

On the third plate only a sample volume of 50L was applied. Growth of 15 colonies was observed on the third plate (only a sample of 50L).The colonies were in white colour. Five of the colonies were transferred to five individual tubes.

Coming down to plasmid purification step using a QIAprep spin miniprep kit and a micro centrifuge. The bacterial cultures obtained from overnight incubation tubes were resuspended in 250L buffer P1 and transferred to a micro centrifuge tube.Buffer P1 should be added with RNase to prevent cell clumps If a LyseBlue reagent has been added to the buffer P, the bottle should be shaken vigorously until the LyseBlue particles are completely dissolved. The bacteria should be resuspended completely by vortexing or pipetting up and down to ensure that there was is no cell clumps. Buffer P2 of volume 250L was added and mixed thoroughly by inverting the tube 4-6 times.Vortex should be avoided as it will result in shearing of genomic DNA. The lysis reaction should be no more than 5 min. LysisBlue will turn the cell suspension to blue colour, but the solution have not turned blue color. Still we are able to see the DNA on agarose gel. If the Blue color appears the solution should be mixed until it becomes clear. Buffer N3 of volume 350L was added and mixed immediately by inverting the tube 4-6 times. Large volumes of solution may require mixing up to 10 times. The solution will become cloudy.A homogenous colorless suspension indicates that the SDS has been effectively precipitated. Centrifugation at 13,000 rpm for 10 minutes will form a compact white pellet.Supernatant was collected and applied to QIAprep spin column by pipetting. Centrifugation was done 60 seconds and the flow through was discarded.The QIAprep column was washed by adding 0.5ml buffer PB and centrifuged for 60 seconds. Again the flow through was discarded. This step was performed to remove trace nuclease activity.QIAprep spin column was washed by adding 0.75ml buffer PE and centrifugation was performed for 60 seconds. The flow through was discarded and an additional centrifugation was performed for one minute. The QIAprep column was placed in a pure 1.5 ml 15. Microcentrifuge tube. Buffer EB of volume 50L was added to elute DNA and allowed to stand for 1min and centrifugation was done for one minute. After the cloning process was finished Agarose gel electrophoresis was performed to ensure the presence of DNA. The same concentration was used that was discussed before and the below figure shows the detection of DNA under UV light.Nano drop method was performed after confirmation of DNA to calculate the concentration of DNA.

Nano Drop method

A drop of DNA cloned on a Nano drop machine will show the concentration on a computer with special software installed.

1. First the tow ends of a Nano drop machine should be wiped with a tissue to prevent it from contamination.

2. Then the test was performed with TBE buffer and it served as a blank solution and it should show zero concentration as there will be no DNA.

3. Then the Nano drop was performed with DNA samples.

4. Prior to the testing of every sample the machine should be wiped and tested with TBE buffer which served as a blank.


Sequencing is the process of finding the primary structure of an unknown molecule. It gives us the detail at molecular level by giving the information about molecules involved in the given biological sample.

After Nano drop method, Samples were diluted that showed highest concentration and were sent to Barts and the London/William Harvey Research Institute / Genomic centre/ London. The fallowing primers were used in sequencing



2.5 Results and Discussion

The ice nucleating showed ice nucleating temperatures. The samples that showed temperatures ranging between -2 to -5 were considered as samples that have ice nucleating bacteria in them. As temperatures that were between -2 to -5 were the bacterial species ice nucleates water (Thomas L.Kifet, 1988).


Graph1: showing ice nucleation results of sample 1.

X-axis= Temperature at which peat samples have shown ice nucleation versus different sample treatments.

Graph2: showing ice nucleation results of sample

Graph3: Showing ice nucleation results of sample 3.



Untreated samples are raw samples with no treatments

37C sample was incubated overnight

37C L sample was incubated treated with lysozyme and incubated overnight

60C and 90C samples was heated for 10mins

From the graphs we can observe that untreated sample showing low temperature ranges and increased slightly with 37C incubation indicating the presence of biological material. The Lysozyme treated samples have shown decrease in temperature except for the DEB of sample2. Samples that were treated at 60C have decreased further indicating that most of the micro organisms have not survived at that temperature. Coming down to samples treated at 90C showed tremendous drop in temperatures. The cells are treated with Lysozyme to disrupt the cell wall so that ice nucleating protein can be released from the cell.

2.6Sequencing results

Coming down to the final result i.e. sequencing they showed mainly Chromobacterium in them. The fallowing results were obtained when the sequences were cited in using BLAST (basic local alignment search tool).

By this we can understand that micro organisms have not survived at those temperatures. Bacillus sp and -proteobacteria(Chromobacterium)were found in ice nucleating bacteria (Barry J.Fuller et al., 2004).

The sequences were aligned as forward and reverse sequences. The sequencing results mainly showed Chromobacterium and it was one of the bacterial genera in peat. Other bacterial samples such as Lysinibacillus, uncultured bacterium were also detected. But the uncultured bacterium may have been detected due to the contamination of the samples. Chromobacterium.v belong to family -proteobacteria.

We have expected methaotrophs and methanogenic bacterial species. But, we have found icenucleating bacteria in our peat sample by extracting DNA from peat sample fallowed by PCR, Gel electrophoresis, Cloning, and Sequencing. We have expected to find new bacterial species and expected of in analyzing UOM (unidentified organic matter) which will be of great importance for further work. But, we are unable to analyze UOM due to lack of knowledge on it and time constrains.

We have no found the new species in the examined peat sample, Chromobacterium, Lysinibacillus (Williums and Crawford 1983)as the main bacterial species in the peat sample from Thrusley. During the first DNA extraction we have not seen DNA on the agarose gel; this may be due to the less concentration of the DNA or due to lower concentration of the gel. We have failed to extract DNA through heat shock method and concentration kit method.

But when the samples were inoculated on the agar plates we have observed bacterial growth, from which DNA extraction was performed by Phenol chloroform method. But before doing DNA extraction the peat samples were also tested for Ice nucleating bacteria and the plates shown bacterial growth were also tested for ice nucleating bacteria. The plates that showed temperatures ranging between -2 to -5 were used for DNA extraction as the temperatures shown are the temperature for ice nucleating bacteria (Thomas L.Kifet, 1988). So the bacteria obtained can be presumed as ice nucleating bacteria.

The bacteria that showed ice nucleation was only an assumption and further research of using techniques particular for ice nucleating bacteria can be of great importance in finding ice nucleating bacteria as ice nucleation is latest trend in keeping food frozen and saving energy (Jingkun Li and Tung-Ching Lee,1995).

The sequencing results showed uncultured bacteria which may be a contamination, because when environmental blast did showed gut bacteria. And great care should be taken when handling samples. Should wash our hands and gloves should be used all the times. The whole experiment should be performed in aseptic conditions.

Chromobacterium is a gram negative rod shaped bacteria. It undergoes anaerobic respiration. It produces cyanide which plays a great importance in bioremediation and (Carepo et al). Infection of chromobacterium occurs usually through cut wounds (chen et al 2002).

The chromobacterium formed like a mat structure when cultured on malt extract agar instead of individual colonies. One may think the mat structure as a contamination. The other way of finding ice nucleation bacteria should be well studied and for further research.

2.8 Conclusion

Chromobacterium, Lysinibacillus, was discovered in the extracted peat sample and there is strong evidence that these are bacterial genera found in peat. Ice nucleation provided good ice nucleation temperatures ranging between -2 to -5 but all the plated samples of the peat core failed to show the bacteria. This should be studied more in detail to find the ice nucleating bacteria and that can be applied potentially in food biotechnology that saves a great amount of energy. The obtained results showed bacterial species that can be used in bioremediation.

The obtained results can be used further to study ice nucleating bacteria and whether Chromobacterium is methanogenic or methanotrophic. This will be a great importance in finding does the bacteria is involved in methane production. As methane as a potent green house gas and global warming as a big concern further study will be interesting.

2.9 References

1. Cloning kit, Invitrogen, Renfrew, PA49RF, UK.

2. David Sands, 2004, Ice nucleation active Bacteria in the Water cycle, Plant sciences and plant Pathology, Montana State University., Bozeman, USA.

3. Elias Anastassopoulous, May 2006, Agar plate freezing assay for the in situ selection of transformed ice nucleating bacteria. School of Agricultural technology, Department of plant production, 41335 Larissa, Greece.

4. Gabor Vali, 1999, Ice nucleation theory, summer colloquium.

5. Jingkun Li and Tung-Ching Lee, 1995, Bacterial ice nucleation and its potential application in the food industry, Centre for advanced food technology and the institute of marine and costal sciences, Cook college, Rutgers University, New Brunswick, USA.

6. Luka Ausec, Barbara Kraigher, Ines Mandic-mulec, 2009. Differences in the activity and bacterial community structure of drained grassland and forest peat soils, University of Lijubljana, Jamnikarjeva, Slovenia.

7. Tehomas L.Kieft, 1988, Ice nucleation activity in lichens, Department of biology, New Mexico Institute of mining and Technology,Socorro, New Mexico.

8. V.R.Depres, J.F.Nowoisky, M.Klose, R.Conrad, M.O.Andreae, and U.Poschl, 2007, Characterization of primary biogenic aerosol particles in urban, rural, and high-alpine air by DNA sequence and restriction fragment analysis of ribosomal RNA genes. Biochemistry department, Max Plank institute of Chemistry, Becherweg 27. 55128, Germany.

Text books

1. The Biology of Peat lands, Hkan Rydin and John Jeglum, 2006, Oxford University press, New York, USA.



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