its a research paper and I have to review it and grade it with well stated comments I’ve attached the research p

its a research paper and I have to review it and grade it with well stated comments I’ve attached the research paper, peer review form, and proposal grade sheet


Biology Science Foundation
BSF FORM 1
PROPOSAL EVALUATION FORM
PROPOSAL NO.
INSTITUTION
PRINCIPAL INVESTIGATORS:
BSF PROGRAM: GLOBAL AND REGIONAL
ENVIRONMENTAL CHANGE
TRIAD NO:
TITLE:
Please evaluate this proposal using the criteria given to you in the lab manual. Please use only the space in this form.
You may wish to xerox this form before printing onto it in case alignment errors need to be corrected.
Overall Rating:  Excellent  Very Good  Good  Fair  Poor
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For Official Use Only:
PROPOSAL GRADE SHEET
Section
Aspect Graded
Cover Sheet
Table of Contents
Project Summary
Introduction
Background
Objectives
Research Plan
Significance
Literature Cited
Biographical
sketches
Budget
Format
TOTAL GRADE
Comments:
Cover Page Complete
Title (descriptive?)
Pages Numbered, etc.
Contains Summary of All Sections
Clarity of Writing
Significance of Proposal Clear
Qualitative (other)
‘Big Picture’ Rationale Developed for Study
Rationale for Focusing on Specific
Factor/Species/Genotypes Developed
Qualitative (how it reads, etc.)
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citations, etc.)
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history/process (adequate citations, etc.)
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Qualitative (other)
Clearly Stated Purpose/Questions
Design, Replication, Factors, Levels
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Dependent Variables (What? How measured?)
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Qualitative (clarity of plan, writing, etc.)
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Qualitative
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Compliance with requested proposal format
Points
Possible
2
2
2
3
3
3
2
3
3
2
5
5
5
3
5
6
5
5
5
5
3
3
2
3
2
3
3
3
2
2
100
possible
Points
Awarded
Cover Sheet For Proposals To The Biology Science Foundation
Program Announcement/Closing Date
For Consideration by
BSF Organization Unit
Global and Regional Environmental Change BSF-19-1 / Feb.
2, 2019
Ecology and Evolution
Program
Title of Proposed Project
The role of an active soil microbial community in mitigating the acid mine drainage stress on
Salix Purpurea
Requested Starting Date
February 20th, 2019
Investigators
1. Molly Roberts
2. Rand Al-Fatlawi
3. Faris Alsaggaf
Proposed Duration
March 29th, 2019
Signature
Molly Roberts
Rand Al-Fatlawi
Faris Alsaggaf
Biology 321, Laboratory Section
C08
Teaching Assistant: Joseph Carrara
Triad No: 20
Date to Resubmit
Authorization
Date Approved
Authorization
Table of Contents
I.
Project Summary…………………………………………………………………………..3
II.
Project Description………………………………………………………….…………3-15
A.
Introduction……………………………………………………………………..3-5
B.
Background……….………….…………….………….…………….…………3-15
1.
Salix Purpurea………………………………………………5
2.
Acidic Water on Salix Purpurea……………………………5-6
3.
Microbial Soil on Salix Purpurea…………………………..6-7
4.
Microbial Soil and Acidic Water on Salix Purpurea………..7
C. Objectives, Hypothesis, or Questions…………………………………………………7-8
D. Research Plan…………………………………………………………………….…9-10
1.
Experimental design……………….……………..…………..9
2. Methods…………………………….…………………………9-10
3. Safety precautions……….……………………………………10
4. Statistical Analysis……….…………………………………… 10-11
5. Research Schedule……….…………………………….………11
E. Expected significance……….………….………………….……………………..……12
F. Literature Cited……….………….…………….……………………………………12-1
III.
Biological Sketches……….………………………………………….………….……15-16
IV.
Budget……….………….…………….………….……………………………………16-19
1. Budget Explanation……….………….……………………….18
2. Equipment Supplies……….………….……………………….18
3. Computing Services……….………….……………………….19
4. Facilities……….………….…………….………………………19
5. Special Equipment……….………….…………………………19
I.
Project Summary
Mining is an essential industry, especially east of the Mississippi, that contributes to
economic and social development, however, the stress implemented on vegetation is inevitable.
The proposal study will attempt to identify the role of an active soil microbial community in
mitigating the acid mine drainage (AMD) stress on the model species, Salix Purpurea. The
specific stressor that will be investigated is pH of acid mine water. Salix Purpurea will be grown
in an autoclaved sterile and microbial rich soil while being introduced to average natural
rainwater with a pH of 5.6 and common acid mine water with a pH of 3.5. The effects on root
mass and length, leaf count and quality, chlorophyll content, and overall length of the plant will
be studied. The relationship between an active soil microbial community and its ability to
compensate for acid mine water will aid in the understanding of vegetative development, which
impacts CO2 production that ultimately provides further insight on the greenhouse effect.
II.
Project Description
A. Introduction
Plants are critical aspects in maintaining the stability of life and play a major role in food
chain and global ecology. Unlike humans and animals, plants are photoautotroph which means
they have the ability to derive their own energy for food synthesis (Huner., et al. 2012). Since
plants are photoautotrophs, humans and animals depend on them completely as a food source.
Using photosynthesis, plants help increase O2 levels in the atmosphere, provide food, and
maintain major ecological functions (Fernando, 2012). On the other hand, plants release carbon
dioxide in the atmosphere as they respire. This plays a major role in greenhouse effect and global
warming. Recent studies show that the amount of carbon dioxide released by plants has
increased by 30%. It is also expected that more carbon dioxide emissions will occur as the
3
temperatures continue to rise (Huntingford, Atkin, et al., 2017). Plants can not only be found in
forests and areas of high vegetation, but also in modern cities and deserts. The study of plants is
considered very hard and complex due to the different phases and changes a seed goes through in
its cycle to becoming a plant (Fernando, 2012).
Acidic water is a result of nitrogen monoxide and sulfur dioxide emitted from mines
reacting together with the water and vapors in the atmosphere. As acid mine water gets deposited
in soils, it will largely affect the composition, length, roots, and stems of plants (Lal, 2016). It is
highly considered a rate-limiting factor to plant growth, due to the high amounts of ions it
contains that contribute to toxicity. Therefore, acidic water has been known to significantly
deplete the growth rate of plants. The main ions that contribute to toxicity are Al3+ and H+ (Kidd
and Proctor, 2001).
Microbial soil is a host to animals, protists, bacteria, and fungi along with the plant root
system. It is the primary source of nutrients to all of these organisms and microorganisms. This
combination of microorganisms and plants can create diverse relationships in which they can
benefit each other, symbiotically, or may cause disadvantages to one another, competitive,
(Jacoby, Peukert, et al., 2017). The microbial soil can aid in growth of plants by beating
pathogenic strains, increasing the amount of nutrients found in the soil, and controlling plant
signaling hormones (Verbon and Liberman, 2016).
Interactions between factors of the microbial consortia are essential for the compensation
of acidic mine water and the microbial community. For instance, the metabolic pathways within
the soil microbial community aid in the maintenance of pH homeostasis and metal resistance
mechanisms (Baker and Banfield, 2003). The microflora community within soils will commonly
retard adverse effect implemented by the acidic pH by compensating the nutrient imbalances
imposed on vegetation. Therefore, it can be expected that the effects of acidic mine water on
plant growth will be mitigated by the microbial soil (Ledin and Pedersen, 1996).
B. Background
1. Salix Purpurea
Salix Pupurea, also known as purple willow, has a set of characteristics which makes it a
model organism representing vegetation as a whole and a primary organism of choice for this
experiment. It grows in a wide range of habitats, acquires an extensive root system, withstands
high concentrations of toxic elements, and degrades soil contaminants (Pagé, Yergeau et al.,
2015).
Plants of the Salix species were also used as herbal medicine in older civilizations. It was
believed that these plants worked like medicine alleviating pain and treating several conditions.
From a scientific point of view, the willow or Salix species displays aspirin-like properties such
as anti-inflammation (Mahdi, 2010).
It is expected that when treated with acidic water, the growth of Salix Purpurea will not
be highly affected as it will help degrade contaminants and toxic elements lessening their effect.
2. Acidic Water on Salix Purpurea
PH is the measure of hydrogen and hydroxide concentrations. Water with a pH lower
than 7 is said to be acidic, and higher than 7 is said to be basic (Covington, et al., 2009). Water is
a major element in plant growth as it enters plants’ cells and aid in a lot of cellular activities. The
acidity of water will alter the activities it can perform in a cell and thus alter plant growth and
physical structure (Allawy, 1957). Acidic water can decrease the number of nutrients available
for plant absorption causing a slower deteriorated growth. A study done on Pinus Strobus
showed opposite results. Although the 2.3 pH water caused a loss in the amounts of potassium,
calcium, and magnesium present in the soil, the plant’s seedlings productivity was boosted up
during the period of experimentation which was 20 weeks. Reasons for that may be linked to
fertilization (Wood and Bormann, 1977). Another study performed on hardwood showed that all
species treated with a pH of 2.0 showed retarded growths and no germination while species
treated with a pH of 3.5 to 5.0 showed an increased seedling growth (Fan and Wang, 2000). The
results of both experiments support the expected results for the effect of acidic water on Salix
Purpurea.
3. Microbial Soil on Salix Purpurea
Microbial soil is a soil containing microorganisms like bacteria and fungi. A lot of
plants growing in microbial soils develop a symbiotic relationship with the fungi in the soil
which yields benefits for both. It has been found that microbial soil can protect plants against
diseases, help plant withstand drought and stressful conditions, and also improve plant nutrition
(Jayne, 2012). Plant growth promoting rhizobacteria (PGPR) is a type of bacteria that grows in a
lot of microbial soils. Theses bacteria influence a better growth for their host plants. Some PGPR
boost plant growth by increasing their ability to absorb nutrients, by fighting pathogens, or
increasing root growth (Vessey, 2003). Phosphorus is an element that contributes to slower
growth rate for plants. It is present in both microbial and sterile soils. Phosphate solubilizing
microorganisms (PSMs), however are only present in microbial soils, and they aid in providing
phosphorus to plants in more ecological and efficient way (Gyaneshwar, Naresh Kumar, et al.,
2002).
Sterilized soil is a soil that lacks the existence of microorganisms. Sterilized soil can
affect plant growth because it is an altered soil with different physicochemical structure and
composition than normal microbial soils (Salonius, et al., 1967). A study shows that plants
grown in a sterile soil displayed an increased root mass and wheat seedlings weight than plants
grown in a non-sterile soil (Tareq, Samina, et al., 2014). Another study shows that during heatsterilization of soil, toxins can form and retard growth of plants and alter their physical
structures. It shows that microorganisms in microbial soils act as detoxifying agents to these
toxins and therefore boost plant growth (Rovira and Bowen, 1966). Furthermore, it would be
expected that the microorganisms in microbial soil will compensate for the hazards acid rain
might cause on the growth of Salix Purpurea.
4. Microbial Soil and Acidic Water on Salix Purpurea.
There are little to no studies conducted that tested the effects of both microbial
soil and acidic water on plant growth. One study shows the effect of acidic water on the bacterial
and fungal communities present in the microbial soil. It was found out that in a pH range of 4,
the bacterial community increased in number by a double while the fungal communities were not
affected (Rousk and Bååth, 2010). This supports the expected findings that microbial soil will
support the plant and its growth against the stressor, which is going to be acidic water. It also
demonstrates that the low pH of water will increase the bacterial communities in the soil which
will provide more support and boost plant growth.
C. Objectives, Hypothesis, or Questions
Question 1: Will an active microbial rich soil community mitigate the stress imposed by acid
mine water to Salix Purpurea ?
At the conclusion of the experiment, measurements of the root tip to the apex, the crown
to the apex, the mass and length of the root system, and chlorophyll concentrations will be
obtained. These values will provide quantitative data of each treatment group in order to identify
whether, or not, the overall health of the plant when introduce to acid mine water was impacted
by the soil media. It is important to obtain measurements above and below the soil in order to
identify where and if plant depletion occurs. It was hypothesized that when introduced to acid
mine water plants grown in microbial soil will have an overall higher value in each of the
quantitative measurements compared to the plants in the sterile soil.
Question 2: Does the pH of water cause significant growth defects and negative health effects on
the model organism, Salix Purpurea ?
With the qualitative measurements of each treatment groups a one tailed, T-test will be
ran in order to obtain a p-value to identify the probability of whether the results occurred by
chance or due to the acidic pH water. Identifying if the pH of water causes significant growth
and health effect is imperative to the results of this study. We hypothesized that when using a
significance level of 0.05 the p-values of the quantitative measurements will be less than 0.05,
meaning there is less than a 5% chance that the growth effects occurred by chance instead of
being derived from the acidic water.
Question 3: Does the quality of the Salix Purpurea deplete from acid mine water exposure?
Along with qualitative measurements, the study will also evaluate the overall quality of
health of the plant species. These observed effects of the plant will provide insight on whether
this plant will be able to thrive and reproduce. It was hypothesized that the Salix Purpurea
exposed to acid mine water, pH 3.5, will have obvious signs of depleted health and unstable
stems, poor budding, and brittle leaf production compared to the Salix Purpurea that’s watered
with natural rainwater, pH of 5.6.
D. Research Plan
1. Experimental Design
Two factors will be investigated throughout the durations of the experiment: the impact
of an active microbial rich soil community compared to a sterile soil on plant health when
introduced to acid mine water and natural rainwater. A model organism, Salix Purpurea, will be
used as a representative species for vegetation as a whole. The plants will be potted in either a
microbial rich soil or a sterile soil. The water source for the plants will have two distinct pH
values 3.5, to represent acid mine water, and 5.6, to represent natural rain water.
2. Methods
Salix Purpurea plants, Fish Creek genome, were grown hydroponically. These plants will
be provided and obtained from the Life Science Building greenhouse at West Virginia
University. A total of forty plants will be used; each will be potted in 5×6 inch pots. Twenty of
the plants will be planted in sterile soil and twenty will be planted in microbial rich soil. Ten
plants from the twenty, within the sterile soil, will be watered with acidic water (pH 3.5) while
the other ten plants will be watered with natural rainwater (pH 5.6). This same watering
procedure will be implemented on the twenty plants potted within the microbial rich soil. In
order to obtain the desired pH values for the water sulphuric acid will be added to water and the
pH will be tested using pH test strips. Throughout the entirety of the experiment all plants will be
assessed three times a week and watered to keep the soil moist. All plants will be subjected to
room temperature and equal light exposure. At the completion of the experiment the following
measurements will be recorded: root tip to apex, the crown to the apex, total root length and
mass, total chlorophyll content, and overall plant quality of health.
Chlorophyll concentration procedure: To obtain chlorophyll content, pour 5 cm3 of
Dimethyl Sulfoxide (DMSO) onto half-gram samples of C leaves without any leafstalk, then
incubate it for an hour in 65 °C to be cooled. After the cooling process, the content of
chlorophyll was determined spectrophotometrically (UV–VIS spectrophotometer, Jasco V 530)
using a 20-fold samples diluted DMSO and chlorophyll at three wavelengths 480, 649 and 655
nm. Calculate the chlorophyll a, chlorophyll b, and total chlorophyll a+b contents using the
Arnon’s formulas (Borowiak, Klaudia, et al. 2015).
3. Safety Precautions
Acidic water, with a pH of 3.5, will created and used throughout this experiment
therefore all experimenters will follow standard precautions when handling and watering the
plants. These standard precautions include: lab apron, gloves, eye protection, no loose articles of
clothing, and long hair will be tied back. Also, the acidic water (pH of 3.5) will be kept isolated
from the rainwater (pH of 5.6) to avoid any cross contamination between treatment groups.
4. Statistical Analysis
In order to determine if the impact from acid mine water on the Salix Purpurea’s growth
rates, while being grown in different soil medias, is by chance a one-tailed T-Test will be
performed. The two factors are soil type and water acidity, using SAS JMP program we will use
statistical data, fit model and ANCOVA to conduct a graph to study the relationship between
solid types and water acidity looking over the chlorophyll contents. A bar graph will be made to
show the T-test performed on the data we will collect to study the significance of the experiment.
For this experiment, a
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