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i‘ffl‘JOW
I LIBRARY
Mimigan State
I lnive rsity This is to certify that the
*———- ~ . thesis entitled

ISOTOPOMER EFFECTS ASSOCIATED WITH
NITRIFICATION AND DENITRIFICATION: IMPLICATIONS
FOR THE GLOBAL NITROUS OXIDE CYCLE

presented by
Adam J. Pitt

has been accepted towards fulfillment
of the requirements for the

Master of Environmental Geosciences

7/034 t/Tlfljil/I’V
yflor proTessor’s Signature
‘ W370:

Date

 

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PLACE IN RETURN BOX to remove this checkout from your record.
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6/01 cJCIFICJDatoDuopss-pt 15

ISOTOPOMER EFFECTS ASSOCIATED WITH NITRIFICATION AND
DENITRIFICATION: IMPLICATIONS FOR THE GLOBAL NITROUS OXIDE
CYCLE
By

Adam J. Pitt

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF ENVIRONMENTAL GEOSCIENCES

Department of Geological Sciences

2003

ABSTRACT

ISOTOPOMER EFFECTS ASSOCIATED WITH NITRIFICATION AND
DENITRIFICATION: IMPLICATIONS FOR THE GLOBAL NITROUS OXIDE
CYCLE

By

Adam J. Pitt

Nitrifying and denitrifying bacteria carry out a vital role in soil systems by
regulating supplies of inorganic nitrogen. Nitrifying and denitrifying bacteria also affect
the Earth’s atmosphere profoundly through the addition of the greenhouse gas, N20. To
better understand the role of nitrification and denitrification in atmospheric greenhouse
gas production we need a reliable method to distinguish N20 formed from these two
processes. Such information will be particularly valuable in agricultural environments
that can be managed to foster one process or the other. The intramolecular distribution of
15N in nitrous oxide (isotopomer) is emerging as a new tool in defining the sources and
sinks of this trace gas (Popp et al., in press; Toyoda and Yoshida, 2000; Toyoda et al.. in
press, Perez et al, 2001) and is commonly expressed in terms of site preference
(difference in S'SN between the central and outer N atoms). Using laboratory cultures of
whole soil microbes and pure cultures of an N20-producing denitrifier, Pseudomonas
c/tlororaphis (ATCC #43928), it was demonstrated that the isotopomer fingerprint of
N20 derived from denitrification is unique from that of nitrification (Christensen and
Tiedje, 1988). Furthermore, it was demonstrated that in agricultural soils the
consumption of N20 during denitrification has no affect on site preference, but in
deciduous forest soils an isotopomer effect is observed. With these results we are now
poised to begin to apply isotopomers to apportion the relative contribution of N20

derived from nitrification and denitrification in agricultural soils.

DEDICATION
This work is dedicated to my grandparents Pinkus Rolnitzky, Alexander Pitt, and
Margaret 0. Pitt who passed before this work could be completed. I thank them for all

their hard work and dedication to my family.

iii

ACKNOWLEDGEMENTS
I would like to thank Drs. Nathaniel Ostrom, John Breznak, Hasand Gandhi,
Robin Sutka, and Tim Bergsma for all of their invaluable experience and advice
throughout the course of this work. I would like to especially thank my committee
chairperson, Dr. Peggy Ostrom, for her patience and ability to take a chance on someone
she hardly knew. I would also like to thank my parents Edward and Celina Pitt for their
love and support, my grandmother Mary Rolnitzky for her amazing inspiration, and

Rachel Frumkin who is my future.

TABLE OF CONTENTS

List of Tables ......................................................................................... vi
List of Figures ........................................................................................ vii
Introduction ............................................................................................ 1
Thesis ................................................................................................... 2
Appendices
Appendix A
Methods ........................................................................... 10
Appendix B
Supplemental Graphs ............................................................ 14
References ............................................................................................ 18

LIST OF TABLES

Table 1 Average Site Preference for reactions leading to N20 production ................ 3
Table 2 Fractionation factors for N20 reduction (Reaction D) in soil mesocosms ...... 5

*CAT, conventional agricultural (replicatesl , 2, 3); HCS, historically
cultivated successional; DF, deciduous forest

vi

LIST OF FIGURES
(Images in this thesis are presented in color.

Figure 1 Reaction resulting in N20 production and reduction. a, Production of N20 by
nitrifying bacteria by oxidation of hydroxylamine and b, reduction of nitrite
during nitrifier-denitrification. c, N20 production during nitrification and d, its
subsequent reduction ...................................................................... 3

Figure 2 N20 Concentration and isotopomer site preference for denitrification
experiments. a, Production of N20 and b, site preference expressed relative to
the natural log of the ratio of the observed concentration, C, to that of the initial
concentration, Co, in triplicate cultures of Psuedomonas chlororaphis. c,
Consumption of N20 and (I, site preference in soil mesocosms from three
replicate plots with a history of conventional agricultural treatrnent(CAT1,
CAT2 and CAT3). e, Consumption of N20 and f. site preference in soil
mesocosms from a historically cultivated successional field (HCS) and a
deciduous forest (DF). Regression equations for site preference vs. In (C/Co)
are as follows: b, PCl, y = -7.0+ 1.8x; PC2, y = -8.5+ 0.01x; PC3, y = -13.7+
5.4x d, CATl, y = -0.4 - 2.4x; CAT2, y = 1.9 - 0.6x; CAT3, y = -0.2 + 3.9x f,
HCS, y = 4.4 - 1.2*x; DF, y = 3.1- 20.4*x ............................................. 6

Figure 3 6'5 N and 6'80 vs. 1n (C/Co) for conventional agricultural treatment
mesocosms. a, r2 = 0.4052; r = 06366, p = 0.0045; y = 0414880127 -
2.8579479*x b, r2 = 0.0035; r = 00595, p = 0.8147; y = 13.6945294 —
0.36836952*x c, r2 = 0.9154; r = 09568, p = 0.0002; y = 0.955740909 -
6.75726686*x d, r2 = 0.8137; r = -0.9020, p = 0.0022; y = 16.5163472 -
13.8830931*x e, r2 = 0.6045; r = 07775, p = 0.00001; y = 0.338244567 —
5.28180685*x 1‘, r2 = 0.5114; r = 07151, p = 0.0001; y =14.6636102 -
8.63820581*x ............................................................................. 14

Figure 4 815 N and 5'80 vs. In (00;) for historically cultivated successional (HCS) and
deciduous forest (DF) mesocosms. a, r2 = 0.3562; r = 05969, p = 0.0114; y =
0.0803007579 - 5.67524563*x b, r2 = 0.3672; r = -0.6060, p = 0.0099; y =
12.6993928 - 13.6296028*x c, r2 = 0.8590; r = 09268, p = 0.00000003; y =
0.906057787 - 14.621457‘x d, r2 = 0.8845; r = 09405, p = 0.000000007; y =
14.7188548 - 38.6903078‘x ............................................................ 15

Figure 5 6'5 N and 8180 vs. ln (C/Co) for Pseudomonas chlororaphis (PC) cultures. a, r2
= 0.9838; r = 0.9919, p = 0.0009; y = -15.5253953 + 6.89719754‘x b, r2 =
0.9817; r = 0.9908, p = 0.0011; y = 24.252223 + 8.71316266’x c, r2 = 0.9833;
r = 0.9916, p = 0.0009; y = -16.1674224 + 10.2834111*x d, r2 = 0.9920; r =
0.9960, p = 0.0003; y = 24.8183474 + 9.17864406*x e, r2 = 0.6670; r =
0.8167, p = 0.0473; y = -19.3910815 + 7.08615027*x f, r2 = 0.9721; r = 0.9860,
p = 0.0003; y = 19.9194665 + 9.89224398*x ......................................... 16

vii

INTRODUCTION

Nitrous oxide emissions from agriculture are a significant contributor to global
greenhouse gas emissions and can, in fact, contribute more to radiative forcing of climate
change than CO2 (IPCC, 2001; Robertson e! 01., 2000). Increasing emissions of N20
over the past century result from human-induced changes to microbial nitrification and
denitrification. While these processes can be managed in agriculture, identification of
their relative importance to N20 flux is unclear. Traditionally, stable isotopes have been
used for apportionment but they have not constrained the N20 budget better than other
approaches (IPCC, 2001, Sutka et a1., 2003). Recent field studies used isotopomeric site
preference of N20 (difference in 6'5N between the central and terminal nitrogen atoms of
N= =0) to differentiate N20 arising from nitrification versus denitrification (Perez et al.,
2001; Yamulki et al., 2001). However, accurate apportionment between reactions using
site preference requires examining individual processes in pure culture. Previous work
evaluated site preference during nitrification (by nitrifying and methanotrophic bacteria)
(Fig. 1, reaction A) and during nitrifier-denitrification (Fig. 1, reaction B) (Sutka et al.,
2003). Here I evaluate site preference during denitrification (Fig. 1, reactions C, D) and
show that site preference is a robust indicator of microbial origins of N20 in agricultural
soils. Trends in site preference during N20 reduction (Fig. 1, reaction D) within
mesocosms revealed differences between deciduous forest and agricultural soil, implying
important differences in microbial community structure and/or function between

managed and natural soils.

Bacterial nitrifiers produce N20 by either oxidation of hydroxylamine (Fig. 1,
reaction A) or reduction of nitrite (Fig. 1, reaction B) (Wrage et al., 1999). These two
pathways impart different site preferences on the N20 produced (Table 1) (Sutka et al.,
2003) making it possible to distinguish which process was involved in N20 production.
Most denitrifying bacteria produce and consume N20 (Fig. 1, reactions C, D). However,
it is unclear if N20 produced by nitrite reduction during nitrifier-denitrification (Fig 1,
reaction B) differs in site preference from that of N20 produced during denitrification
(Fig. 1, reaction C) and if there is any change in site preference during consumption of
N20 (Fig 1, pathway D) that could confound our ability to distinguish nitrifier-
denitrification from denitrification.

I used Pseudomonas cltlororaphis to evaluate site preference Of N20 production
during denitrification (Fig 1, reaction C), because this bacterium lacks the enzyme to
reduce N20 to N2 (nitrous oxide reductase). Thus, it will only express isotopomer effects
during N20 production from nitrite without influences associated with consumption of
N20. P. chlororaphis cultures showed N20 production over time (Fig. 2a) and no
correlation between concentration and site preference (Fig. 2b). A Rayleigh model
(Mariotti et al., 1988) indicated no fractionation in site preference during production.

F urthermore, site preferences for N20 production via nitrite reduction by P. chlororaphis
and N. curopaea were virtually identical (Fig. 1, pathway C and B, respectively; Table 1).
This suggests that the two organisms share similar mechanisms for nitrous oxide
production; and that irrespective of whether or not a nitrifier or denitrifier is involved

there is a unique site preference for N20 produced by nitrite reduction.

IQ

 

Denltn'ftcation

 

 

A C

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

D
Nitrification N03‘
_.__N.2.O. E
A E l Nitrifier-denitrification
i I B : *
NH3 —* NHZOH E‘EEN02'—* NO ——' N20 E ------------ N,
L .......... ,_.| I1

 

 

 

‘Not regarded as environmentally significa rt

Figure 1. Reactions resulting in N20 production and reduction. A, Production of N20 by
nitrifying bacteria by oxidation of hydroxylamine and B, reduction of nitrite during
nitrifier—denitrification C, N20 production during denitrification and D, its subsequent
reduction.

Table 1 Average Site Preference for reactions leading to N20 production.

Ave.
-2.3 1.9

-8.3 3.6
-8.1 +/- 3.4

 

My next objective was to detemiine if consumption of N20 during denitrification

(Fig. 1, reaction D) influences site preference. This was done by monitoring site

preference during N20 reduction in anaerobic soil mesocosms (agricultural, successional
field and deciduous forest soils). Following procedures of Bergsma et a]. (2002), I
verified that N20 was not being produced in the mesocosms. Briefly, prior to initiating
the experiment, the mesocosms were purged with N2 and, after two weeks, the headspace
was sampled for N20; N20 was not detected indicating that production of N20 was not
significant. An N20 headspace was then established in all mesocosms and N20 was
subsequently consumed over time (Fig. 2c, 6).

In the process of generating isotOpomer results, fractionation factors (a) for 8N
and 5130 of N20 were established using a Rayleigh model (Table 2) (Marriotti et al.,
198 8). Traditional approaches have applied fractionation factors to apportion N20
produced from either nitrification or denitrification (Perez et al., 2001). However, the
results show that fractionation factors for oxygen and nitrogen vary markedly between
replicate soil treatments (CAT 1,2,3) and are, therefore, not conservative tracers of
denitrification (Table 2). Fractionation was lowest (Table 2) in the mesocosm (CAT 1)
with the highest water filled pore space (saturated vs. 91%). This is consistent with the
observation that diffusion limits the expression of fractionation, resulting here from
differences in the magnitude of water filled pore space (Brandes and Devol, 1997).
Since variation in fractionation factors limits their application for N20 source

apportionment, site preference is an important alternative.

Table 2 Fractionation factors for N20 reduction (Reaction D) in soil mesocosms.

 

 

 

 

 

 

 

Soil mesocosm el’N 8180
treatment type'

CAT] -2.9 -0.4
CAT2 -6.8 -13.9
CAT3 -5.3 -8.6
HCS -5.9 -13.6
DF -14.6 -38.7

 

 

 

 

*CAT, conventional agricultural (replicatesl , 2, 3); HCS, historically cultivated
successional; DF, deciduous forest

In agricultural and historically cultivated successional soils, no change in site
preference with reduction in N20 concentration was observed (Fig. 2d,t). I demonstrated
that: (I) site preference can distinguish N20 produced by denitrification (Fig. 1 reaction
C) and nitrifier-denitrification (Fig. 1, reaction B) from that produced during nitrification
(Fig. 1, reaction A) (Table 1); and (2) reduction of N20 to N2 does not obscure site
preference imparted by denitrification in agricultural soils (Fig. 1d). Thus, site
preference provides a robust indicator of the process producing N20.

Unlike other mesocosms, those composed of deciduous forest soils showed a
change in site preference with a decrease in N20 concentration (Fig. 20. The associated
fractionation factor (slope) was large (-20.4 960) (Fig. 2t) and I believe differences in the
fractionation for site preference between these soils and agricultural soils reflect
differences in microbial community structure. Buckley and Schmidt (2001) showed that
microbial community structure differed between fields that were cultivated and those
with a history of conventional tillage. Our data reflect differences in process associated

with these differences in microbial communities.

 

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time (min) In (010.)
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c .
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time (h) In (010.)
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0 4 8 12 10 20 24 28 40 -0.8 -0.0 -0.4 -02 0.0 0.2
time (h) In (00.)

Figure 2. N20 concentration and isotopomer site preference for denitrification
experiments. a, Production of N20, and b, site preference expressed relative to the
natural log of the ratio of the observed concentration, C, to that of the initial
concentration, Co, in triplicate cultures of P. chlororaphis. c, Consumption of N20 and
(I, site preference in soil mesocosms from three replicate plots with a history of
conventional agricultural treatment (CATl, CAT2 and CAT3). e, Consumption of N20
and f. site preference in soil mesocosms from a historically cultivated successional field
(HCS) and a deciduous forest (DF). Regression equations for site preference vs. In
(C/Co) are as follows: b, PCl, y = -7.0+ 1.8x; PC2, y = -8.5+ 0.01x; PC3, y = -13.7+ 5.4x
d, CATl, y = -0.4 - 2.4x; CAT2, y =1.9 - 0.6x; CAT3, y = -0.2 + 3.9x f, HCS, y = 4.4 -
1.2*x; DF; y = 3.1- 20.4*x.

Toshida and Toyoda (2000) were the first to demonstrate the use of isotopomers
for N20 source apportionment by identifying the origins of N20 in the troposphere. Our
research has focused on individual N20-producing microbial reactions. Our data indicate
that denitrification imparts the same site preference on N20 whether it is performed by a
nitrifier (nitrifier-denitrification) or traditional denitrifier (denitrification) (Table 1). Site
preference imparted by denitrification was distinct from that associated with
hydroxylamine oxidation (Sutka et al., 2003) (Table 1). Furthermore, N20 reduction
during denitrification does not alter the site preference imposed on N20 during nitrite
reduction in agricultural and historically cultivated successional soil mesocosms (Fig.
2d,t). Our results and those of Sutka et al., (2003) suggest that site preference
distinguishes nitrification (Fig. 1, reaction A) from denitrification (Fig. 1, reactions B,C)
in agricultural soils. In contrast to the agricultural soil mesocosms, changes in site
preference during N20 reduction in deciduous forest soil mesocosms (Fig. 20 may reflect
a difference soil microbial community structure attributed to land-use history (Buckley
and Schmidt, 2001).

Approximately 80% of the current annual increase in atmospheric N20 derives
from agricultural soils (Veldkamp etal., 1998). Unlike other ecosystems, nitrification or
denitrification can be directly managed in agricultural systems. Previously, without the
means to distinguish the relative importance of nitrification from denitrification, we have
not been able to manage soil microbial processes to mitigate N20 flux. The ability to
apportion sources of N20 in agricultural soils with isotopomer site preference now
provides this possibility and. thus, has profound implications for the management of

global N20 emissions.

APPENDICES

APPENDIX A

Methods

Site Description and Soil Collection

Approximately 4 kg of soil was collected bytaking 20 cores from the upper 25 cm of soil
from each of three different treatment plots from the replicated series of cropped and
unmanaged ecosystems at the Kellogg Biological Station/Long Term Ecological

Research main site (http://lter.kbs.msu.edu) in southwest Michigan. Treatments included

 

conventional agricultural treatment (CAT), historically cultivated successional field
(HCS) and a deciduous forest (DF). Conventional agricultural tillage treatment crop
yields are equivalent to average yields for the USDA Central Region. A 4 mm sieve was
used to homogenize soil. Aliquots were covered and allowed to air dry for ca. three
weeks.

Mesocosm Construction

One hundred grams of dry soil was added to mesocosms (1 L glass Mason jars bearing
lids fitted with butyl rubber septa) and packed to a volume of ca. 80 ml. Water was
added to achieve ca. 85% water filled pore space (Bergsma etal., 2002). However,
heterogeneities in the soil resulted in differences in water filled pore space. We verified
that N20 production was not occurring by following the methods of Bergsma er a1.
(2002). Sealed jars were amended with 500 pl of pure N20 at atmospheric pressure,
delivered with a gas tight syringe (Hamilton).

Sample Collection and Analysis

500-uL headspace samples were taken and stored in vacutainers previously purged with

pure N2 and brought to atmospheric pressure. The gas samples were analyzed on a multi-

collector Micromass Isoprime Mass Spectrometer interfaced with a continuous flow

Trace Gas Inlet System for separation and purification of N20. This mass spectrometer
has been constructed with collectors to simultaneously measure the 5 masses of interest
for N20 isotopomers; 30, 31, 44, 45 and 46. The S'SN, 5'80 and SUN“ values are
obtained from the ratio of the 45:44, 46:44 and 31 :30 ion beam ratios, respectively.
Corrections are applied for the contribution of 170 to masses 31 and 45 and for the minor
rearrangement of '5N between the or and 0 positions within the ion source (Brand 1995.
Toyoda and Yoshida, 1999; Breninkmeijer and ROckmann, 2000; Sutka et al., 2002).
The value of ZS'SN‘3 is calculated given that 8'5N is the average of 5'5N“ and

SISNB Toyoda and Yoshida, 1999; Breninkmeijer and Rdckmann. 2000).

P. chlororaphis Cultures

Pseudomonas chlororaphis was cultured from a frozen stock (ATCC 43928) provided by
J.M. Tiedje, Michigan State University and maintained on Tryptic Soy Broth (TSB;
Difco) amended with 5 mM KNOj. Concentrated cell suspensions made from starter
cultures provided sufficient concentrations of N20 for mass spectrometric analysis. These
suspensions were derived by inoculating ten 50 mL serum bottles containing 20 mL Of
TSB medium with 0.1 mL of starter culture. After 48 hours of incubation at 22 °C cells
were concentrated (centrifugation: 8,000 g for 10 min at 5 °C). The cell pellet was
washed twice with 20 mL of 0.1 M pH 7.5 phosphate buffer containing 171 uM CaCl2,
and 78.7 uM MgCl2 and resuspended in 20 mL of sterile TSB. For each replicate, a 25
mL butyl stoppered serum test tube (Bellco) was prepared with 3 mL of the concentrated
cell suspension and 1 mL of a 0.01 M KN03 stock solution. The tubes were stoppered
with a N2 headspace and incubated at 22 oC. Nitrous oxide was sampled from the

headspace approximately every 20 minutes using a 100 or 500 uL gas tight syringe. The

ll

gas sample was immediately injected into a Trace Gas system (Micromass) interfaced to

an Isoprime (Micromass) mass spectrometer.

APPENDIX B

13

515N bulk

815N bulk

815N bulk

CAT 1

 

 

 

 

 

0.2

 

 

 

 

_2 J . . .
-0.8 -0.6 -0.4 -0.2 0.0
In (C/Co)
CAT2
6 \fl .
4 .
2 - I ,
1:3
It-
0 _ \
_2 . . . .
-0.8 -0.6 -0.4 0.2 0.0
In (C/Co)
CAT3
6 v

0.2

 

 

 

 

 

-0.8

-0.6

-0.2
In (C/Co)

-0.4

0.2

5180

8180

8180

 

 

 

 

 

 

 

 

 

 

CATI
24 -
20
16 . 1
.3! .‘0
12- o 5 ‘0 °
0.8 -0.6 -0.4 '02 0.0 0.2
In (C/Co)
CAT2
24 '
20 '
16 '

12 u 4
-0.8 -0.6 -0.4 '02 0.0 0.2
In (C/Co)

CAT3

 

 

 

 

 

-0.4 -0.2 0.0 0.2

In (C/Co)

-0.6

Figure 3 SN and 6180 vs. In (C/Co) for conventional agricultural treatment mesocosms. a,
r2 = 0.4052; r = 06366, p = 0.0045; y = 0414880127 - 2.8579479'x b, r2 = 0.0035; r = -
0.0595, p = 0.8147; y = 136945294 - 0.36836952‘x c, r2 = 0.9154; r= 09568, p = 0.0002;
y = 0.955740909 - 6.75726686‘x d, r2 = 0.8137; r = 09020, p = 0.0022; y = 165163472 -
13.8830931'x e. r2 = 0.6045; r = 07775, p = 0.00001; y = 0338244567 - 5.28180685'x

t, r2 = 0.5114; r= 07151, p = 0.0001; y = 146636102 - 8.63820581'x

 

 

 

 

 

 

 

 

 

 

 

HCS HCS
32 - O
’9
28 -
O . .
x
.8 8 24 -
Z .-
59 "° 20 - ’
(O
16 - ‘9
9 O) .
0
12 .
_2 I I A A a A A a a
-1.0 -0.8 -0.6 -0.4 -0.2 -1.0 -0.8 -O.6 -0.4 -0.2 0.0 0.2
In (C/Co) In (C/Co)
DF DF
\ 32 u \ 4
6 . .t‘g . ....
.l 28 * 4
i‘ j!
3 4 . 1g 0 24 I I 4
e °—° "
1— 0° . 1
0° 2 . . 20
I I
16 -
0 - I
12 E

 

 

 

 

 

 

_2 A A A A A
-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2
In (C/Co)

-1.0 -0.8 -O.6 -0.4 -0.2 0.0 0.2
In (C/Co)

Figure 4 WM and 5‘80 vs. In (C/CO) tor historically cultivated successional (HCS) and
deciduous forest (DF) mesocosms. a, = 0.3562; r = 05969, p = 0.0114; y = 0.0803007579 -
5.67524563'x b, r2 = 0.3672; r= 06060, p = 0.0099; y = 126993928 - 13.6296028'x

c, r2 = 0.8590; r = 09268. p = 000000003; y = 0906057787 - 14.621457'x d, r2 = 0.8845;

r = 09405, p = 0000000007; y = 14.7188548 - 38.6903078'x

 

f‘j‘vf'vvva

   

815N bulk
is

 

p
p
P
b
h

vvvvvvvvvvvvvvv

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1
1
4
4
1

 

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
In (C/Co)

PC2

 

815N bulk

 

 

 

 

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

In (C/Co)
PCS

 

815N bulk

 

 

 

 

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

In (C/Co)

8180

8180

8180

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Flgure 5 815N and 8180 VS. In (C/Co) for Pseudomonas Chlomraphis (PC) cultures. a, I’2 = 0.9838; r =
0.9919, p = 0.0009; y = -15.5253953 + 6.89719754‘x b, r2 = 0.9817; r = 0.9908, p = 0.0011; y =
24.252223 + 8.71316266*x c, r2 = 0.9833; r = 0.9916, p = 0.0009; y = -16.1674224 +
10.2834111‘x d, r2 = 0.9920; r = 0.9960, p = 0.0003; y = 24.8183474 + 9.17864406'x e. r2 =
0.6670; r = 0.8167, p = 0.0473; y = -19.3910815 + 7.08615027*x r, r2 = 0.9721; r = 0.9860. p =
0.0003; y = 19.9194665 + 9.89224398‘x

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