Team:HK GTC/Human Practices
Integrated Human Practices
Interviews with Professors
Interview with Professor Stanley Lau, HKUST
According to Professor Lau’s opinion, the water quality of Hong Kong isn’t extremely good, as Hong Kong is a city and activities on land will always make an impact on local water quality, for example, seawater in Hong Kong appears green because nutrients from land are flushed into the ocean. The water quality around the Eastern coast is better, while waters around the Western coast has more suspended particles discharged from Zhujiang. He suggested that we can refer to the marine water quality objective the HKSAR government made, which divides Hong Kong’s waters into 9 sites, but it needs updating. during the 1980s (industrial era), the government carried out a water quality administration to measure the amount of E. coli and quantify by 4 levels by monitoring the health condition of volunteers after full body immersion at 9 local beaches. Professor Lau also pointed out that although reclamations led to some areas of water disappearing recently, overall Hong Kong’s water quality has been improving these years.
Professor Lau gave us a brief summary during the interview regarding how bacteria diversity in the ocean is affected by marine pollution. Foreign bacteria either survive or die in the ocean. If they survive then they will be competing against local bacteria or cyanobacteria, which may lead to a decrease in the oxygen volume in the sea. Large, continuous changes will also appear due to pollution, bacteria composition will be seriously affected, which may lead to a change in biogeochemical processes and the overall ecosystem due to a bottom-up effect. Pathogens once affect domestic animals, such as humans and pets, may infect marine creatures too once in the ocean.
Professor Lau has been making attempts to track water pollution by using gene markers. Different bacteria come from different pollution sources and are unique organisms, therefore its gene marker and the amount can be tracked, and the seriousness of pollution can be estimated. As for biological approaches to reduce pollution, the professor pointed out that currently the biggest obstacle is that a large amount of persistent organic matter, such as antibiotics and sunscreen, which cannot be degraded in regular water treatment, are present in waters. Plus, government regulations don’t help too much, because in order to avoid government regulations, manufacturers simply change the compound structure while the property of the pollutant remains. Professor Lau suggested that activated sludge could be used to degrade antibiotics.
Regarding our project on PET plastic degradation, the professor gave us some suggestions and advice during the interview. In order to degrade PET plastic completely to simple and inorganic molecules, more than one kind of enzyme or bacteria may be required. As for mutant designing, he advised us to do modeling instead of trial and error as it would be less time-consuming. Plus, he suggested that we should repeat others’ experiments before experimenting on original thoughts, as we can learn techniques and gain experience from it. He also reminded us to be aware for details which are sometimes not mentioned in papers which may result in a completely different experiment result if left out. He thinks PETase would be a long term solution to tackle plastic pollution, and it would be more effective and suitable if executed in a large, singular event.
If biological approaches are really used to tackle pollution problems, the professor thinks that the possible effects brought to the marine ecosystem depend on the situation. He suggested two ways of biological approach: one is to allow bacteria to digest all harmful compounds and then they will simply die and be decomposed after they finish digesting due to a lack of energy source. Another possible method is to use natural on-site bacteria which has a small population, then they will die after consuming all pollutants, so there won’t be severe damaging effects on the ecosystem.
Soon in August we will have the opportunity to visit Professor Lau's lab in order to learn more about microbiology and marine biology. Also, after the interview, our team would be more aware of the biological effects our project may bring to the environment and ecosystem. We may also carry out modelling to simulate real-life scenarios, and integrate other enzymes in the digestion process of PET plastic.
Lab Visit to Prof. Lau's Lab, Aug 21st
Introduction
The students visited Professor Stanley Lau’s Ocean Science lab in The University of Science and Technology. They also briefly visited the Biosciences Central Research Facility, which houses a lot of state-of-the-art apparatus. In total, 12 team members attended the event. Clare, the lab assistant of Professor Stanley Lau was the contact and led us through the laboratory.
The purpose of the visit is to learn more about advanced apparatus and take a look at the laboratory of a professor specializing in microbiology. We hope to understand the experiments they’re doing and to know how they do the experiments.
The visit
After we entered the lab, there was initially a presentation on what Professor Lau and his team were working on, which is the detection of Fecal Coliform bacteria in water to find out the water quality and a selective method to find from which animal the bacteria come from, which is also the pollution sources. Hong Kong uses Escherichia coli to monitor the water quality of beaches and grade them on a scale of 1-4 according to the E. coli concentration in water.
Then we went to see the essential apparatus of the lab: These includes light microscopes, gel tanks for DNA gel electrophoresis, selective agar plates with colonies of different colours indicating the strain of bacteria, and membrane filters that extract bacteria from the water so the morphology of the bacterial colonies can be observed and identified.
Thereafter, we progressed to see the more recent equipment they have, which is a droplet digital PCR machine, spectrometer, and nanodrop spectrophotometer. The ddPCR is used to directly quantify and amplify nucleic acids precisely as they assort DNA molecules into separate droplets before PCR reaction. The Nanodrop spectrometer is able to find the light absorbance of different wavelengths of a single drop of solution and is widely used to determine the concentration of substances in a solution.
Subsequently, we observed the biohazard cupboards. Heterotrophs, autotrophs and RNA all have their own biohazard cupboard to avoid cross-contamination, and they are all equipped with UV lights to sanitize equipment and the environment. In the same room there were more large-scale centrifuges.
We were then taken to the autoclave room where they sanitize all the types of equipment with high-pressure water vapor and then to dry them out in a large oven.
After that, 7 of us were taken to the Biosciences Central Research Facility. Inside, they saw Quantitative real-time PCR (Q-PCR) machines, which is exceptionally useful for monitoring amplification of DNA molecules and quantify them. We also saw Next-Gen Sequencing technology, which can sequence multiple genes quickly at the same time. Mass spectrometers that can quantify known materials or identify unknown ones. Also a cell sorter, and a slicing machine to precisely slice samples.
Conclusion
We showed our interests and actively participated in the visit. The visit gave us the opportunity to understand various types of equipment in the laboratory and gain knowledge about procedures and equipment. It also reinforced our connections.
Lab Equipment:
1--384 well-plate Quantitative PCR
2-- Computer Analysis
3-- Gel Imaging system
4-- Droplet Digital PCR
5--Lab bench
6-- Omnilog Microarray System
7-- Cryostat
8-- Cell Sorter
Interview with Professor Christelle Not, HKU
First Interview
Microplastic pollution in water is a very serious problem in Hong Kong, comparing to other countries in Europe as the plastic per liter of seawater in Hong Kong is much higher than that in Europe. The plastics polluting oceans in Hong Kong are also different from those in Europe, as the plastics found in samples from surface manta trawls are Styrofoam and Polystyrene, which does not match the research results in Europe. However, there is no actual data that reflects what the specific sources of pollution are.
Prof. Not has brought students to study seawater and quantify microplastics. She has also realized there are only a few papers or information sources on microplastics, and there are contradictions between different papers. Plastic consumption has caused severe pollution in the ocean, as microplastic ingestion by marine organisms can be observed. According to investigations, microplastics can be found in bodies of half of the mullet fish. Also, almost all crabs in mangroves contain debris. Moreover, more microplastics are present in the oceans after rain. This shows that microplastics in the sea mostly come from local sources like rivers in cities. Prof. Not has also found out that the degradation of plastics generally occurs on beaches, and that tides usually bring microplastics away from sand to the water. Plastic microbeads are a giant problem in today’s oceans since there aren’t any solutions to remove microbeads from the ecosystem. Physical methods such as using nets may work but it is very time consuming and hard.
Currently, there is no policy from HKSAR government to restrict the use of microplastics and other microbeads. They may be still planning for such policies, however, the government lacks scientific data on marine pollution due to microplastics. Prof. Not states that banning the usage of single-use plastics is not suitable as it will cause mass disagreement within the community. She thinks that the government should limit the usage of single-use plastics instead.
PET is a plastic that is harmful to the environment. Due to PET being highly malleable and very resilient, it is very commonly used. The large amounts of PET disposed to the environment further increases its physical and chemical impacts on the environment. Prof. Not thinks that biotechnologies such as PETase may produce potential and effective solutions and that they should work on plastics with any sizes. However, it is unclear whether the same solution works for contaminated plastics, such as food containers. Also, enzymes are specific and selective, and a single enzyme may not work for multiple plastics. However, Prof. Not isn’t sure whether PET can be used in sedimentation/plastic sinks if bacteria using it as foods are deposited in the area because it depends greatly on the environment (such as factors like oxygen concentration). Considering cost-effectiveness, Prof. Not is not sure how PETase compares with other methods to relieve the problem of plastic pollution, as it depends on how much it costs to maintain the PETase in the environment.
Prof. Not predicts that PET will be more common for usage as PETase provides a method to reduce pollution specifically from PET. It is also going to allow better waste management.
Lastly, responding to the gap in survey results, Prof. Not thinks that some people are ignorant while others are knowledgeable. The public should be exposed to more education about plastics, and acknowledge how to stop polluting, as plastics are non-biodegradable. Activities to reduce pollution by plastics are only done on a voluntary basis and a large portion of the public does not care about the problems of plastic pollution. Affecting the viewer’s emotions(eg. by using movies) may be a solution as it can cause a change in habits of the viewers.
Second Interview
According to Professor Not, the reason plastics are currently commonly used is that it is easily available, so the method of using regulations to limit the public use of plastic by making it less affordable and acquirable can be used to reduce plastic pollution at the source. The increasing availability of substitutes is also a prime way to reduce the usage of plastics, and that the plastic problem should be examined and tackled from 2 points: the source and the waste already in the environment.
In regard to our project, she said that the use of PETase should never be in the ocean as it is a genetically modified organism, and thus has unknown effects on the host ecosystem. The usage of the enzyme shall be restricted to controlled environments and plastics should be extracted physically from the ocean before treatment. She said that in order to see if this technology could be fielded, a field test of a larger scale should be conducted, which required large amounts of funding and manpower.
She exclaimed that the main risk of the project is that the PETase could escape the closed system and be released into the wild as an invasive species. The effect cannot be predicted due to the complexity of the ecosystem, and thus, this technology should be used with care and attention.
She said that the enzyme could easily be used in waste and water treatment plants around the globe, and the industry may become interested in our enzyme once it becomes marketable and effective. Compared to common ways of treating plastics, such as recycling and incineration, the enzyme has much less restriction and could be utilized with lower cost and higher efficiency.
According to Professor Not’s studies, the amount of plastic pollution in organisms depend on physical parameters. For example, scavenging crabs show a larger amount of plastic pollution in them than specialized crabs. The effect of hydraulic action, ultraviolet light, humidity and wave motion on the fragmentation of large plastics to microplastics can also be further investigated. Biofilms on the plastic yet to be investigated on its effect on degradation of plastic waste in the natural environment, which is essential information for physical removal of plastics from the natural environment (e.g. targeting plastics with a higher rate of fragmentation).
She offered to distribute our surveys to her students to provide us with a stronger collaborative effort and a larger variety pool in the subjects of the surveys. With this information, we can deduce the effects of education and generational differences in plastic pollution and reduction of use. She also informed us about a future project she plans to work on investigating the effect of biofilm on the degradation of plastic and how it affects the natural environment.
Through interviews with professors, we obtained a lot of suggestion that can be integrated in our project. During Professor Lau’s interviews, he showed concern over how the engineered bacteria could compete with native bacteria and cyanobacteria and cause a lack of oxygen in the marine environment. He suggested that either the gene can be inserted into native bacteria with a small population to control plastic pollutants without severely damaging the environment. He also suggested using modelling to predict the effect of the enzyme and its mutants, and to integrate enzymes from a consortium of bacteria into a system to completely degrade products of the enzyme. He said that future tests of the enzyme need to be of a large scale which utilize economies of scale to reduce costs per treated plastic and increase attractiveness to potential investors. This influenced how we designed our applied modelling in future perspective on how we could use the enzyme in a wastewater treatment scenario. In the model that we proposed, we used multiple enzymes in the form of immobilized enzymes in filters. These enzymes would ensure complete biodegradation of enzyme products. We also proposed using common industrial methods such as elutriation, sludging tank with centrifugal force, which separate particles based on their size to treat large amounts of water quickly, in order to have a higher cost per treated plastic ratio. Prof. Lau also suggested modelling for mutant design to have a prediction on how the mutant would perform. He told us that introducing foreign matter into the marine environment is illegal and could very easily unbalance the ecosystem, so leakage of the enzyme into the natural environment is a problem. We can solve this by making the system closed with only inorganic non-toxic substances being discharged. Because he suggested that bacteria could not be released into the environment, this means that a cell lysis approach of purifying the enzymes is recommended, as the purification process using His-tag will be much cheaper, and the digestion process doesn’t have to take place in bacterial culture, which creates easier treatment of outflow of digested solution. Professor Not also suggested that a purified protein approach in wastewater treatment is of high feasibility. Therefore, we adopted cell lysis and enzyme purification approach rather than using secretion tags which transport a protein from the interior of an E.coli cell to the extracellular medium to obtained purified recombinant PETase for PET degradation. Prof. Not also suggested that we carry out surveys to investigate public knowledge of plastic pollution, and to raise public awareness with public engagements, both of which we integrated into our Human Practices. The first survey we carried out confirmed that people didn’t have much knowledge of the plastic problem, and the second one confirmed that the availability of the plastic products are the biggest reason they are used so much. Professor Not indicated interest in the 2nd survey results and that increasing availability of substitute products are enough to massively help the plastic problem. She also expressed that she is interested in how university students behave in correlation to secondary students in order to observe the effect of different generations have on the problem. She offered to help us distribute the surveys to her marine science class, which allow our surveys to have higher variability and sample size. Integration
How the insights from the Professor’s interview integrated into our project