From blue crab to green chitin A sustainable extraction method using bacterial fermentation

As a first step, to remove surface impurities and excess of soft tissue a preliminary cleaning is carried out by washing cycles in demineralized water, followed by a drying process in the oven for 10 hours at 70°C.

The shell is then coarsely ground, this process allowes maximum reaction yield and optimises the contact surface. Subsequently, the raw product obtained is introduced into a specially designed bioreactor where the biotransformation phase begins. Inside the reactor there is the broth (Lactose Broth), and an addition of peptone as a nutrient for bacterial growth, then the pH is adjusted to about 6.5 using an acid solution. This pH values is optimal for the enzymatic activity of microorganisms. The temperature is constantly monitored and maintained between (34-37)°C and glucose is added to provide energy to support the metabolic activity of microorganisms.

When bacteria consume glucose by glycolysis, they produce ATP, which is the basic unit of cellular energy.

The energy produced in this process allows the synthesis of essential molecules for bacterial growth, such as nucleotides, fatty acids, proteins and other cellular molecules. Moreover, glucose influences bacterial metabolism by regulating gene expression and promoting the production of enzymes and other factors involved in the growth and survival of bacterial cells.

The addition of Lactobacillus plantarum as a fermentation agent allows the start of the first fermentation phase (demineralisation). During this phase, which lasts 4 days, the system is constantly oxygenated and stirred to ensure proper mixing of the reagent and a uniform distribution of nutrients and microorganisms within the bioreactor.

After 4 days, the second bacterial species - Bacillus subtilis - is added to the reactor, to start the deproteinization phase. Additional broth is also incorporated to provide the newly added bacteria with enough nutrients. This bacterial species, owns specific metabolic capacities, that contributes significantly to the conversion of the initial substrate into the desired compounds. During this second four-day phase a series of biochemical reactions occur, culminating in the formation of the desired compounds.

After this biotransformation phase, the resulting compound is subjected to a purification process to ensure that the final product is of high purity and quality. This process includes additional washing in distilled water and the extraction of pigments (carotenoids) through a treatment with ethyl alcohol. At the completion of this process the product is dried at 40°C for about 20 hours to ensure a complete removal of residual humidity.

The final sample was analyzed by IR spectrophotometry [Figure 2]. A comparison was carried out between samples obtained by different extraction methodologies:

1) A biological process which employed a single bacterial species (Bacillus subtilis) followed by a HCl 0,1 M treatment for 6 hours,

2) A biological process which employed two different bacterial species ([1] Bacillus subtilis and [2] Lactobacillus plantarum) followed by a treatment with HCl for 6 hours,

3) A process that employed the same two different bacterial species but in reverse order ([1] Lactobacillus plantarum and [2] Bacillus subtilis) with a subsequent treatment in diluted CH₃COOH for 12 hours,

4) A chemical process carried out only with NaOH and HCl, with a duration of 6 hours per phase, to replicate what is reported in literature.

All the processes mentioned above were followed by a final purification of the obtained chitin with pure ethyl alcohol to ensure the remotion of any pigment residues (the alcohol was then distilled and the pigments were used for secondary tests). As a result of these analyses, it was possible to compare the effectiveness of the aforementioned processes used for the extraction of chitin, in relation to their environmental impact. The observation focused on the discrepancies of characteristic peaks likely caused by possible impurities or incomplete states of demineralization or deproteinization. Initially it was observed how the demineralization phase had more variations compared to deproteinization. Given that, we focused our approach to wards improving the effectiveness of demineralization through the use of specific bacteria such as Lactobacillus plantarum, the manipulation of pH and changes in the properties of the culture medium improving performance and reducing residual carbonates present in the final product. Comparing the IR analyses, a promising similarity in the spectrum was found, with significant changes in some molecular characteristics. Peaks obtained are discussed in detail in [Figure 3] and in the graph below [Figure 4]. In conclusion it was possible to observe different molecular structures through an approximate microscopic observation of the chitin obtained with the four different methods, [Figure 9, Figure 10, Figure 11 and Figure 12].

Following the extraction of chitin from the blue crab exoskeleton and its spectrophotometry analysis, it can be observed how the production of chitin through bacterial fermentation results more environmentally sustainable compared to the chemical extraction. However, structural chemical-physical changes directly caused by the different production processes were identified. Variations in various peaks of the FT-IR graphs were observed, most likely caused by enzyme activity and the specific environmental conditions within the bioreactor. Significant changes in the amino functional groups and the carbonyl (C=O) groups, particularly around 1660 cm⁻¹, suggest that crystalline alterations could be the potential result of interactions between different enzymes in the bioreactor, thereby introducing variations in the levels of deacetylation and decarbonization of the final product. Changes in the peaks were associated with C-N and C-O-C bonds; they were respectively observed at approximately 1380 cm⁻¹ and 1030 cm⁻¹, indicating possible interactions with bacterial metabolites. The aforementioned variations highlight the molecular adaptability of the biopolymer to biological treatments. Additionally, images captured using optical microscopy reveal variations in the texture of the final product, with bacterial chitin showing a more wrinkled texture compared to the smoother appearance of chemically produced chitin, indicating a difference in crystallization. A key objective in this research was optimizing the process from an ecological perspective, aiming for minimal environmental impact. The production of chitin through bacterial processes is easily reproducible and cost-effective, due to the rapid reproduction rate of the bacterial species involved in the process. In addition to that, the process is highly sustainable, since it leverages reactions that naturally occur in bacterial metabolism. In contrast to this production method, large-scale industrial chitin extraction relies on highly polluting chemical reagents; laboratory evidence shows that purifying approximately 5g of chitin utilizing chemical solvents requires not only over 200 ml of NaOH and an additional 200 ml of HCl, but also larger quantities of water, used to eliminate excess reagents in multiple washes. On the other hand, the microorganisms can produce multiple chitin stocks, resulting in a much more economical and ecological production, with minimal product waste. While limited in several aspects, this research aims to provide a tangible demonstration of how an environmental emergency that causes significant damage to both regional biodiversity and the economy can be intelligently and innovatively repurposed. As the second most abundant biopolymer on Earth after cellulose, chitin is a fundamental resource in multiple sectors and should not be wasted. With current technologies, we can exploit this resource in extremely eco-sustainable ways.