Reusing recyled oranges to lower our production costs
One of the biggest challenges with scaling up bacterial cellulose (BC) production and producing an alternative packaging product at an industrial scale is the cost of Hestrin-Schramm (HS) media, the conventional BC growth media used to produce BC. In fact, this media represents up to 65% of total production costs (1).
Although the increased production costs associated with Cellucoat may be justifiable in a shifting packaging market where sustainable alternatives are becoming increasingly desirable, this remains an important area of improvement. Fortunately, this issue is not isolated to our project: given the growing scope of BC applications in the packaging and biomedical fields, the potential for fruit waste as a media supplement is a relatively well-researched phenomenon (2). This inspired our fruit waste media subproject, where we explored the potential of enzymatically-digested navel oranges to supplement HS media and reduce BC production costs (3).
After meeting with several industry stakeholders, including John Kelly and Christopher Clark, we realized that our BC production system needed work: while packaging companies may accept a 3- or 4-fold cost increase to shift to more sustainable materials, a product costing more than this would be significantly more difficult to market. We also learned that food waste is inevitable in the produce production pipeline, and that there are already systems in place to direct this byproduct to secondary industries. As such, we wanted to assess if fruit waste could be a viable supplement to HS media, both to reduce costs, and to integrate our underlying value of sustainability through a cradle-to-cradle design.
One of the key components of BC growth media is glucose, which is also a significant contributor to its high cost. By breaking down the polysaccharide components of the fruit mass - including cellulose and pectin - with enzymes, the resulting glucose can be used as a low-cost alternative to glucose in HS media. Hydrolysis is a reaction wherein the complex sugars in fruits are broken down into monosaccharides, which can serve as fermentable sugars for bacteria such as K. xylinus, our BC producer. Although chemical and physical hydrolysis methods exist, enzymes have demonstrated a superior binding affinity to substrates than inorganic alternatives, and were thus selected for this project.
The long-term purpose of this subproject was to demonstrate a proof of concept for the use of other fruits, specifically discarded fruit components, as a means to supplement BC growth. To assess the effects of various common fruit waste, including pulp, peels and juice, we separated our oranges accordingly. When selecting enzymes for hydrolysis, we used cellulase, pectinase and invertase, or sucrase. This selection was based on the composition of oranges and fruits in general.
The experimental design of this subproject was inspired and adapted from literature. Although enzymes are relatively expensive, our scoping of literature revealed that fruit processing requires relatively small levels of enzymes. We decided to use 1% solutions of our respective enzymes and add appropriate volumes of this solution for our varying orange components.
For the hydrolysis, we utilized 24-hour water baths to hold our fruit waste at the most enzymatically efficient temperatures. We can then freeze our treated fruit waste or immediately combine it with HS media to create our fruit waste media which we can send to be autoclaved. We have utilized a handheld refractometer to test the levels of glucose of control samples, cellulase/pectinase-treated samples, as well as cellulase/pectinase/invertase-treated samples. Apart from making the FWM we have also looked at how well it grows our BC and if there any physical properties of the BC that gets altered because of it. We have tests for BC growth as well as co-culture growth and a series of corresponding uniaxial tests to analyze physical properties.
One frequent question about the FWM subproject addresses concerns about simultaneously reducing food waste and using it in our system. Our answer is two-fold. First, as with all attempts to reduce waste, completely removing it is very difficult and requires immense fundamental changes to how we think and interact with our food. As such, the idea that we might reduce food waste to the point of not having enough to recycle is a far-off problem. Secondly, from our HP we learned that due to a number of factors such as produce quality and appearances, there is a large amount of fruit waste before they even hit the shelves. As such, there seems to be a supply we can always tap into our cradle-to-cradle design.
First for FWM comes the enzymatic treatment of the navel oranges. Using our refractometer we were able to quantify how much glucose we could produce from the other sugar-based components. We took measurements of our untreated (control) samples, cellulase and pectinase (CP) treated, and CP and invertase treated samples. We first made a standard curve from our refractometer to compare Brix% measurements to glucose levels in grams per millilitres. Then we could take measurements and use them to analyze the success of our treatments.
For orange juice, we were able to increase glucose from our control to CP and invertase treatment by 5.74%. For orange peels, we increased glucose by 46.28%, and for pulp, by 28.63%. The actual concentration of glucose for juice, peel and pulp we were able to achieve ended up being 0.1283 g/mL (±0.0185), 0.0156 g/mL (±0.0021), and 0.01842 g/mL (±0.0014) respectively.
Figure 1. Glucose concentration of orange juice samples after enzymatic treatment. Each treatment was for 24 hours. Each measurement (n=3) was taken with a refractometer and converted to a g/mL value.
Figure 2. Glucose concentration of orange peel samples after enzymatic treatment. Each treatment was for 24 hours. Each measurement (n=3) was taken with a refractometer and converted to a g/mL value.
Figure 3. Glucose concentration of orange pulp samples after enzymatic treatment. Each treatment was for 24 hours. Each measurement (n=3) was taken with a refractometer and converted to a g/mL value.
With a better picture of the amount of glucose we were working with we could perform growth tests to understand how our FWM affects the growth of E. coli and BC.
Figure 4. BC growth in a co-culture, grown in varying Juice FWM (15%, 30%, 45%) with daily feedings. Each measurement (n=3) was taken with a scale.
Figure 5. BC growth in a co-culture, grown in varying Peel FWM (15%, 30%, 45%) with daily feedings. Each measurement (n=3) was taken with a scale.
Figure 6. BC growth in a co-culture, grown in varying Pulp FWM (15%, 30%, 45%) with daily feedings. Triplicates used. Each measurement (n=3) was taken with a scale.
Figure 7. E. coli growth in a co-culture, grown in varying Juice FWM (15%, 30%, 45%), with daily feedings of 1.767 mL of the respective FWM media. Each measurement (n=3) was taken with a spectrophotometer at OD600 of a 400μL media aliquot.
Figure 8. E. coli growth in a co-culture, grown in varying Peel FWM (15%, 30%, 45%), with daily feedings of 1.767 mL of the respective FWM media. Each measurement (n=3) was taken with a spectrophotometer at OD600 of a 400μL media aliquot.
Figure 9. E. coli growth in a co-culture, grown in varying Pulp FWM (15%, 30%, 45%), with daily feedings of 1.767 mL of the respective FWM media. Each measurement (n=3) was taken with a spectrophotometer at OD600 of a 400μL media aliquot.
Analyzing the general trends of these graphs we see that for BC growth we see more growth with lower concentrations of FWM as is an expected result. We also see that Juice FWM produced more BC than Peel FWM, and both produced more BC than Pulp FWM. This is expected from our refractometer results showing the treated orange juice having a higher concentration of glucose than either the hydrolyzed pulp or peel.
For E. coli growth, generally 15% FWM provided better growing conditions than 30% and 45%. We generally see a peak number of E. coli on day 4 for peel and pulp, with a peak for juice around days 3-4. The number of E. coli decreases which is expected from our models as the rate of substrate consumption by E. coli is higher than K. xylinus.
In the production of bacterial cellulose, media has been defined as the most important factor. This is evident as culture media accounts for about 30% of the total production cost (Figure 10). Hence, using fruit waste media (FWM) was for the sole purpose of reducing the production costs of bacterial cellulose to make it more economically advantageous. To validate that our modified media accomplishes its designated purpose, we conducted cost analysis on the lab level comparing the costs of the traditional HS media to our modified media consisting of HS media and FWM.
The method we used in conducting cost analysis involved calculating the costs of making a gram of BC from the media used in the lab. As the FWM experiments were conducted for both monocultures of just K. xylinus and co-cultures with E. coli, we performed the analysis on all iterations of the experiments.
The analysis took into account the cost of producing the BC being based on the cost of the media. Hence, we documented the costs of all the materials used in producing the media and scaled their costs down to the amount of media actually used in each iteration of the BC growth experiment. Then, we calculated the cost of producing it per gram of BC by dividing the costs of the media by the BC yield for that experiment, thus resulting in the unit cost of producing a gram of BC. This value was then used to determine the cheapest media used in producing BC.
Figure 10. Overview of the costs analysis of several growth strategies. Legend - Juice (JU), Pulp (PU), Peel (PL)
From our experiments and analysis, we discovered that glucose makes about 33% of the media costs. Hence, if we could reduce that cost, then we would be able to reduce the overall costs of production by 33%. Using fruit waste reduces our costs significantly, and we predicted that replacing all of the glucose would be the most efficient method for production. However, from our experiments and analysis, we realized that the BC sample grown in a co-culture using only 45% Pulp FWM had the lowest cost of production per gram of BC.
Figure 11. The percent difference of the several production iterations of BC in our lab. Legend - Juice (JU), Pulp (PU), Peel (PL), Co-culture (C) and Monoculture (M)
From these results, we realized that we were able to reduce the cost of production actually by 60% instead of 33% compared to the cheapest production of BC in pure HS media. We can also conclude that since this media still contains HS media, reducing the amount of HS and still maximizing BC yield could still help us further reduce the costs of BC production. Ultimately, reducing the costs would make Cellucoat more economically advantageous. We have also performed a full financial analysis on the industrial scale production of BC using a modified media of 45% FWM which can be found on our Entrepreneurship page. Therefore, this proves that our FWM performs its aim of reducing the costs associated with the production of BC.
With a better understanding of our FWM and how it fairs compared to regular BC a future step would be to test more fruit, this could be done by using different fruits individually or more practically using a mixed “fruit salad” that would better emulate a process that would be used on a larger scale. Still, we will look to isolate waste components like we have done into juice, peel, and pulp since the type of waste that might be found in the real world may be different. We would look at methods to homogenize our fruit waste to allow consistent FWM batches. Another piece this could help with would be to reduce the number of particulates in our FWM. Our physical treatment process did help but particulates could still be seen in the media which would affect media consistency.
More tests with larger varieties of enzyme cocktails can also be tested to truly make the most of our fruit waste. As well we could experiment with the levels of enzymes, method of delivery, and even order of enzymes to understand the effects they may have. All these could be useful in optimization. Further methods of breakdown could be useful to look into as well, perhaps heat or chemical treatments could be of use in breaking down stronger sugar-based compounds found in our fruit waste such as peels and rinds.