The Death Zone Poison
Background
What is Eutrophication?
Eutrophication is a process caused by the gradual increase in the
concentration of phosphorus, nitrogen, and other plant nutrients in the
biosystem, which are introduced into the ecosystem primarily when streams
wash away soils from land. Debris, products of reproduction, and dead
terrestrial organisms are then deposited in water bodies as they enter the
ecosystem. The overabundance of nutrients in water bodies results in
harmful environmental effects. As the organic materials break down into
nutrients, the productivity or fertility in the ecosystem increases. A
great concentration of algae and microscopic organisms in the water bodies
feed on the nutrients, growing, propagating, and forming unsightly algae
blooms on the water surface. When the scum on the water surface blocks
light penetration, plants produce less oxygen for underwater life.
Moreover, dead algae are decomposed by bacteria and produce putrid odors
or even release toxins in the water. The bacteria will consume oxygen in
the water during the decomposition process and deplete the oxygen needed
to sustain life. Ultimately, without oxygen, bodies of water become "dead
zones."
Fig. 1 An image of marine polluted by the phytoplankton bloom
Why are excess phosphates a problem?
Ammonia (NH3), Ammonium (NH4+), nitrate (NO3-), and phosphates are the
primary forms of nitrogen and phosphorus in water bodies. Phosphate is a
limiting nutrient, which means only limited solubility of phosphates is
required for organism growth in aquatic ecosystems. In other words, excess
quantities of phosphorus will result in problems of water quality such as
eutrophication and harmful algal growth.
In the past two decades, human activities exacerbated the release of
phosphorus. Discharges of agricultural and industrial wastes, applications
of inorganic fertilizers, along with increased soil erosion and landslides
from fields result in accelerating phosphorus transport into aquatic
systems. As described, restricted nutrient levels can often limit the
productivity of bacterioplankton and algae. However, organic and inorganic
phosphorus from soils has increased in the water system due to land
erosion. As a result, the overall nutrient concentration in the water
bodies rises to unnaturally high levels. Bacteria and algae, thus, are
allowed to flourish and deplete oxygen in the system.
Fig. 2 Pollution of water bodies by chemical fertilizers
A Global Issue
Given the widespread extent of water quality degradation caused by
nutrient enrichment, eutrophication has been and continues to be a major
global challenge. A study by the United Nations Environment Programme
indicates that 30~40% of lakes and reservoirs around the world have been
affected by eutrophication. Evidently, it poses a serious threat to the
aquatic ecosystem, yet current solutions are either ineffective or
extremely costly. Tainted drinking water supplies and the degradation of
recreational opportunities, for example, can be detrimental to human
health and well-being. Eutrophication as well contributes to the spread of
gastrointestinal and dermatological diseases such as conjunctivitis and
hypoxia. In the US alone, the combined costs of the treatments for
eutrophication in freshwaters are approximately $2.2 billion annually
(Dodds, et al).
Current Solutions
Eutrophication is one of the major environmental issues today. There have
been studies that provide great notions and methods that can be applied to
solve the problem of eutrophication.
According to Natural Resources 235, the best way to cope with
eutrophication is to reduce and manage the amount of fertilizers humans
use for agriculture. The use of fertilizers is one of the major reasons
behind eutrophication. However, there are already a lot of dead zones,
which are bodies of water that are hypoxic. Therefore, taking preventative
measures would not be the most idealistic way to cope with the current
condition. Most of the 27 reservoirs in Taiwan are affected by
eutrophication (Lo Chi & Jake Chung). Mainly, the Taiwanese government has
two current solutions to deal with eutrophication. The two methods being
Multi-soil Layering (MSL) and Biomanipulation.
Multi-soil Layering is a system introduced by Japanese researchers to
decrease pollution and effectively remove phosphorus in the bodies of
water (“水庫優養化剋星─多層複合濾料(MSL)”). MSL contains two layers to
filter and extract phosphate. This system has the benefits, such as the
use of natural materials and high efficiency. The total phosphorus (TP)
removal efficiency is approximately 92% to 99.2% (Chia-Chun Ho & Pei-Hao
Wang). In Taiwan, recently, MSL has been successfully implemented in
Longtan Pond, Taoyuan. The system is placed near or juxtaposed to the
reservoir, ensuring the cleanliness of the pond. Unfortunately, the cost
of this system comes at a relatively high price. It is estimated that
the whole cost is about $10,000 USD (Chia-Chun Ho & Pei-Hao Wang).
Compared to the average cost for Annual Pond Maintenance Cleaning, which
is about $2,000 USD, it is relatively expensive. Though this MSL is
great, the main downside is the cost.
The other solution implemented by the Taiwanese government leans toward
a biological approach, which is biomanipulation (歐士豪).
Biomanipulation is the use of ecological techniques to solve
environmental problems. By reducing the number of planktivorous fish,
the density of large cladoceran zooplankton increases; hence, the growth
of algae is reduced. (L.-A. Hansson, C. Brönmark). This method is known
as the fish-stock biomanipulation. The cost, efficiency, and time of
this approach is unforeseeable due to the environmental variability. The
time needed to clear the water was four weeks in this fish-stock
biomanipulation, which is efficient and effective in this example. In
spite of that, the flaw that holds this method back is its lack of
instability because biomanipulation is dependent on the environment.
Some environments are suitable for biomanipulation, but others may not
(Gerard ter Heerdt & Michiel Hootsmans). Therefore, the success rate of
operating biomanipulation is uncertain.
To summarize above, both MSL and biomanipulation are considerable
solutions to eutrophication. The major problem of MSL is the high price;
meanwhile, biomanipulation lacks stability. Due to those problems, we
designed our project to modify the MSL system into a better version.
Our Solution
Our approach to solving eutrophication is to engineer cells of
Escherichia coli
to decline the external phosphorus level, which is the main reason behind
eutrophic waters. The plan is to overexpress target genes, specifically
Organophosphate Hydrolase (OPH) and an antisense mRNA for phoU (AsPhoU),
that hydrolyze organic phosphate into inorganic phosphate (Pi), catalyze
Pi transport, and polymerize Pi into Polyphosphate (PolyP), which can
ultimately be stored in the bacteria to reduce the overall amount of
phosphate in the surrounding bodies of water.
Fig. 3 phosphate uptake in the presence and absence of AsPhoU
Through overexpressing OPH, our E.coli is able to hydrolyze
organophosphates, such as Paraoxon-methyl, into p-nitrophenol (pNP) and
Dimethyl phosphate (DMP) (Grimsley JK). Glycerophosphodiesterase (GpdQ)
then hydrolyzes DMP into inorganic phosphate for later use
(“Glycerophosphodiester Phosphodiesterase Gpdq.”).
By producing AsPhoU, we are able to eliminate phoU expression, thus
enhancing PstSCAB expression. The PstSCAB proteins then transport Pi into
the bacterial cytoplasm via the ABC-type transport systems – with PstS as
the periplasmic Pi binding protein, PstC and PstA as integral membrane
proteins, and PstB as the ATP binding protein (William R. McCleary).
Furthermore, the low concentration of Pi induces the expressions of PhoB
and PhoR genes from the Phosphate regulons. The histidine kinase sensor
PhoR subsequently catalyzes the activation of the response regulator PhoB,
whereas the activated PhoB positively regulates the PstSCAB transcription
operon and enhances Pi intake into
E. coli
, as explained above (William R. McCleary).
After Pi is intaken,
E. coli
activates expressions of PhoB and PhoR genes from the Phosphate regulons.
The histidine kinase sensor PhoR subsequently catalyzes the activation of
the response regulator PhoB, whereas the activated PhoB positively
regulates the PstSCAB transcription operon and enhances Pi intake into
E. coli
, as explained above (William R. McCleary).
Lastly, Polyphosphate kinase (PPK) is responsible for the synthesis of
polyP in E.coli (Ye Zhu). As a result, excess polyphosphate is stored in
bacteria, therefore diminishing the concentration of phosphate in the
water body, reducing the overgrowth of algae, and inducing the amount of
oxygen available for aquatic ecological growth.
Inspiration
At the earliest stage of research, we came up with ideas spanning an
impossibly wide area of expertise, from diagnostics and therapeutics to
food and nutrition. Yet the more we expanded the scope of our topics, we
realized that there is no better place to start than from our own living
environment. We were astonished to learn that sixteen out of the
twenty-six reservoirs in Taiwan are affected by the over-enrichment of
nutrients (水質處生物組). Through synthetic biology, we hope to approach
such a multifaceted issue from a different angle and develop a creative
but feasible solution.
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