Introduction

Our home, Macau, is renowned for its concentrated and well-preserved cultural heritage sites. The Historic Centre of Macao, which has a collection of over 20 sites, was inscribed on the UNESCO World Heritage List in 2005 because it “bears witness to one of the earliest and longest-lasting encounters between China and the West, based on the vibrancy of international trade.” [1]

This year, we are innovating and invigorating HERITAGE CONSERVATION in Macau using synthetic biology. It is our mission to protect our city’s heritage buildings, ensuring their long-term sustainability in the face of a rapidly changing climate.



Environmental factors is a major contributor to the deterioration of historical heritages. SALT EFFLORESCENCE happens commonly in concrete buildings, especially in Macau, once a seaside village, where most of the soil is rich in sea salt. The hydrated salt is gradually carried to up to the walls via capillary action. Once the hydrated salt reaches the surface, water evaporates and the salt crystal remains, creating cracks and damages on the structures.

Figure 1. Hydrated salt accumulating on building surfaces.


Macau’s rapid urban development and proximity to industrial areas contribute to high levels of air pollution. Sulfur oxides (SO₂ and SO₃) react with moisture in the air to form acid solutions containing sulfite, which will then become acid rain via precipitation, leading to SULFATION. It ultimately transforms calcium carbonate in building materials into calcium sulfate dihydrate (gypsum), which is softer and more prone to erosion.

Figure 2. Erosion on building surfaces due to sulfation.

Environmental factors is a major contributor to the deterioration of historical heritages. SALT EFFLORESCENCE happens commonly in concrete buildings, especially in Macau, once a seaside village, where most of the soil is rich in sea salt. The hydrated salt is gradually carried to up to the walls via capillary action. Once the hydrated salt reaches the surface, water evaporates and the salt crystal remains, creating cracks and damages on the structures.

Macau’s rapid urban development and proximity to industrial areas contribute to high levels of air pollution. Sulfur oxides (SO₂ and SO₃) react with moisture in the air to form acid solutions containing sulfite, which will then become acid rain via precipitation, leading to SULFATION. It ultimately transforms calcium carbonate in building materials into calcium sulfate dihydrate (gypsum), which is softer and more prone to erosion.

There are several solutions in Macau nowadays aiming to tackle the issue, most of which face severe limitations. Hence, our project this year aims to develop a sustainable, bioengineered solution to protect vulnerable heritage sites from environmental degradation. Our integrated approach addresses two primary mechanisms of deterioration, acid erosion and salt efflorescence, using a three-step solution: prevention, repair and adhesion.


Current solutions and limitations associated

There are several established solutions in Macau being employed on heritage protection at this time:

1. Plastic Polymer Coatings

What is Plastic Polymer Coating (PPC)? It is the application of a coating on the surface of the historical buildings.

Figure 3. Mechanism of current plastic polymer coating method.
Advantages:

1. Plastic polymer coatings offer protection against environmental degradation. By forming a durable, water-resistant barrier, they help shield historical building surfaces from moisture, pollutants and other factors.


Disadvantages:

1. The coatings will become viscous under high temperature, humidity or strong ultraviolet radiation conditions due to the breakage of chemical bonds[5].Hence pollutants adhere, forming a GRAY-BLACK SHELL.

Figure 4. Building surfaces forming a grey-black shell due to pollutants.

2. The coatings on the surface of the building will eventually peel off due to environmental stress, which plaster on the wall itself and its substrate will peel off together, causing severe widespread damage.

Figure 5. Building surfaces peeling off due to environmental stress.

2. Nanolime Coating

Nanolime coating involves spraying calcium hydroxide nanoparticles suspensions.

Advantages

1. When calcium hydroxide is exposed to carbon dioxide, insoluble calcium carbonate is formed, which creates hard coatings after consolidation, filling cracks on building surfaces.

Figure 6. Nanolime coating mechanism. [11]

Disadvantages:

The HIGH COST of the building materials and manual labour, as well as LOW effectiveness limited the production of nanoparticles suspension.[6]


Our proposal

Our project this year aims to develop a sustainable, bioengineered solution to protect vulnerable heritage sites from environmental degradation. Our integrated approach addresses two primary mechanisms of deterioration, acid erosion and salt efflorescence, using a three-step solution: repairing, prevention and adhesion


Repairing: Neutralising Acid erosion

Climate change has been becoming more severe in the past few decades. Acid rain also aggravated, following the intensifying climate change. This causes the sulfates and sulfites to remain on the building surfaces after precipitation, leading to acid erosion. Sulfate reduction can help alleviating this issue, as this reaction pathway reduces sulfates and sulfites to the amino acid--cysteine, thereby removing the key contributor to acid erosion. We overexpressed the sulfate-reducing strain in E. coli to enable the bacteria to operate in the sulfate reduction pathway. However, the efficiency of this process is low. Therefore, we employed two genes from the EamA transporter family, YdeD and YhaM to enhance the overall efficiency. The former encodes a transmembrane protein that exports cysteine out of bacterial cells, while the latter encodes the enzyme cysteine desulfidase, which is responsible for the breakdown of cysteine into hydrogen sulfide, pyruvate, and ammonium. Both of the genes reduce the amount of intracellular product, i.e. cysteine, in the reduction pathway, thus accelerating the overall reaction, which forms more cysteine. As a result, the sulfates and sulfites deplete faster, and the reaction efficiency is improved. The three byproducts-- hydrogen sulfide, pyruvate, and ammonium supports the engineered bacteria’s continuous function—through energy and redox support, and adaptability—via flexible nitrogen and carbon assimilation, under diverse environmental conditions.


Figure 7. Mechanism of increasing the efficiency of sulfate reduction pathway by removing cysteine.

Prevention: Crack-filling Biosilica

Biosilica is the silicon dioxide yielded by living organisms. Silicatein from monomeric silicon compounds, such as silicic acid, can catalyse biosilica formation. The chemical inertness and thermostability of this material corroborate its suitability for historical heritage conservation.

As a result, we utilised E. coli engineered to produce silicatein, an enzyme that catalyses the formation of biosilica from tetraethyl orthosilicate[9]. More importantly, the enzyme does not produce E. coli-viability-inhibiting ammonium/carbonate pollutants.

We are utilising E. coli engineered to produce silicatein, an enzyme that catalyses the formation of biosilica from tetraethyl orthosilicate[9]. More importantly, the enzyme does not produce E. coli-viability-inhibiting ammonium/carbonate pollutants.


The generated continuous, micrometer-thick biosilica coating, creates a cohesive structure with building particles, thereby filling micro-cracks, reducing surface porosity, restoring the material's structural integrity, and increase resistance to acid rain. The coating has the potential to remediate the damage done on the layer itself by reapplying silicatein, enabling a renewable protective system akin to living materials.

Adhesion: Surface adhesion

For effective application, we harnessed INP and csgA as an adhesion factor. The INP gene, sourced from Pseudomonas syringae, facilitates protein expression on the bacterial cell surface that will cause it to attach strongly to the building surfaces. Its highly repetitive structure enhances stability, allowing bacteria to remain attached to building walls despite environmental stressors like wind and rain.

Meanwhile, the CsgA gene, found in Escherichia coli, plays a crucial role in curli fiber production. These fibers form a tough, resilient coating that strengthens the attachment of bacterial cells to the building structure[10]. The synergised INP and csgA gene ensures firm bacterial adhesion to building surfaces, with a pH-responsive trait that results in increased adhesive properties under more acidic conditions.

Meanwhile, the CsgA gene, found in Escherichia coli, plays a crucial role in curli fiber production. These fibers create a durable and resilient coating, reinforcing the attachment of bacterial cells to the structure[10]. The synergised INP and csgA gene ensures firm bacterial adhesion to building surfaces, with a pH-responsive trait that results in increased adhesive properties under more acidic conditions.


References

[1] https://whc.unesco.org/en/list/1110/

[2] https://www.epa.gov/acidrain/what-acid-rain

[3] Karim, Md & Islam, Md. Hamidul & Morshed, Syed. (2016). Influence of Salt Efflorescence on Rendering Mortar of Brick Masonry Walls.

[4] Mehta, Prashant. (2010). Science behind Acid Rain: Analysis of Its Impacts and Advantages on Life and Heritage Structures. S. Asian J. Tourism and Heritage.

[5] Caramitu AR, Ciobanu RC, Lungu MV, Lungulescu EM, Scheiner CM, Aradoaei M, Bors AM, Rus T. Polymeric Protective Films as Anticorrosive Coatings-Environmental Evaluation. Polymers (Basel). 2024 Aug 1;16(15):2192. doi: 10.3390/polym16152192. PMID: 39125219

[6] Tzavellos S, Pesce GL, Wu Y, Henry A, Robson S, Ball RJ. Effectiveness of Nanolime as a Stone Consolidant: A 4-Year Study of Six Common UK Limestones. Materials (Basel). 2019 Aug 22;12(17):2673. doi: 10.3390/ma12172673. PMID: 31443366

[7] Kopriva, Stanislav & Koprivova, Anna. (2004). Plant adenosine 5′-phosphosulphate reductase: The past, the present, and the future. Journal of experimental botany. 55. 1775-83. 10.1093/jxb/erh185.

[8] Nakatani, Takeshi & Ohtsu, Iwao & Nonaka, Gen & Wiriyathanawudhiwong, Natthawut & Morigasaki, Susumu & Takagi, Hiroshi. (2012). Enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from S-sulfocysteine increases L-cysteine production in Escherichia coli. Microbial cell factories. 11. 62. 10.1186/1475-2859-11-62.

[9] Vigil TN, Schwendeman NK, Grogger MLM, Morrison VL, Warner MC, Bone NB, Vance MT, Morris DC, McElmurry K, Berger BW, Steel JJ. Surface-displayed silicatein-α enzyme in bioengineered E. coli enables biocementation and silica mineralization. Front Syst Biol. 2024 May 30;4:1377188. doi: 10.3389/fsysb.2024.1377188. PMID: 40809133

[10] DeBenedictis EP, Liu J, Keten S. Adhesion mechanisms of curli subunit CsgA to abiotic surfaces. Sci Adv. 2016 Nov 18;2(11):e1600998. doi: 10.1126/sciadv.1600998. PMID: 28138525; PMCID: PMC5262458.

[11] Gherardi, F., & Maravelaki, P. N. (2022). Advances in the application of nanomaterials for natural stone conservation. RILEM Technical Letters, 7, 20–29. doi: 10.21809/rilemtechlett.2022.159.