<p>The Unicamp_Brazil team was established based on the premise of employing synthetic biology to solve local environmental challenges, providing a significant contribution to society. We began with visits to waste management facilities and initially identified polystyrene accumulation in landfill as a pressing matter that demanded attention. We designed a project for the bio-conversion of polystyrene into bioplastics such as PHA or PHB, however, upon calculating the negative environmental impact of polystyrene transport, we realized that the environmental costs would outweight the potential benefits of the proposed bioconversion. Thus, we returned to the planning stage and identified another massive source of pollution in our region: residues from orange juice production.</p>
<p>Searching for ways to up-cycle this biomass, we came across a research group using orange pulp as a substrate for bacterial cellulose (BC) production by the bacteria <i>Komagataeibacter rhaeticus</i> AF1. BC is chemically very similar to its plant counterpart but synthesized in a purer state, which allows many downstream applications in areas as diverse as packaging or treatment of wounds. The strain <i>K. rhaeticus</i> AF1 was isolated from kombucha tea. Due to its high capacity of producing BC from unconventional sources (such as orange pulp), its genome was sequenced¹, but the strain had never been genetically manipulated.</p>
<p>The Unicamp_Brazil team was established based on the premise of employing synthetic biology to solve local environmental challenges, providing a significant contribution to society. We began with visits to waste management facilities and initially identified polystyrene accumulation in landfill as a pressing matter that demanded attention. We designed a project for the bio-conversion of polystyrene into bioplastics such as PHA or PHB, however, upon calculating the negative environmental impact of polystyrene transport, we realized that the environmental costs would outweight the potential benefits of the proposed bioconversion. Thus, we returned to the planning stage and identified another massive source of pollution in our region: agroindustry residues.</p>
<p>Searching for ways to up-cycle this biomass, we came across Dr. Hernane Barud´s research group using orange pulp as a substrate for bacterial cellulose (BC) production by the bacteria <i>Komagataeibacter rhaeticus</i> AF1. BC is chemically very similar to its plant counterpart but synthesized in a purer state, which allows many downstream applications in areas as diverse as packaging or treatment of wounds. The strain <i>K. rhaeticus</i> AF1 was isolated from kombucha tea. Due to its high capacity of producing BC from unconventional sources (such as orange pulp), its genome was sequenced¹, but the strain had never been genetically manipulated.</p>
<p>In June we decided to take on the challenge of domesticating <i>K. rhaeticus</i> AF1 and developing a pipeline spanning the cultivation of orange pulp to creating new biomedical applications for BC. Following the synthetic biology principles of design-build-test-learn, we designed, executed, and evaluated experiments from pulp to new BC applications. As Unicamp_Brazil is a diverse and multidisciplinary team, we worked on the optimization of culturing conditions, metabolic modeling as a guide for genetic manipulations, development of transfection protocols, design and construction of a toolbox with plasmid backbones suitable for <i>K. rhaeticus</i> AF1 engineering, design of low-cost induction system, construction of a low-cost dual-stage bioreactor, development of molds for custom BC sheet production, successful tests of adhesion of human cell lines to BC membranes, and, last but not least, dissemination of synthetic biology to the wider community. </p>
<hr>
<h1>Sustainability</h1>
<p>The world demands sustainable technologies in order to prosper, and one of science's means of achieving this is by using synthetic biology techniques. Cellulopolis is a project designed to meet demands from several markets, with the production of pure bacterial cellulose (BC) obtained from genetically-modified strains. Cellulose (CL) is a naturally occurring biodegradable polymer that has properties that allow its applicability as a fibrous scaffold for skin, cartilage, and vein, among others, as well as being an excellent alternative in the treatment of burns. Obtaining CL from plant sources by traditional methods is cumbersome due to difficulties in purification, genetically modified organisms have been the most viable alternative in this regard. Some microorganisms produce CL naturally, as is the case with <i>Komagataeibacter rhaeticus</i> AF1 strain, found in a fermented beverage popularly known as Kombucha.</p>
<p>Genetic engineering can optimize the metabolic pathways of various microorganisms. In our project, we performed computational simulations of key features in the BC synthesis pathways and identified potential targets for modification. Thus, we are genetically engineering, for the first time, <i>K. rhaeticus</i> AF1 by using Modular Cloning (MOCLO) compatible transcription units designed to increase BC production with minimal impact in biomass accumulation, so as to not give a selective advantage to non-BC producing strains. This will be achieved through a bimodal growth/production strategy, as will be discussed below.</p>
<p>In addition, we are producing BC in a culture media derived from orange juice production residues, in view of the negative environmental impacts caused by the waste generated from this process in Brazil, which is the largest producer in the world, as is illustrated in Figure 1. Our project aims to sustainably increase the production of BC, aided by strain engineering employing a genetic toolkit we are adapting for <i>Komagataeibacter</i>, thus making BC more accessible for the treatment of burns, other injuries, or further applications.</p>
<p>Brazil is the largest producer of orange juice in the world, as is illustrated in Figure 1, which generates large amounts of waste with negative environmental impacts. We decided to work on a bacteria strain capable of producing the BC in a culture media derived from orange residue. Our project aims to sustainably increase the production of BC, aided by strain engineering employing a genetic toolkit we are adapting for <i>Komagataeibacter</i>, thus making BC more accessible for the treatment of burns, other injuries, or further applications.</p>
<figcaption>Figure 1. Dimensions of orange juice production in Brazil. Image adapted from (CITRUSBR, 2021).</figcaption>
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<p>A good alternative to plant cellulose is bacterial cellulose, which has the same chemical structure as the plant counterpart, but without “contaminants” such as hemicellulose, lignin, and pectin. This easily moldable material has a three-dimensional network structure of cellulose nanofibrils in the form of a ribbon, which is capable of efficiently absorbing and retaining water.</p>
<p>This BC is produced by bacteria in a natural and purer way than by plants, being a very versatile natural biomaterial that can have its production optimized through bacterial engineering techniques. Bacteria can convert about 50% of carbon into cellulose, varying according to growth conditions, and naturally produce it for various purposes, such as protection and preservation of nutrients. The properties of cellulose vary according to the bacterium that produces it and the substrates available in the cultivation medium.</p>
<p>There are several possible applications for bacterial cellulose, including in the medical field, where it can be widely used in the treatment of wounds and burns, as artificial skin during healing. However, this process has a high cost and pulp production is not very efficient, making it necessary to seek measures for the development of modified strains in order to increase pulp production, and also to lower the costs involved in this process. Bacterial cellulose can also have applications in food, cosmetics, and bioethanol areas, not forgetting its use as a scaffold for the growth of cells, tissues, and organs.</p>
<p>The standard medium for cultivation of <i>Komagataeibacter</i> is HS (Hestrin; Schramm, 1954), which we employed during most of our work. In parallel, we also performed tests cultivating <i>Komagataeibacter rhaeticus</i> AF1 in media containing waste from the orange juice industry. This is interesting due to the fact that Brazil produces over 16 million tons of oranges annually with 60% of this biomass being discarded. Hence, the optimization of the production of bacterial cellulose from orange waste can provide a noble destination for these residues, reducing their environmental impact.</p>
<p>The standard medium for cultivation of <i>Komagataeibacter</i> is HS (Hestrin; Schramm, 1954), which we employed during most of our work. In parallel, we also performed tests cultivating <i>Komagataeibacter rhaeticus</i> AF1 in media containing waste from the orange juice industry. In parallel, we also learned from BioSmart Nanotechnology to cultivate Komagataeibacter rhaeticus AF1 in media containing waste from the orange juice industry. This is interesting due to the fact that Brazil produces over 16 million tons of oranges annually with 60% of this biomass being discarded. Hence, the optimization of the production of bacterial cellulose from orange waste can provide a noble destination for these residues, reducing their environmental impact.</p>
<hr>
<h1>Inspiration</h1>
<p>The idea for a waste management project arose from research and brainstorming within the team, which started its journey in 2021, with an intense interest in making a real impact on the paths of synthetic biology in Brazil and the desire to transform waste into high-value products. Our goal until May 2021 was to engineer bacteria to digest polystyrene and convert it into PHA or PHB, in a project named Styropolis (video).</p>
<p>We designed a strategy whereby that goal could technically be achieved, however, through diverse insights obtained by stakeholders we learned that, even though polystyrene is recyclable and could potentially be used as a carbon source for bioplastic production, its low density leads to high transportation costs per kg of raw material, creating a negative environmental impact that would detract from our “green” goals. This is discussed in greater detail in the Human Practices section.</p>
<p>As previously mentioned, Brazil is the largest orange producer in the world and we are in the State of São Paulo, where 78% of the country's production is concentrated, so, in June 2022 our work was completely re-designed towards a project with a more concrete application. Inspired by the Imperial_College iGEM team (<i>K. rhaeticus</i> iGEM strain sequencing and toolkit development) and by collaborators from the city of Araraquara (BC production from orange juice as a culture medium), we decided to develop a strain capable of producing cellulose on a large scale from orange residues, overcoming the most diverse obstacles, through synthetic biology. The Cellulopolis project was born!</p>
<p>As previously mentioned, Brazil is the largest orange producer in the world and we are in the State of São Paulo, where 78% of the country's production is concentrated, so, in June 2022 our work was completely re-designed towards a project with a more concrete application. Inspired by the Imperial_College iGEM team (<i>K. rhaeticus</i> iGEM strain sequencing and toolkit development) and by collaborators from BioSmart Nanotechnology, we decided to develop a strain capable of producing cellulose on a large scale utilizing based on Komagataeibacter rhaeticus AF1, through synthetic biology. The Cellulopolis project was born!</p>
<p>BioSmart Nanotechnology is a research and development company that, in collaboration with JBT Corporation, works on the Food Waste project, which consists of the reuse of agroindustry residues to produce BC (a technology proprietary of the consortium Uniara, BioSmart Nanotechnology, and HB Biotec).</p>
<hr>
<h1>Challenges</h1>
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<p>Bacterial cellulose is produced by several groups of bacteria and among them is <i>Komagataeibacter</i>, with one of the most efficient productions of this polymer. <i>K. xylinus</i> is the most studied bacterium of the genus and its metabolism has been well studied and described, being used as a model for the study of cellulose production. In the present work, we focused our efforts on the related species <i>Komagataeibacter rhaeticus</i> AF1, a producer of the fermented tea (kombucha), which was isolated similarly to <i>K. xylinus</i>, as a producer of cellulose. The iGEM project was hosted by a yeast laboratory, therefore we carried out several experiments to gain experience with bacterial growth and BC production, aiming at an optimization of cellulose synthesis through the cultivation conditions, including the composition of the culture medium itself. The literature describes genetic engineering efforts to develop <i>Komagataeibacter</i> with improved BC synthesis, however, it is unclear if engineered strains are stable in large-scale production conditions. Thus, we would like to contribute to the BC research community by building new parts and adapting methods and protocols, aiming at a more efficient cellulose synthesis.</p>
<p>As <i>K. rhaeticus</i> AF1 is not a standard model organism, there are no protocols adapted for this specific bacterial strain, so the Unicamp_Brasil team worked with experimental conditions and protocols to allow the cultivation and genetic manipulation of the locally isolated strain. We performed experiments to identify optimal incubation temperatures for <i>K. rhaeticus</i> AF1, looking for the most suitable temperature to maximize its growth. We also made use of waste from the production of orange juice, normally discarded, seeking to contribute to the reduction of the environmental impact caused by these, in addition to the media commonly used in laboratories, such as Hestrin-Schramm (HS). In addition, titrated the concentration of different antibiotics in the growth media in order to determine the minimal concentration required to discriminate between wild-type and transformed strains. This required extensive research into optimized protocols for closely related species and a long period of testing with many variations in parameters, which culminated in an adapted set of instructions for handling and genetically manipulated <i>Komagataeibacter rhaeticus</i> AF1. </p>
<p>As <i>K. rhaeticus</i> AF1 is not a standard model organism, there are no protocols adapted for this specific bacterial strain, so the Unicamp_Brasil team worked with experimental conditions and protocols to allow the cultivation and genetic manipulation of the locally isolated strain. We performed experiments to identify optimal incubation temperatures for <i>K. rhaeticus</i> AF1, looking for the most suitable temperature to maximize its growth. In addition to the media commonly used in laboratories, such as Hestrin-Schramm (HS). In addition, titrated the concentration of different antibiotics in the growth media in order to determine the minimal concentration required to discriminate between wild-type and transformed strains. This required extensive research into optimized protocols for closely related species and a long period of testing with many variations in parameters, which culminated in an adapted set of instructions for handling and genetically manipulated <i>Komagataeibacter rhaeticus</i> AF1. </p>
