Bioreactor

A Novel Proposal

The first design for the bioreactor

Features

• Optimized biocontainment and mass transfer of the substrate.
• Automated Temperature control.
• Automated pH and minerals via sensor feedback composition control.
• Provision for OD and flow rate analysis.

Details

The bioreactor was designed to degrade air-borne hydrophobic substrates and halocarbons and release purified air.

To achieve the same, we proposed the water-insoluble mineral Kaolinite to provide a surface for degradation via the adsorption of the halocarbons and bacteria.

This setup may help ensure the bacteria can access halocarbons from the surface they're adsorbed onto and the appropriate mineral environment and medium to disperse waste in the solution.

The bioreactor has a bio-containment mechanism based on Kaolin's excellence in adsorbing bacteria and quaternary ammonium biocides, eliminating any escaping bacteria.

The bioreactor is drawn with a mechanism to maintain its mineral composition and pH, minimizing the toxicity of the byproducts of the reaction.

Our design of the bioreactor proposed to optimize the following: 

1. The flow of Halocarbon substrate entering the reactor [coupled with O2 supply] (l/h)
2. Product of mass-transfer coefficient for Halocarbon and gas-phase specific area (/h)

Sources for design inspiration

• Designing Principles: Tekere, M., 2019, 'Microbial Bioremediation and Different Bioreactors Designs Applied', in E. J.  -Lopes, L. Q.  Zepka (eds.), Biotechnology and Bioengineering, IntechOpen, London. 10.5772/intechopen.83661.
•  (HualongWu and Yenli Chang et al., adsorption of P. Putida onto Kaolin at different growth phases ) 

Biocontainment

• A novel, cost-effective biocontainment method was proposed, inspired by the containment prowess of the human nostril.
• A plastic mesh was thought to resemble nostril hair and Kaolin, a clay mineral, as the absorbing substrate playing the role of mucus.
• Quaternary amine salts were chosen as the active biocide that will absorb into the Kaolin alongside bacteria and lyse the adsorbed bacterial cells.

Components of biocontainement proposal

The primary advantage of such a design comes from its cost efficiency. All components are readily available, very cheap, and the design is easy to construct.

Kaolin is available at 0.037 USD/kg, and the quat salts are widely used as stock disinfectants in labs and hospitals. The filter costs a minute fraction of the most commercially available filters and is most suitable for coarse filtration.

If the need arises, the design can be scaled to cover the reactor and used for BSL 2 reactors.

Background

The principal variables in this design, accompanied by previous research on the same, are as follows:

• The extent of deposition of Kaolin onto steel: (R. Nahji et al., a study of electrodeposition of Kaolin onto steel)
  Rachida, Najih & Abdelilah, Chtaini & Oulfajrite, Hassan. (2011). Preparation and Physicochemical Characterization of Natural Phosphate and Kaolin Coatings in Stainless Steel. Portugaliae Electrochimica Acta. 29. 39-45.
• The kinetics and the extent of adsorption of the bacteria onto the Kaolin: (J.B. Gunnison and M.S. Marshall et al., comparison between bio-adsorbents like Kaolin, CaCO3 and charcoal) https://journals.asm.org/doi/epdf/10.1128/jb.33.4.401-409.1937, (Hualong Wu and Yenli Chang et al., adsorption of P. Putida onto Kaolin at different growth phases ) https://www.sciencedirect.com/science/article/pii/S0009254114004471
• The extent of adsorption of the biocide onto the Kaolin: (Jiang Hao et al., study on the extent of adsorption of different quaternary ammonium salts onto Kaolin) https://www.sciencedirect.com/science/article/abs/pii/S2095268613000499
• The possible change in bactericidal properties after adsorption: (Benjamin Justus Hayde et al., study to decipher change in biocidal properties of quats after adsorption onto different clay minerals like Kaolin and smectite) https://pubmed.ncbi.nlm.nih.gov/32958787/ 

Alternatives to Kaolin

We have an alternative adsorbent for Kaolin, CaCO3, which has mild bactericidal properties. CaCO3 was not chosen in lieu of its poor adsorbance concerning Kaolin. The relevant research on using CaCO3 as the adsorbent is as follows.

• A Yamanaka et al. study on the adsorbing ability of porous calcium carbonate compared to the mineral version demonstrated by incorporation in chewing gum and used to adsorb oral bacteria. https://pubmed.ncbi.nlm.nih.gov/11212584/
• Email Chibowsky et al., a study of the deposition of calcium carbonate on various substances (stainless steel, glass, etc.) under quiescent conditions and the presence of magnetic fields at different temperatures. CaCO3 adhered firmly onto surfaces like stainless steel under quiescent conditions with no MF and room temperature. https://pubmed.ncbi.nlm.nih.gov/14568055/

Standardization

Modification of Proposal to fit manufacturing norms and achieve degradation and cost efficiency.

Why do we need to standardize?

• The proposed model had been designed from first principles and focused on optimally fulfilling all our proposal requirements but has no pre-existing data.
• Standardization of existing models has to precede any hardware or financial evaluation based on a literature survey and professional guidance.

Timeline for standardization process

Airlift Bioreactor Model

Description: Gas agitated bioreactors where the injection of a gas stream into a riser compartment causes the reaction broth to circulate to the interconnected downer compartment with no gas phase.

An example of Airlift Design

(Picture credit: Negi, B.B., Sinharoy, A. & Pakshirajan, K. Selenite removal from wastewater using fungal pelleted airlift bioreactor. Environ Sci Pollut Res 27, 992–1003 (2020).)

Draft for Adaptation to Airlift

Proposal for Airlift adaptation

First standardization due to the proximity of the design to the proposal.

1. Widely used for immobilized cells. Hence, the kaolin model fits right in.
2. Energy-efficient mass and oxygen transfer due to convective movement.
3. Known for very low shear stress, and hence shall preserve the fragile kaolin layer
4. Relatively simple to implement and scale up, with little to no emissions.

Had very poor degradation efficiency and higher cost (41%, from Peilun Xua Yang Wei Nana Cheng Sujing Li Wei Li  Tianjiao Guo  Xiangqian Wang w et al., 2019 Evaluation on the removal performance of dichloromethane and toluene from waste gases using an airlift packing reactor) than BTF and the RBC and hence was discarded in their favour. 

Moreover, studies on using the same to process halocarbons (e.g., DCM) are sparse compared to the latter standardizations. 

Biotrickling Filter Model

Description: A bio-trickling filter combines a biofilter and a scrubber. The bacteria responsible for decomposition are immobilized on a carrier or filter material. The filter material consists of synthetic foam or structured plastic packing. In our case Kaolin.

An example of Biotrickling Design

(Picture credit: Valdebenito-Rolack, E.; Díaz, R.; Marín, F.; Gómez, D.; Hansen, F. Markers for the Comparison of the Performances of Anoxic Biotrickling Filters in Biogas Desulphurisation: A Critical Review. Processes 2021, 9, 567.)

Draft for Adaptation to BTF

Proposal for BTF adaptation

The second standardization was chosen due to extensive literature on its use to process halocarbons.

• Widely used for immobilized cells. Hence, the kaolin model fits right in.
• Favourable reported mass and oxygen transfer.
• The filter itself uses little energy (< 1 kWh/1 000 m³). Only a small recirculation pump is needed for the vaporization of water. Most usage can be attributed to the ventilator.
• Claimed to be the most efficient per unit cost at average flow rates in some sources (~100m3/h).

The following shortcomings led to the BTF being discarded in favour of the RBC:

• Fluctuations in composition and load of incoming air have serious consequences for the yield.
• Toxic and high concentrations of waste components are common problems. Generating caustic waste products like HF and HCl in halocarbons seeping into the biomass and producing dead zones.
• Packing can become blocked by biomass or due to the pressure of the column above.
• Generates biomass-rich wastewater continuously, which is a serious pollutant.

Rotatory Bio-contactor Model

Description: Rotating biological contactors (RBC), also called rotating biological filters, are fixed-bed reactors consisting of stacks of rotating disks mounted on a horizontal shaft. The microbial community is alternately exposed to the atmosphere and the wastewater, allowing both aeration and assimilation of dissolved organic pollutants and nutrients for their degradation.

Note: The team has conducted experiments to study the degradation efficiency of Putida in 

An example of Rotatory Biocontactor Design

Proposal for RBC adaptation

Example of Solid State Design

The third standardization was chosen due to its well-known efficiency, robustness, and ability to process wastewater and VOC pollutants in airstreams. 

It was thought that it even took care issue of the non-solubility of the substrate in water, as it seemed to allow gaseous uptake by biofilm in the upper half. 

There were an overwhelming number of advantages and almost no disadvantages to it until it was pointed out by Professor G. Suraishkumar that the biofilms it uses are itself an aqueous phase. Unfortunately, that will severely impact the gas transfer rates of the halocarbons, which are insoluble in water.

In the literature survey, it seemed to be the most cost and degradation-efficient bioreactor for remediating pollutants out of a gas or wastewater flow.

Advantages

1. It is mechanically straightforward, and skilled technical labour will not be required. (Source https://www.evoqua.com/en/evoqua/products--services/aerobic-wastewater-treatment/rbc/rotating-biological-contactors/)
2. RBCs are commonly manufactured in India (so we have cost-effective, domestic market supply available) https://connect2india.com/Rotating-Biological-Contactor-suppliers.
3. It is a common part of biotech and bio-hardware courses given in India; hence, skilled experts specializing in its manufacture and maintenance are easily available.

An article described RBCs as resistant to stress as the biofilm (of Putida F1) was shown to evolve into a community culture that was very robust, more efficient, and versatile and grew quickly with increasing supply, as remarked in the cited literature.

It was seen quite interestingly on increasing stress of incoming gas, the colonized biofilm showed greater robustness, and the biofilm increased in thickness, compensating for the increased substrate inflow!

(Reference: Vinage I., Rudolf von Rohr, Biological waste gas treatment with a modified rotating biological contactor. Ι. Control of biofilm growth and long-term performance. Bioprocess Biosyst Eng 26, 69–74 (2003). )

Solid State Design Model   

Description:  Solid state design is a class of upcoming bioreactors with widespread application from fermentation to bioremediation. At the forefront of research in interest in bioreactor design, solid state reactors are easy to construct, monitor and maintain and give high reactor efficacies per unit volume due to the active element being the area of the reactor instead of volume, which can be highly optimized.

Example of Solid State Design

(Picture credit: Manan, M.A., Webb, C. Newly designed multi-stacked circular tray solid-state bioreactor: analysis of a distributed parameter gas balance during solid-state fermentation with influence of variable initial moisture content arrangements. Bioresour. Bioprocess. 7, 16 (2020))

Draft for Adaptation to Solid State Design

Final proposal for implementation

The RBC model had one major fault: the mass transfer of the gas to the bacteria would remain low due to negligible dissolution of the gas in the biofilm.

This finally led to a solid-state design wherein the bacteria would be allowed gaseous uptake.

A small reversal of the standardization techniques followed so far allows for a creative, robust design optimized for gas bioremediation.

It may be noted that a pattern of shifting towards "drier" bioreactors emerges from the airlift model to the final solid-state design.

This design, thus, optimizes all three necessary parameters:

1. Gas transfer rate to bacteria
2. Degradation efficiency
3. Cost efficiency

Alternation

Need

As established in the theoretical basis, the bioreactor must operate in a cycle between an anaerobic reduction phase and an aerobic oxidation phase to complete degradation.

Background

We discovered that high BOD wastewater processing units use solid-state reactors to alternate between aerobic and anaerobic conditions for treatment. Some examples are slaughterhouses, dairy, and, rather surprisingly, the Azo dye industries.

A very insightful work on the same has been cited below: 

(Talaiekhozani, Amirreza, A Review on Different Aerobic and Anaerobic Treatment Methods in Dairy Industry Wastewater (March 30, 2019). Goli A, Shamiri A, Khosroyar S, Talaiekhozani A, Sanaye R, Azizi K. A review on different aerobic and anaerobic treatment methods in dairy wastewater. Journal of Environmental Treatment Techniques 2019;7(1):113-41.)

Model for Alternation

• There are two alternative methods two achieve alternation, one with high capital cost and one with high running costs.
• The first involves sequencing two separate bioreactors with fixed aerobic and anaerobic conditions, and the other involves flushing a single reactor alternatively with nitrogen.
• In both cases, the porting will be controlled by a halocarbon-level sensor which shall track the progress of the reaction.

The first is the only one quoted in the literature survey, and the second one is our proposal. The performance analysis can only be truly established by building lab prototypes of both designs.

Finalization

With great help from Professor G. Suraishkumar from IIT Madras, the design was finalized after a series of 4 virtual meetings. Additions to the final design include the following:

• A resin-coated plastic top to monitor the culture conditions of the solid-state reactor
• A novel spiral + cone design prevents any "shade" onto the culture plates beneath each level, enabling easy monitoring and effectively utilizing the volume.
• A 'flushing' port for easy cleaning and maintenance
• Batch input design 

3D render of solid state bioreactor

Prospects

The whole process of designing a bioreactor from first principles, then standardizing it and finally innovating on the standardized design has led to a model which is both in line with industrial norms (enough to even comment on its finances without construction) and yet novel in it's working and the more nuanced features.

We hope to regenerate interest in the field of VOC bioremediation using our proposed model and combat halocarbons and other pollutants using the same. 

Financial Model

A study on the economic viability and impact of a hypothetical setup based on survey data.

A Case Study

To rationalize the use of our engineered organism, an industrial application was needed. We surveyed several industries dealing in bioreactors and arrived upon the foam industry as a major emitter that can curb its emissions by utilizing our proposed reactor.

We conducted a thorough survey of the foam manufacturing and recycling process and have centred our financial model for the bioreactor on the figures obtained therein.

Foam Recycling as a source of Indirect Emissions

Direct emissions are emissions from the source plant of the device while it is in the manufacturing stage. Indirect emissions are emissions from the manufactured device that takes place elsewhere other than the manufacturing plant, also known as energy-inefficient emissions.

Placement of Bioreactor in manufacturing plants will lead to the processing of only the direct emissions, which is quite less as compared to indirect emissions

https://www.green-cooling-initiative.org/country-data#!total-emissions/all-sectors/absolute

In our case study, we obtained indirect halocarbon emissions during foam recycling generated in the grassroots waste repurposing industry in Bangalore Suburbs.

Foam is blown using halocarbons. These gases are stored in the foam on compressions as bubbles are released into the atmosphere. This case study is unique in targeting decentralized halocarbon emissions by small-scale manufacturers.

Data Obtained

Stage 0: Raw plastic waste is collected from specific collectors that sell it on the market. (Sell for ₹10/kg)

Stage 1: These aggregators source the plastic from the collectors and segregate it by type and colour. (Sell for ₹20/kg).

Stage 2: The segregated plastic waste is washed and broken down into chunks with a grinder. The plastic is then melted and converted into granules. (Sell for ₹40/kg). Foam is compressed into hales and sent for repurposement directly. The compression releases all the stored halocarbons in the foam.

Stage 3: The granules are then coloured and melted to make finished products like buckets, pans, etc.

Proposal: Adding our bioreactor to Stage 2 processing shall curb the majority of halocarbon emissions from this source.

Solid State Design

The cost analysis for the proposed bioreactor is difficult, the design being novel. 

The structure looks similar to industrial stirred fermenters; thus, the cost for the chassis is assumed to be the same. The 1500L estimate, it comes out as 3.5L on the Indian market. 

An additional cost will be generated for the porting mechanism and maintenance of a nitrogen supply. While alternating bioreactors are not mainstream, such a strategy comes up in slaughterhouse wastewater treatment, and such cases were surveyed from the literature to get a cost estimate.  

Finances of Alternation

The first case is equivalent to building two bioreactors and thus puts the estimated cost of the establishment at 3.5L (aerobic 1500L chassis) + 4.5L(anaerobic 1500L chassis) + [1.5L] gas sensor, porting, pumps and other miscellaneous costs + 35,000: Solid State culture setup

As our case study model requires, we expect a setup cost of under 10L to handle 17-18kg of gas per day. 

The second case would require 3.5L for the chassis and [1.5L] under miscellaneous costs, but the running cost is expected to be much higher as a vacuum pump and nitrogen flush would have to be operated every half cycle.

Costs for Chassis have been noted from selling prices on IndiaMart. The approximate assembling cost of 1.5L was calculated by Sagar Bidwe, a bioreactor design engineer from Vicitore Solutions and Equipment LLP.