An Integrated Micro-Device System for Coral Growth and Monitoring

Podsumowanie

This protocol describes the development of a modular controllable micro-device system that can be applied for the long-term culturing and monitoring of sea corals.

Streszczenie

Corals are fundamental organisms in marine and coastal ecosystems. With the advancement of coral protection research in recent years, precise control of the coral culture environment is highly in demand for coral conservation and study. Here, we developed a semi-closed coral culture micro-device system as a multi-functional platform, which can provide accurate and programmable temperature control, a sterile initial environment, long-term stable water quality, an adjustable dissolved oxygen concentration, and a customized light spectrum for corals. Owing to the modular design, the coral culture system can be upgraded or modified by installing desirable new modules or removing existing ones. Currently, under appropriate conditions and with proper system maintenance, the sample corals can survive for at least 30 days in a healthy state. Furthermore, due to the controllable and sterile initial environment, this coral culture system can support research into the symbiotic relationship between corals and associated microorganisms. Therefore, this micro-device system can be applied to monitor and investigate sea corals in a relatively quantitative manner.

Wprowadzenie

The deterioration of coral reef ecosystems has been occurring worldwide over the past 70 years. Considering all the major coral areas across Central America¹, Southeast Asia^2,3,4,5,6, Australia^7,8, and East Africa⁹, the global coverage of coral reefs has halved since the 1950s¹⁰. This mass loss of coral reefs has resulted in ecological and economic problems. For example, by tracing the presence/absence and abundance of all kinds of coral-dependent fishes for 8 years, researchers concluded that the coral decline has directly caused a substantial decrease in fish biodiversity and abundance in Papua New Guinea¹¹. This result proved that the coral decline can not only undermine coral reef-based biological systems but also reduce fishery incomes.

Over decades of field surveys, including direct monitoring, remote sensing, and data comparison, the scientific community has identified several factors causing the mass coral decline. One major reason for the mass coral decline is coral bleaching caused by high seawater temperatures^12,13. By combining bleaching and meteorological records, scientists have concluded that coral bleaching is happening more frequently in El Niño-Southern Oscillation phases¹⁴. Another reason for the coral decline is ocean acidification. Owing to the increased CO₂ concentration in both the atmosphere and seawater, calcium carbonate dissolves faster than before, causing downscale net coral reef calcification¹⁵. Indeed, it has been concluded that when the CO₂ concentration in the atmosphere reaches above 500 ppm, tens of millions of people will suffer, and the coral reefs will be at risk of significant deterioration and symbiodinium detachment^16,17. There are other factors that can also affect coral survival, such as inshore pollutants causing or accelerating coral decline. Researchers in Hawaii measured the carbon, oxygen, and nitrogen isotopes in corals, along with the dissolved inorganic carbonate and the related nutrients (NH₄⁺, PO₄^3-, NO₂⁻, and NO₃⁻), and concluded that pollution from the land magnified the coastal acidification and bioerosion of corals¹⁸. Further to pollution, urbanization also endangers coral survival and causes relatively low architectural complexity in corals, as revealed by a study on the coral survival status in Singapore, Jakarta, Hong Kong, and Okinawa. Thus, the impact of anthropogenic stressors and the superimposed effects of climate change are leading to widespread reduced biodiversity on coral reefs and an associated decline in coral ecological function and resilience¹⁹.

It should also be noted that a large number of microorganisms participate in the physiological functions of corals, including nitrogen fixation, chitin decomposition, the synthesis of organic compounds, and immunity²⁰, and these microorganisms should, thus, be included when considering coral reef deterioration. In natural environments, such as coral reefs, many factors cause hypoxic or anoxic conditions, including insufficient water circulation, algal exudate, and algal overgrowth. This phenomenon negatively affects the population distributions of coral and coral-related microorganisms. For example, Vietnamese scientists found that in Nha Trang, Phu Quoc, and Ujung Gelam, bacterial composition in the coral Acropora Formosa could be affected by dissolved oxygen at different locations²¹. Researchers in the United States explored hypoxic or anoxia conditions in corals and found that algal exudates can mediate microbial activity, leading to localized hypoxic conditions, which may cause coral mortality in the direct vicinity. They also found that corals could tolerate reduced oxygen concentrations but only above a given threshold determined by a combination of the exposure time and oxygen concentration²². Researchers in India found that when Noctiluca scintillans algae bloomed, the dissolved oxygen decreased to 2 mg/L. Below this concentration, about 70% of Acropora montiporacan died because of hypoxic conditions²³.

All the abovementioned facts and factors suggest that environmental change leads to the deterioration of coral reefs. To culture and study reef corals under certain conditions, it is important to accurately and comprehensively build up a controllable microscopic environment for reef corals to inhabit. Normally, scientists focus on temperature, light, water flow, and nutrients. However, other features, such as the dissolved oxygen concentration, microorganism abundance, and microorganism diversity in the seawater, are commonly ignored. To this end, our group has explored the possibility of applying small equipment to culture coral polyps in a relatively controlled environment^24,25. In this work, we designed and built up a modular micro-device system for coral culture. This modular micro-device system can provide a controllable micro-environment in terms of the temperature, light spectrum, dissolved oxygen concentration, nutrients, and microorganisms, etc., and has the capacity for expansion and upgrade.

Modules and functions of the device
The micro-device system was inspired by the Berlin system²⁶, but no live rocks are used in the current system. As shown in Figure 1, the current system comprises six main modules, two brushless motor pumps, one gas pump, one flow-through UV lamp, one power supply, certain electronic control components, and the related wires and screws. The six main modules include a seawater store module (with an air pump and temperature sensor), a temperature control module, an algae purification module, a microbial purification module, an activated charcoal purification module, and a coral culture module.

Device architecture
As shown in Figure 2 and Figure 3, the overall micro-device system can horizontally be divided into two compartments with a temperature control module in between. For safety reasons, all the seawater-containing modules and parts are placed in the left compartment, named the culture compartment. The other electronic parts are placed in the right compartment, named the electronic compartment. Both compartments are sealed or packaged within shells. The temperature control module is fixed in a divider plate in between. The shell of the culture compartment includes a baseboard and three screw-fixing plates. This design ensures compartment tightness and facilitates the operation of the system. Additionally, the tightness favors accurate temperature control. The shell of the electronic compartment includes a baseboard, two screw-fixing plates, and one front control panel.

Water circulation
An inner and outer seawater circulation loop connected to the seawater store module was pre-designed. The inner circulation loop successfully connects the seawater store module, temperature control module, flow-through UV lamp, algae purification module, and microbial purification module. This circulation loop aims to provide suitable physiochemical and physiological seawater conditions for the corals, and no frequent maintenance is needed. The algae purification module contains Chaetomorpha algae, which absorbs the extra nutrients (nitrate and phosphate) in the water. The microbial purification module contains the bacterial culture substrate, which cultivates the microbiome to transfer nitrite and ammonium into nitrate for water purification. All these modules need to be replaced only under critical circumstances.

The outer circulation loop successively connects the seawater store module, coral culture module, and activated charcoal module. This circulation loop aims to provide light, tightness, water current, and high seawater quality to the corals. The seawater can be refreshed through a water inlet and a water outlet. Additives are added through a three-way valve, and the seawater sample can also be extracted from this valve for inspection. Air can be pumped in through an air inlet and discharged from an air outlet.

Electronic design
A 220 V AC power supply with a switch and a fuse is used for the whole system. The input power is divided into four branches. The first branch goes to a 12 V DC power supply, which directly powers the heating panel, cooling panel, and cooling fan. This branch also indirectly powers two pumps and two lighting panels through a four-channel DC transformer. The second branch goes to a PID temperature controller. The third branch goes to an air pump power supply. The last branch connects to a UV lamp power supply. A solid-state relay connects the PID temperature controller and the cooling panel in the temperature control module. A regular relay is used to connect the PID temperature controller and the heating panel. The four-channel DC transformer converts the voltage to that required.

There are two control panels on the right part of the system. There are four switches and one controller for the UV lamp on the top panel, including a main power switch, a UV lamp power switch, an air pump switch, and a temperature control switch. The main power switch controls the 12 V power supply of the system.

A PID temperature controller, a cycle timer, a four-channel DC transformer, and a three-channel timer are on the front panel. The PID temperature controller adjusts the water temperature by controlling the heating and cooling panels in the temperature control module. The temperature control module only works when the inner circulation pump is working and the water is flowing past the temperature control module. The cycle timer is connected to the air pump power line. Its purpose is to assign the working time period to the air pump. There is a three-channel timer deployed in the electronic compartment too. This timer controls the work time period for the air pump, coral light, and algae light.

Protokół

The corals used for the present study were Seriatopora caliendrum, which are cultured in our lab. All the corals were kindly provided by the South China Sea Institute of Oceanology, University of Chinese Academy of Sciences.

1. Inspection and startup

NOTE: Each module must be tested for tightness and function individually before assembling the system. Deionized water should be used to test the module's tightness. Commercial details of all the module components are provided in the Table of Materials.

1. Tightness test of the inter-module connection
   1. Connect all the modules and the pumps (Figure 1), and ensure the water flows circularly over the system for at least 30 min.
   2. Check all the seams for possible leakage issues. If any leaking occurs in any of the bonding seams, apply bonding glue from the outside. If any leaking occurs in any of the connection seams, tighten the connection again, and check whether the sealing gasket needs to be changed.
2. Loading
   1. After the tightness test, evacuate and dry the water inside.
   2. Load the appropriate contents.
      NOTE: For example, the bacterial culture substrate is loaded into the microbial purification module, and the Chaetomorpha Algae is loaded into the algae purification module.
3. Assembling and testing the whole system
   1. After loading, fix the modules onto the baseboard using the screws.
   2. Connect the inner circulation modules with the outer circulation ones (without the coral culture module).
   3. For the seawater perfusion, inject the seawater through the water inlet in the seawater store module. When the water level is 3 cm higher than the water inlets of the pumps, switch on the pumps, and continue the injection of the seawater until the inner circulation modules are full of the seawater, with room for air (3 cm high) in the seawater store module.
      NOTE: The seawater is prepared using pure water and sea salt (see the Table of Materials).
4. System test
   1. Turn on all the switches and set both the seawater pump voltages to 9 V. Set the water temperature to 25 °C.
   2. Set the cycle timer to "1 minute on and 1 minute off". Set all three channels of the three-channel timer to "9:00 am on" and "5:00 pm off".
   3. Monitor the system for at least 24 h for any malfunction. If no problem is found, then the system is ready for the next step of the operation.
      ​NOTE: It is important to clear all the bubbles in all the modules except for the seawater store module. The whole system can be slightly lifted and shaken to move the bubbles from the module inlet to the outlet.

2. Establishment of the microbial environment

NOTE: Establishing a coral-friendly microbial environment is necessary before coral transplantation. In order to culture microorganisms in the system, especially in the microbial purification module, the diluted probiotic solution should be added as the microbial source for the nitrification system.

1. Adding the microbiome source
   1. Add 1 mL of the commercial microbiome source solution (see the Table of Materials) into 500 mL of seawater with stirring.
   2. Inject 50 mL of the above diluted solution and 10 µL of the commercial coral nutrition solution (see the Table of Materials) into the circulation system.
2. Microbiome culture
   1. Switch on the inner circulation pump and an air pump to culture the microbiome for 21 days. The microbiome oxygen content requirements determine the on-time and off-time proportions of the air pump.
      ​NOTE: This step aims to culture the seawater purification microbiome and promote coral-beneficial microbiome growth in the system. In this process, the seawater starts to become muddy from the second day to the fourth day after the microbiome injection. After this microbiome culture process, the nutrient degradation capacity in the system should be established. It should be noted that to meet different experimental requirements, different microbiome sources can be used to establish the microbiome environment.

3. Coral transplantation and growth

1. Coral transplantation
   1. Cut the raw coral branches with length scales of 3-5 cm, and then adhere these coral branches onto 3D-printed coral support bases (Supplementary Coding File 1).
   2. Put these coral branch samples back into the original seawater tank for at least 7 days for recovery.
   3. Fix the coral support base onto the rotation unit with glue. Assemble the coral culture module, and connect it to the outer circulation loop.
2. Imaging the coral growth
   NOTE: The coral images over time need to be acquired to evaluate the coral growth. Using a demountable connection makes it convenient to remove the coral culture module from the whole system for imaging. To this end, a mini-photo studio with appropriate illuminating conditions is built. A camera with a macro lens (see the Table of Materials) is used to capture the coral surface morphologies in different periods. The coral rotation unit in the culture module can be operated outside the module using non-contact mode. By rotating the magnetic handle adjacent to the module, full-angle coral images can be captured.
   1. Place the camera on the top of the studio, and capture the images from a vertical view.
   2. Place the coral culture module in the mini-photo studio with the coral positioned in the center and at the bottom.
   3. Capture the coral images by rotating the outside handle.
      ​NOTE: For coral survival reasons, the time period of imaging should be limited to 15 min.

4. System routine maintenance

NOTE: The routine maintenance includes leakage inspection, malfunction inspection, additive addition, and seawater exchange.

1. Leakage inspection
   1. Inspect the baseboard for water stains or droplets. As the system's cover shell is transparent, visually inspecting the water leakage is easy and convenient. This inspection must be performed every day.
2. Malfunction inspection
   1. Ensure that this step includes the inspection of the water temperature, pumps, light voltages, air pump status, and timer status, including visually checking and recording the set water temperature, real-time temperature, transformer output voltages, UV lamp settings, and timer working status. This inspection should be performed every day.
      NOTE: Certain system malfunctions can be diagnosed based on abnormal sounds or unusual temperatures.
3. Additive addition
   NOTE: Additive addition is the process of adding nutrients and other reagents into the system.
   1. For example, extract 10 mL of the seawater using a syringe from the three-way valve between the activated charcoal module and seawater module.
   2. Dissolve the additives in the extracted seawater.
   3. Inject the solution back into the system through the three-way valve. In real cases, the types, amounts, and addition frequencies of the additives are decided by the system seawater quality, considering the experimental requirements.
4. Water exchange
   NOTE: Routine water exchange can reduce toxic concentration and eutrophication in the culture system. If the experimental conditions allow, exchanging the seawater can be a routine operation.
   1. Switch off the power for the whole system, and unplug the power cable for safety reasons.
   2. Remove the coral culture module.
   3. Connect the outside wastewater pipeline to the outlet in the seawater store module.
   4. Rotate the system, and place the system front-side down.
   5. Switch on the outlet. Let the inside seawater flow out of the system.
      NOTE: Do not use any pump to draw the water out since the inside negative pressure may damage the system.
   6. Discharge an appropriate amount of seawater, and switch off the outlet. The amount of discharged seawater is decided by the physiological state of the corals.
   7. Reset the system, and inject the newly prepared seawater into the system through the water inlet.
   8. Install the coral culture module back into the system.
   9. Switch on the system power, and wait until the whole system goes back to normal.

5. Module replacement

NOTE: If any module needs to be replaced due to malfunction or according to the experimental arrangement, it is important to change the module without suspending or negatively affecting the culture experiment.

1. For the seawater store module, algae purification module, microbial purification module, or activated charcoal purification module, switch off the inner circulation pump, and loosen the fixing screws for the module.
2. Disconnect the pipelines between the two joined modules, and disassemble the module to be replaced from the system. The final step is to assemble the new module into the system by connecting the pipelines and retightening the fixing screws.
   NOTE: The replacement of the temperature control module is somehow different. First, all the wires need to be disconnected from the module. The fixing bolts are then unscrewed, and the pipelines are disconnected. Afterward, the heating panel is dismounted, and the module is disassembled from the system. The installation process for the temperature control module is the reverse process.

6. Shutting down the system and restoring the system to its initial state

NOTE: The system will eventually be shut down after the necessary coral culture experiment. The system needs to be restored to its original state.

1. Shutting down the system
   1. Switch off the system power, and unplug the power cable.
   2. Evacuate the seawater inside the system.
   3. Disassemble the modules in the following order: the coral culture module, the activated charcoal purification module, the seawater store module, the algae purification module, the microbial purification module, the UV lamp, the two circulation pumps, and the temperature control module.
2. System restoration
   1. Clean all the modules with pure water and surface active agents (see the Table of Materials).
   2. Sterilize the modules with 3% hydrogen peroxide solution.
      ​NOTE: Do not use any organic solvent to wash the modules.
   3. Dry the modules at 40 °C for 12 h. Ensure that all the water inside the system is evaporated.
   4. Clean all the pipelines and valves using the same surface active agents.

7. Modification for the controlled microorganism environment

NOTE: Aside from the coral culture experiment, for certain special experiments, such as acquiring a controlled microorganism environment in the system, the microbiome species and abundance must be strictly controlled. The most innovative feature of our coral culture system is that the coral's physiological activity can be explored in a specific microbial environment in a relatively closed micro-ecosystem. Performing this function requires a different operating procedure.

1. Pre-sterilization
   1. Sterilize all the modules, pipelines, and valves using 3% hydrogen peroxide solution before assembling the system.
   2. Sterilize the bacterial culture substrate via autoclaving.
   3. Sterilize Chaetomorpha algae with 75% ethanol solution, and dry it using sterile paper.
2. System modification and sterilization
   1. In assembling the system, add an air sterilizing filter (see the Table of Materials) between the air pump and the seawater store module.
   2. Add a water sterilizing filter between the inlet and the three-way valve. This step ensures the air and water injected into the system are sterilized.
   3. Introduce ozone into the system to eliminate the remaining microbiome.
   4. Wash off the remaining disinfectant agents with sterile seawater three times, and inject the sterile seawater into the system.
   5. For only the establishment of the microbial environment, inject the microbiome source solution through the water outlet.
      NOTE: Do not use the water inlet to inject the microbiome source. Other reagents and seawater are still injected through the water inlet.

Wyniki

Temperature control accuracy
The system temperature is normally set to 23-28 °C depending on the coral species. However, as one of the most important factors, temperature fluctuation can strongly affect coral survival. Hence, temperature control accuracy is a decisive factor for the coral culture system. A temperature sensor and an independent data collector with a temperature range from 9 °C to 32 °C can be used to test the temperature control accuracy in the coral culture module. W...

Dyskusje

This coral culture system is designed to simulate and provide a relatively natural or customized microenvironment for corals to be transplanted into and survive. Meanwhile, as a self-developed equipment, this system needs to be reliable, user-friendly, and safe. For example, in terms of temperature control, the seawater temperature should be controlled appropriately based on the daily environmental circumstances. The system was tested by culturing the coral for 1 month, confirming the system's reliability.

Materiały

Name	Company	Catalog Number	Comments
12V DC power supply	Delixi Electric Co., Ltd.	CDKU-S150W	12V12.5A
3% hydrogen peroxide solution	Shandong ANNJET High tech Disinfection Technology Co., Ltd	NULL	NULL
75% ethanol solution	Shandong ANNJET High tech Disinfection Technology Co., Ltd	NULL	NULL
Air pump	Chongyoujia Supply Chain Management Co., Ltd.	NHY-001	NULL
Air sterilizing filter	Beijing Capsid Filter Equipment Co., Ltd	S593CSFTR-0.2H83SH83SN8-A	NULL
Camera	SONY	Α7r4-ILCE-76M4A	NULL
Coral nutrition solution	Red Sea Aquatics Co., Ltd.	22101	Coral nutrition
Coral pro salt (sea salt)	Red Sea Aquatics Co., Ltd.	R11231	NULL
Cycle timer	Leqing Shangjin Instrument Equipment Co., Ltd.	CN102A	220V version
Double closed quick connector	JOSOT Co., Ltd	NL4-2103T	NULL
Flow-through UV lamp	Zhongshan Xinsheng Electronic technology Co., Ltd.	211	NULL
Four-channel transformer	Dongguan Shanggushidai Electronic Technology Co., Ltd	LM2596	NULL
Macro lens	SONY	FE 90mm F2.8 Macro G OSS	NULL
Microbiome source solution	Guangzhou BIOZYM Microbial Technology Co., Ltd.	303	NULL
Mini-photo studio	Shaoxing Shangyu Photography Equipment Factory	CM-45	NULL
PID temperature controller	Guangdong Dongqi  Electric Co., Ltd.	TE9-SC18W	SSR version
Pump (for water)	Zhongxiang Pump Co., Ltd.	ZX43D	Seaswater version
Pure water machine	Kemflo (Nanjing) environmental technology Co, ltd	kemflo A600	NULL
Solid-state relay	Delixi Electric Co., Ltd.	DD25A	NULL
Surface active agents	Guangzhou Liby Group Co., Ltd.	Libai detergent	NULL
Three-channel timer	Leqing Changhong Intelligent Technology Co., Ltd.	CHE325-3	220V version
Water sterilizing filter	Beijing Capsid Filter Equipment Co., Ltd	S593CSFTR-0.2H83SH83SN8-L	NULL