In any scientific endeavor, the paramount importance of safety cannot be overstated. It serves as the essential bedrock upon which all successful and ethical research is built. This commitment spans every facet of our work: from the both immediate and online safeguards of laboratory safety, to the meticulous risk management of project safety, and extending to the long-term ethical considerations of application safety. Upholding the highest safety standards is our shared responsibility—it protects our personnel, preserves the integrity of our data, and ensures that our contributions to knowledge and technology are both responsible and sustainable.
Laboratory safety is the most direct factor influencing the overall security of research projects. Only within a safe experimental environment can personnel carry out their tasks safely and experimental data be properly protected. In this section, we will explore how laboratory safety is safeguarded from three key dimensions: Chemical Safety, Biological Safety, and Security of Experimental Data.
It is inevitable that chemicals in the laboratory represent a potential source of hazards. To maximize the safety of the laboratory environment and protect the health of personnel, we will now discuss the safety aspects of substances with potential risks.
Ethanol Ethanol is an essential laboratory reagent. It is required for preparing 75% ethanol solutions, serves as fuel for alcohol lamps, and is a component in various kits, such as those for silver staining and gel extraction. However, we are fully aware of its flammability and toxicity. Our control measures for ethanol include the following: First, we do not store large quantities of ethanol in the laboratory. At any given time, the laboratory contains no more than 1 liter of anhydrous ethanol and 1 liter of 75% ethanol. Second, the only open flame in our laboratory is located inside a biosafety cabinet (due to an alcohol lamp), alongside various electrical equipment. Therefore, our ethanol stocks are stored in a fire-resistant cabinet under a bench, kept away from any ignition sources, electrical wires, power sockets, and combustible materials. Finally, we use 75% ethanol for cleaning shaker components. Any remaining waste ethanol is disposed of under professional supervision—it is poured into a dedicated liquid waste container. We avoid discharging large quantities of ethanol into the drain at once to prevent potential corrosion.
Methanol Methanol is an essential chemical in our laboratory, as it is required to induce the promoter for laccase expression in Pichia pastoris. We fully acknowledge the associated hazards of methanol, being flammable, volatile, and toxic. Our stringent control measures are as follows: First, our consumption is low, and we do not stockpile large quantities. Only one bottle of methanol is stored in a dedicated metal safety cabinet, kept away from light, ignition sources, electrical wires, and sockets. Access is restricted, with the cabinet key held exclusively by our Principal Investigator. Second, all handling of methanol, even in small volumes, is conducted within a fume hood to minimize vapor release and prevent contamination of the laboratory air. Finally, after any experiment involving methanol, all residual methanol is disposed of into a designated chemical waste container, regardless of the minute quantity. It is never discarded in the general laboratory waste or poured down the drain.
Nucleic acid stain Nucleic acid stains are essential for any DNA gel electrophoresis experiments. However, we are fully aware of their potential carcinogenic risks. Our safety management protocol for these dyes includes the following measures: First, our laboratory has replaced the highly hazardous Ethidium Bromide with the considerably safer alternative, SYBR Safe DNA Gel Stain. Furthermore, we strictly limit inventory, ensuring no more than one single tube (1.5 mL) of the dye is stored in the lab at any given time. Second, a dedicated pipette is assigned exclusively for handling the dye to prevent cross-contamination of other reagents or microbial cultures. Finally, during all pipetting procedures involving the dye, we wear an additional pair of disposable plastic gloves over our standard laboratory nitrile gloves, providing an extra barrier to prevent skin contact.
Perchloric acid Our laboratory does not directly store perchloric acid. However, the initial lignin detection kit we selected did contain reagents comprising perchloric acid. Being fully aware of its strong oxidizing properties, high acidity, and corrosive nature, we proactively sought an alternative lignin detection kit after a single use of the original one. We successfully identified a kit that does not contain any highly hazardous chemicals and promptly disposed of the original kit through an approved chemical waste disposal protocol.
Vinasse As a project targeting the degradation of vinassE, it was necessary for us to use this material to validate the efficacy of our dual-bacterial system. However, we are fully aware of the associated hazards, including its flammability, potential for dust explosions, and risk of microbial contamination. Our control measures are as follows: Since our validation experiments did not require large-scale testing, we maintained minimal stock, storing only five small bags frozen at -20°C. This approach effectively mitigates the risks by preventing direct contact with any ignition sources, electrical wires, or sockets; eliminating the possibility of combustible dust dispersion; suppressing the growth of native microorganisms within the grains; and establishing a physical barrier between these indigenous microbes and the laboratory’s stored microbial strains.
Following the confirmation of our team roster, we immediately conducted a comprehensive laboratory safety training program. This process began with an orientation to the lab’s basic layout by senior team members, followed by an emphasis on critical safety protocols from our Principal Investigator (PI). The detailed training curriculum is outlined below:
1. General Safety Training
| Aspect | Practice |
|---|---|
| Laboratory Layout & Safety Equipment | Familiarization with the locations of safety exits, emergency eyewash stations and showers, fire extinguishers, and first-aid kits |
| Personal Protective Equipment (PPE) Policy | Clear instruction on PPE requirements for different scenarios, accompanied by a demonstration of proper donning and doffing |
| Fundamental Chemical Safety | Introduction to the hazards of specific chemicals used in our lab, with particular emphasis on high-risk substances like ethanol and nucleic acid stains |
| Fundamental Biosafety | Instruction on biosafety levels, aseptic techniques, and core principles for preventing contamination |
| Waste Segregation and Disposal | Clear differentiation and demonstrated procedures for disposing of various waste types (general, chemical, bio-sharps, and bio-infectious waste) |
| Emergency Response Protocols | Institution-mandated training on standardized procedures for responding to common incidents, including fires, chemical spills, and personal injuries |
2. Specialized Equipment Training
| Aspect | Practice |
|---|---|
| Biosafety Cabinet (BSC) | Covered operational principles, misconceptions regarding UV lamp usage, proper cleaning and disinfection procedures before, during, and after operations, and key practices to avoid cross-contamination |
| Autoclave | Included a demonstration of the operational workflow, daily water level checks, selection of appropriate sterilization parameters, safe lid-opening techniques, and routine maintenance |
| Other Equipment | Provided essential safety operating instructions for commonly used equipment such as centrifuges and PCR machines |
3. Project-Specific Safety Training
| Aspect | Practice |
|---|---|
| Chassis Organism Risks | Focused session on the contamination risks associated with Pichia pastoris, spore containment for Trichoderma reesei, and the importance of antibiotic resistance management and biocontainment for engineered bacterial strains |
| Specialized Procedure Safety | Conducted risk assessments and provided standardized operational training for project-specific experimental steps, such as co-culture experiments and vinasse handling |
Information security is a critical pillar of modern laboratory management, extending beyond traditional safety concerns to protect the very integrity and continuity of research. Digital assets—including experimental data, genetic sequences, and proprietary findings—constitute invaluable intellectual property, the loss of which can compromise months or even years of scientific effort. Our team learned this through a significant incident on June 30, 2025. We experienced a severe disruption when critical project data—including foundational literature, gene sequences, and plasmid maps—was suddenly deleted from our central data platform. The incident caused considerable alarm during a group meeting and brought our project work to an abrupt halt.
A subsequent investigation, conducted with the platform operator, traced the cause to a security breach: an attacker had compromised the login credentials of one of our team members and used this unauthorized access to delete our project information. With the kind assistance of staff from the data platform, we were able to recover our lost data in 3 days. Nevertheless, this event served as a profound and costly lesson, highlighting two critical security principles:
The absolute necessity of individual account responsibility Sharing platform credentials, even for convenience, creates a single point of failure and is an unacceptable security practice.
The non-negotiable need for redundant data backups Regardless of a cloud platform’s reliability, data loss remains a persistent risk. We have since instituted a disciplined protocol of maintaining synchronized local backups for all critical data, adhering to the “3-2-1” backup rule.
This incident has fundamentally reshaped our approach to digital stewardship, reinforcing that rigorous information security is not an IT formality but a fundamental component of responsible scientific research.
Our project utilizes three types of Chassis Organisms: Pseudomonas putida, Pichia pastoris, and Trichoderma reesei. In the following section, we will examine the safety aspects of these chassis organisms and outline the key precautions we have taken when conducting experimental procedures with them.
Our project utilizes Pseudomonas putida as a chassis organism. Specifically, we employed two strains: the standard Pseudomonas putida KT2440 and a custom acid-tolerant P. putida KT2440 strain capable of growth at pH 4.5. The following biosafety measures have been implemented for these strains:
Anti-Release Strategies While Pseudomonas putida is not a human pathogen, it is a known fish pathogen. Therefore, when disposing of liquid cultures, we strictly avoid pouring them directly down the drain. Instead, all liquid waste is collected in a dedicated container and treated with sodium hypochlorite effervescent tablets for a minimum of 10 minutes before disposal. This procedure ensures complete inactivation of viable cells, preventing their environmental release and potential harm to aquatic life.
Safety Precautions Against Antimicrobial Resistance Our genetically engineered acid-tolerant P. putida KT2440 strain carries resistance markers for kanamycin and ampicillin. We fully recognize the potential environmental risk related to antimicrobial resistance (AMR) should this strain be released. Consequently, the strain is strictly confined to designated laboratory workspaces and is not permitted to be removed in any form (e.g., liquid cultures, agar plates). Furthermore, we are actively exploring the potential application of suicide plasmid systems for additional biocontainment. For instance, Part BBa_K914014, which enables arabinose-induced cell death in E. coli, presents a promising concept. We propose adapting such a system by transferring the key genetic components into a broad-host-range shuttle vector compatible with P. putida.
Mitigation Strategies Against Acid Tolerance Preliminary investigation into the acid-tolerance mechanism of our engineered strain revealed its capacity to secrete alkaline substances, elevating the medium pH from an initial 4.5 to above 8.0. We have noted this distinctive phenotype and plan to further elucidate the underlying mechanism in subsequent studies. A comprehensive understanding of the specific genetic mutations, to be identified through whole-genome sequencing and comparison against the P. putida KT2440 reference genome, will enable us to develop more targeted and effective safety mitigation strategies.
Our project initially explored the use of Pichia pastoris as a chassis organism. Although it was ultimately concluded that P. pastoris could not stably coexist with Pseudomonas putida, leading to its discontinuation in our final design, a comprehensive biosafety analysis of the related experiments is provided here, given its promising performance in laccase secretion and the detailed experimental data we recorded.
Contamination Concerns While not conventionally classified as a pathogenic fungus, P. pastoris exhibits robust survival and reproductive capabilities, making it a potential source of laboratory equipment contamination. Our team experienced this firsthand when one of our shaker incubators became contaminated. For a period in August, all microbial cultures incubated in this specific shaker resulted in the growth of P. pastoris. After a week, we traced the source to a probable spill of P. pastoris culture. The issue was successfully resolved by thoroughly cleaning the shaker with 75% ethanol, and we had sulfur fumigation prepared as a further sterilization step. This incident served as a valuable lesson, leading to significantly more careful handling procedures forP. pastoris in all subsequent work.
Bactericidal Effect of Pichia pastoris We attempted to co-culture laccase-producing P. pastoris with our engineered acid-tolerant P. putida KT2440 to explore the feasibility of a dual-microbe system for vinasse treatment. However, microscopic examination of the co-culture revealed that P. pastoris had completely eradicated the **P. putida* cells, with only cellular debris remaining. This outcome not only discouraged further pursuit of their coexistence but also highlighted the significant bactericidal activity of P. pastoris. To prevent potential harm to native microbial communities, we implemented a strict decontamination protocol for all P. pastoris* liquid waste. Cultures are collected in a dedicated container and treated with sodium hypochlorite effervescent tablets for a minimum of 10 minutes before disposal, ensuring no viable cells are released into the environment.
AMR Concerns with Engineered Pichia pastoris Our genetically modified P. pastoris strain carries a kanamycin resistance marker. We fully acknowledge the potential environmental risk related to antimicrobial resistance (AMR) should this strain be released. Consequently, the strain is strictly confined to designated laboratory areas and is not permitted to be removed in any form (e.g., liquid cultures, agar plates). Simultaneously, we proactively investigated potential suicide plasmid systems for enhanced biocontainment. Our research on Addgene identified several CRISPR-Cas systems compatible with yeast, which could be induced to create double-strand breaks in the P. pastoris genome, thereby serving as an effective kill-switch mechanism.
Based on our project’s specific context, we have implemented a multi-layered biosafety strategy to address the primary concern associated with our chassis organism, Trichoderma reesei: its highly dispersible spores. Recognizing the potential for contamination, we have established the following containment and safety procedures:
Dedicated Cultivation Facility & Equipment The cultivation of T. reesei is strictly confined to a designated room, which houses two shaker incubators exclusively used for this organism. A key feature of these incubators is their integrated UV sterilization lamps. Following each cultivation cycle, a standard operating procedure is in place to activate the UV lamps, thereby decontaminating the internal environment and minimizing the risk of cross-contamination.
Secondary Physical Containment When cultivating T. reesei on Petri dishes, an additional layer of physical containment is employed. The open Petri dishes are placed inside a larger, sealed glass vessel, which is then placed into the incubator. This measure ensures that any spores escaping the primary culture dish are effectively trapped within the secondary container, significantly reducing the likelihood of contaminating the incubator itself.
Use of an Auxotrophic Strain Our project utilizes the Trichoderma reesei TU6 strain, which is an auxotroph incapable of synthesizing uracil. This genetic modification introduces a biological containment measure, as the strain’s ability to survive and propagate in the environment is substantially reduced without specific nutritional supplements, thereby lowering its overall environmental persistence.
Strict Segregation of Materials All equipment and consumables that come into contact with T. reesei—including pipette tips, culture media, and spore filtration apparatus—are sterilized and designated for its exclusive use. For instance, pipette tips used for T. reesei protocols are never used for general molecular biology work such as plasmid extraction from E. coli. This practice is rigorously enforced to prevent any potential cross-contamination with other experimental systems.
Inherently Low Pathogenicity It is important to note that Trichoderma reesei is not considered a pathogenic fungus in the conventional sense. It does not pose a health risk to laboratory personnel who are immunocompetent and adhere to standard laboratory protocols, including the proper use of Personal Protective Equipment (PPE).
The intended final application of our project is industrial-scale biomanufacturing, a premise that fundamentally shapes our safety strategy. We recognize that while our chassis organisms—specifically the engineered acid-tolerant Pseudomonas putida and Trichoderma reesei—present specific biosafety risks in a laboratory context (such as the potential spread of antibiotic resistance genes or environmental dispersal of fungal spores), the industrial production process itself provides a highly effective and reliable containment solution for these risks.
Physical Inactivation At the terminus of the full production process, the entire fermentation broth—including microbial cells, genetic material, and products—will be directed to downstream processing units. Here, high-temperature sterilization will be employed as a standard procedure. This step will completely inactivate all microorganisms, including the acid-tolerant P. putida and the resilient spores of T. reesei. Consequently, any processed fermentation waste will be entirely free of viable engineered microbes, fundamentally eliminating the risks of environmental release and horizontal gene transfer.
Process Containment and Biocontainment The entire production process will occur within a fully closed bioreactor (fermenter) system. This design not only optimizes production conditions but also acts as an effective physical barrier, minimizing the potential for the release of live cells—such as via aerosols—during operation. Transfer of material to the inactivation unit will only occur post-fermentation.
Understanding of Project-Specific Risks Our ability to implement such direct and effective safety measures stems from a profound understanding of our project’s specific characteristics: First, our target product is succinate, not the microbial biomass itself. This allows for the inactivation of microorganisms at the process endpoint without affecting product recovery and purification. Secondly, physical conditions like high temperature and pressure are standard in industrial chemical processes, contrasting sharply with biomedical applications that often depend on the functionality of live cells.
Warning on Potential Misuse We explicitly emphasize that our entire biological system—including the co-culture strategy and engineered strains—is specifically designed and optimized for a controlled industrial production environment. Direct application in medical or open environments is strictly prohibited: Any attempt to repurpose our engineered strains for direct human therapeutics, agricultural release, or other open-environment applications is extremely dangerous and expressly forbidden. In such scenarios, the crucial physical inactivation step is absent, and the inherent risks associated with the environmental persistence and spread of the chassis organisms would become uncontrolled.
