Description:FORM 2
THE PATENTS ACT, 1970
( 39 of 1970)
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(Section 10 and Rule 13)

“System and method for Cost-Effective Mass Production of Rhodospirillum Rubrum using Polypropylene Bubble Cans’’

BIOFAC INPUTS PRIVATE LIMITED, Indian, Unit-I : Plot No. 74C, Anrich Industrial Estate IDA ,
Bollaram, Hyderabad -502325. Telangana State

The following specification particularly describes and ascertains the nature of this invention and
the manner in which it is to be performed
FIELD OF THE INVENTION :
The present invention relates to the field of microbiology, specifically to the cost-effective cultivation of Rhodospirillum rubrum. The present invention provides an optimized medium and cost-effective cultivation method for the large-scale production of R. rubrum using cost-effective materials (in high-density polyethylene (HDPE) containers) instead of conventional cultivation methods.
BACKGROUND OF THE INVENTION:
A PBR is any device or system that supports the culture of photosynthetic organisms using light. There are disadvantages to both types of PBR systems. In any type of algal/photosynthetic bacterial production light penetration, light path and surface area to volume ratio all affect the density that can be achieved:
• The further light can penetrate into a culture, the more algal cells can be sufficiently lit to divide and grow, but as cell density increases, light is absorbed over a shorter distance within the culture.
• A shorter light path through cultures is beneficial to density as it allows light to reach a larger proportion of the culture, before being absorbed by the algae.
• Higher surface area to volume ratio is beneficial for the same reason.
All these factors have critical implications for the design of any system and shape how open and closed systems are used. In general open systems tend towards larger volumes, with lower density and closed systems tend toward higher densities with lower volumes.
Rhodospirillum rubrum is a gram-negative, purple non-sulfur bacterium with diverse metabolic capabilities. Morphologically, R. rubrum is a spiral-shaped bacterium, classified as a spirillum (plural: spirilla). R. rubrum has several promising applications in biotechnology, including:
• Accumulation of polyhydroxybutyrate (PHB) precursors for bioplastic production.
• Generation of biological hydrogen as a renewable fuel source.
• Serving as a model organism for studying energy conversion from light to chemical energy and the regulatory pathways of nitrogen fixation.
• hydrogen production, and
• Biosynthesis of coenzyme Q10 and single-cell protein.
However, its large-scale cultivation presents several challenges, including specific oxygen requirements, pH control, light intensity needs, and complex nutritional requirements. It also functions as a single-cell protein source and produces bacteriochlorophyll and carotenoid pigments, such as spirilloxanthin, which aid in light absorption during anaerobic fermentation.
Despite these advantages, large-scale production of R. rubrum is hindered by its complex metabolic requirements, sensitivity to environmental conditions, and the high cost of photo bioreactors. The present invention overcomes these challenges by introducing a cost-effective medium formulation and an alternative cultivation method using HDPE bubble cans.
Photosynthesis in Rhodospirillum rubrum is genetically suppressed under aerobic conditions. However, once oxygen is depleted, R. rubrum promptly initiates the synthesis of its photosynthetic apparatus, including membrane proteins, bacteriochlorophylls, and carotenoids, allowing it to become photosynthetically active. R. rubrum utilizes bacteriochlorophylls, which absorb light at wavelengths between 800 and 925 nm, whereas chlorophyll a absorbs light at a maximum wavelength of 660 to 680 nm. Additionally, R. rubrum is capable of nitrogen fixation, meaning it can produce and regulate nitrogenase, an enzyme complex responsible for converting atmospheric nitrogen (dinitrogen) into ammonia. However, exposure to ammonia, darkness, or phenazine methosulfate inhibits nitrogen fixation.
Though there are various beneficial applications of R.rubrum, various challenges are faced in the mass production due to its diversified metabolic activities and environmental sensitivities. The key sensitivities in cultivating the R.rubrum include the requirement of oxygen, pH, and intensity of the light and complex nutritional requirements. Though the traditional approaches of mass production of R.rubrum include the usage of photo bioreactors with computational fluid dynamics, the cost is too high.
One of the prior art means discloses Model-based high cell density cultivation of Rhodospirillum rubrum under respiratory dark conditions disclosed in Biotechnol Bioeng, 2010 Mar 1;105(4):729-39. The potential of facultative photosynthetic bacteria as producers of photosynthetic pigments, vitamins, coenzymes and other valuable products has been recognized for decades. However, mass cultivation under photosynthetic conditions is generally inefficient due to the inevitable limitation of light supply when cell densities become very high. The previous development of a new cultivation process for maximal expression of photosynthetic genes under semi-aerobic dark conditions in common bioreactors offers a new perspective for utilizing the facultative photosynthetic bacterium Rhodospirillum rubrum for large-scale applications. Based on this cultivation system, the present study aimed in determining the maximal achievable cell density of R. rubrum in a bioreactor, thereby providing a major milestone on the way to industrial bioprocesses. As a starting point, we focus on aerobic growth due to higher growth rates and more facile process control under this condition, with the option to extend the process by an anaerobic production phase. Process design and optimization were supported by an unstructured computational process model, based on mixed-substrate kinetics. Key parameters for growth and process control were determined in shake-flask experiments or estimated by simulation studies. For fed-batch cultivation, a computer-controlled exponential feed algorithm in combination with a pH-stat element was implemented. As a result, a maximal cell density of 59 g cell dry weight (CDW) L(-1) was obtained, representing so far not attainable cell densities for photosynthetic bacteria. The applied exponential fed-batch methodology therefore enters a range which is commonly employed for industrial applications with microbial cells. The biochemical analysis of high cell density cultures revealed metabolic imbalances, such as the accumulation and excretion of tetrapyrrole intermediates of the bacteriochlorophyll biosynthetic pathway.
Another prior art means is disclosed in Dynamic modeling of Rhodospirillum rubrum PHA production triggered by redox stress during VFA photoheterotrophic assimilations is disclosed in Journal of Biotechnology, Volume 360, 10 December 2022, Pages 45-54.
Polyhydroxyalkanoates (PHA) represent an environmentally friendly alternative to petroleum based plastics for a broad range of applications from packaging to biomedical devices. In the prospect of an industrial PHA production, it is highly valuable to accurately control the incorporation of different repeating units into the polymer, to produce a polyester with specific material characteristics. In this study, we develop macroscopic dynamic models predicting the polymer production and composition when mixtures containing up to four volatile fatty acids (VFA) are used as substrates. These models successfully reproduce the sequential (and preferential) substrate consumption and polymer production/reconsumption patterns, experimentally observed during biomass growth, thanks to simple kinetic structures based on Monod and inhibition factors. These models can serve as a basis for numerical simulation and process analysis, as well as process intensification through model-based optimization and control.
Drawbacks of the prior art:
Rhodospirillum rubrum (R. rubrum), being a photosynthetic, nitrogen-fixing bacterium, has valuable applications (e.g. bioplastics production, hydrogen production, bioremediation). However, traditional cultivation methods (like batch photo bioreactors or fermenters) are costly due to factors like: Expensive media (carbon sources, vitamins, trace elements);Energy-intensive lighting for photosynthesis; Complex harvesting and downstream processing (especially if aiming for high-density cultures).
Conventional photobioreactor-based cultivation of Rhodospirillum rubrum is hindered by high capital and operational costs, as well as the necessity for sophisticated control systems to maintain optimal growth conditions. These factors limit the scalability and economic feasibility of mass production.
The mass production of Rhodospirillum rubrum is currently limited by the high costs and technical complexities associated with conventional cultivation methods. Traditional systems typically utilize photo bioreactors, which are expensive to build, operate, and maintain, particularly at commercial scales. Furthermore, these methods require sophisticated control systems to precisely regulate key environmental factors such as light intensity, temperature, aeration, and nutrient levels. This relies on complex infrastructure and tight operational controls which significantly increases production costs, making large-scale cultivation economically unfeasible for many applications.
1)High Operational Costs:
Conventional methods for the cultivation of Rhodospirillum rubrum often rely on phototrophic growth under controlled lighting conditions, which significantly increases operational costs due to the high energy demands of continuous illumination. Additionally, the requirement for specific anaerobic and nutrient-rich media further adds to the cost burden, making large-scale production economically challenging.
2)Requirement for Sophisticated Control Systems:
Traditional cultivation processes necessitate sophisticated control systems to regulate environmental parameters such as light intensity, temperature, aeration, and nutrient supply. This increases both the technical complexity and operational costs, making the process less accessible for large-scale or cost-sensitive applications.

Current methods for cultivating Rhodospirillum rubrum are costly and complex, relying on expensive photo bioreactors and sophisticated control systems to maintain optimal growth conditions. These challenges make large-scale, cost-effective production difficult, highlighting the need for simpler, lower-cost methods that still achieve high cell densities.

Therefore, there is a pressing need for a cost-effective, scalable cultivation method that achieves high cell density while minimizing reliance on expensive infrastructure that reduces production costs and complex operational controls. The present invention is a cost effective medium and process for mass production of Rhodospirillum rubrum.
Therefore, there is a need for a cost-effective system and method for the mass production of R. rubrum that reduces production costs while maintaining high cell density.
Objects of the invention:
The principle object of the present invention is to provide a cost-effective system and method for the mass production of R. rubrum that reduces production costs while maintaining high cell density.
Another object of the present invention is to increase microbial nutrition through the development of a low-cost, nutrient-rich medium suitable for large-scale cultivation of Rhodospirillum rubrum.
Another object of the present invention is to develop an efficient, scalable cultivation process using HDPE water bubble cans as a low-cost alternative to conventional and expensive photobioreactors.
Another object of the present invention is to optimize key growth parameters, including carbon source, nitrogen source, and inoculum percentage, to maximize bacterial yield and culture productivity.
Summary of the invention:
Accordingly, a cost-effective system and method for the mass production of R. rubrum that reduces production costs while maintaining high cell density. In present invention, instead of photobioreactors, Polypropylene clear bubble cans are used. The present invention provides a cost-effective method for the mass production of Rhodospirillum rubrum by:
1. Developing an optimized nutrient medium containing malic acid and yeast extract.
2. Utilizing HDPE water bubble cans (20 L capacity) as a low-cost alternative to photobioreactors.
3. Establishing an efficient cultivation process under direct sunlight for enhanced bacterial growth.
The invention enables large-scale R. rubrum production without requiring expensive computational fluid dynamics-based photobioreactors, making the process commercially viable for industrial applications.
Description of the drawings:
Fig 1 shows process flow of the large-scale R. rubrum production without requiring expensive computational fluid dynamics-based photobioreactors.
Fig 2 shows Photograph of the grown Rhodospirillum rubrum culture.
Detailed description of the invention with respect to drawings:
The present invention provides a cost-effective, scalable, and efficient method for the mass production of Rhodospirillum rubrum. By replacing expensive photobioreactors with HDPE water bubble cans and optimizing growth parameters, this invention makes large-scale bacterial cultivation economically viable for various industrial applications.
In another embodiment, a cost-effective method for the mass production of R. rubrum that reduces production costs while maintaining high cell density. In present invention, instead of photobioreactors, Polypropylene clear bubble cans are taken and cleaned with 70% isopropyl alcohol and weighed 20 L equivalent nutrients (Malic acid 10 g/L and Yeast extract 5 g/L) in 18 L of water is optimized. Thus the nutrient solution is sterilized at 121oC for 15 min at 15 psi. 10% of the Rhodospirillum rubrum inoculum is taken in each can and the cans are filled till the brim with sterilized nutrient solution (optimized solution). The cap is put for the cans and sealed with the plastic stretch films. Finally the cans are kept in the direct sunlight for 15 days.
The present invention includes the following steps:
Methodology followed:
Sourcing of Rhodospirillum rubrum strain:
The Rhodospirillum rubrum DSM 107 microbial strain was sourced from DSMZ, Germany. The Rhodospirillum rubrum was revived and subcultured on the selective medium with the composition:
Rhodospirillum rubrum DSM 27 medium:
Component Quantity
Yeast extract 0.3 g
Na₂-succinate 1 g
(NH₄)-acetate 0.5 g
Fe(III) citrate solution (0.1% in H₂O) 5 ml
KH₂PO₄ 0.5 g
MgSO₄ x 7 H₂O 0.4 g
NaCl 0.4 g
NH₄Cl 0.4 g
CaCl₂ x 2 H₂O 0.05 g
Vitamin B12 solution (10 mg in 100 ml H₂O) 0.4 ml
Trace element solution SL-6 (see below) 1 ml
L-Cysteinium chloride 0.3 g
Resazurin (0.1%) 0.5 ml
Distilled water 1000 ml
pH Adjust to 6.8
Boil, bubble with nitrogen, dispense 10 ml into 15 ml tubes, autoclave at 121°C for 15 min, inoculate using sterile syringes, and incubate under tungsten lamp light.
Rhodospirillum rubrum DSM 27 Medium Composition (per liter) is comprising of; 0.3 g of Yeast extract; 1 g of Na₂-succinate, 0.5 g of (NH₄)-acetate; 5 ml of Fe(III) citrate solution (0.1% in H₂O); 0.5 g of KH₂PO₄; 0.4 g of MgSO₄ x 7 H₂O, 0.4 g of NaCl; 0.4 g of NH₄Cl: 0.05 g of CaCl₂ x 2 H₂O; 0.4 ml of Vitamin B12 solution (10 mg in 100 ml H₂O); 1 ml of Trace element solution SL-6; 0.3 g of L-Cysteinium chloride; 0.5 ml of Resazurin (0.1%); 1000 ml of Distilled water and pH adjusted to 6.8. The medium was prepared by boiling, bubbling with nitrogen, dispensing into tubes, autoclaving at 121°C for 15 min, and incubating under tungsten light.
Trace Element Solution:
Component Quantity
ZnSO₄ 0.1 g
MnCl₂ 0.03 g
H₃BO₃ 0.3 g
CoCl₂ 0.2 g
CuCl₂ 0.01 g
NiCl₂ 0.02 g
Na₂MoO₄ 0.03 g
Distilled water 1000 ml

Trace Element Solution SL-6 Composition (per liter)
• ZnSO₄: 0.1 g
• MnCl₂: 0.03 g
• H₃BO₃: 0.3 g
• CoCl₂: 0.2 g
• CuCl₂: 0.01 g
• NiCl₂: 0.02 g
• Na₂MoO₄: 0.03 g.
2. Optimization of Growth Parameters
• Experiments were conducted to determine optimal carbon, nitrogen, and inoculum concentration levels for maximizing bacterial growth.
Optimization of medium:
Selection of carbon source: Preliminary selection of the carbon sources was carried out based on the research literature.
Table: 1 Selection of carbon sources

Carbon Source Replicate 1 (CFU/ml) Replicate 2 (CFU/ml) Replicate 3 (CFU/ml) Average SD
Sodium pyruvate @ 3 g/L 1.62E+08 1.39E+08 1.49E+08 1.50E+08 1E+07
Sodium succinate @ 1 g/L 9.00E+07 1.31E+08 1.09E+08 1.10E+08 2E+07
Ammonium acetate @ 0.5 g/L 2.00E+08 2.54E+08 2.06E+08 2.20E+08 3E+07
Malic acid @ 4 g/L 3.78E+08 4.05E+08 3.87E+08 3.90E+08 1E+07

This table summarizes the results of a comparative study assessing the growth of Rhodospirillum rubrum using different carbon sources in the growth medium. The growth was measured in terms of colony-forming units per milliliter (CFU/ml) across three independent replicates for each carbon source. The average CFU/ml and the standard deviation (SD) were calculated to assess the consistency of the results.
• Sodium Pyruvate (3 g/L): The average bacterial count was 1.50 × 10⁸ CFU/ml, with a standard deviation of 1 × 10⁷ CFU/ml, indicating moderate growth consistency.
• Sodium Succinate (1 g/L): The bacterial growth averaged 1.10 × 10⁸ CFU/ml, with a standard deviation of 2 × 10⁷ CFU/ml, showing relatively more variation among replicates.
• Ammonium Acetate (0.5 g/L): This carbon source supported higher bacterial growth, with an average of 2.20 × 10⁸ CFU/ml and a standard deviation of 3 × 10⁷ CFU/ml.
• Malic Acid (4 g/L): Malic acid showed the highest bacterial growth, with an average of 3.90 × 10⁸ CFU/ml and a low standard deviation of 1 × 10⁷ CFU/ml, indicating both high efficiency and consistency as a carbon source for Rhodospirillum rubrum.
These results suggest that malic acid is the most effective carbon source among those tested for cultivating Rhodospirillum rubrum, followed by ammonium acetate, sodium pyruvate, and sodium succinate.

Figure 1. Graph showing the total microbial count in various carbon sources
The graph shows the effect of different carbon sources on the biomass production of Rhodospirillum rubrum, measured in colony-forming units (CFU/ml). The carbon sources tested include:
• Sodium pyruvate @ 3 g/L
• Sodium succinate @ 1 g/L
• Ammonium acetate @ 0.5 g/L
• Malic acid @ 2.5 g/L
The y-axis represents the CFU/ml, ranging from 0 to 45,000,000 (4.5 × 10⁷). The x-axis displays the carbon sources and their respective concentrations.
Observations:
1. Malic Acid @ 2.5 g/L resulted in the highest biomass yield, reaching approximately 4 × 10⁷ CFU/ml. This indicates malic acid as the most favorable carbon source under the tested conditions.
2. Ammonium Acetate @ 0.5 g/L showed moderate growth, with CFU/ml values around 2 × 10⁷.
3. Sodium Pyruvate @ 3 g/L also supported moderate growth, with CFU/ml around 1.5 × 10⁷.
4. Sodium Succinate @ 1 g/L resulted in the lowest biomass yield, approximately 1 × 10⁷ CFU/ml.
Conclusion:
The graph highlights that malic acid is the most effective carbon source for biomass production in R. rubrum among those tested. This is likely due to its role as an intermediate in the TCA cycle, supporting efficient energy metabolism and cell growth. The other carbon sources—ammonium acetate, sodium pyruvate, and sodium succinate—show progressively lower biomass yields, indicating comparatively less efficient utilization by R. rubrum.

Optimization of Malic acid (Carbon source):
Table 2. Optimization of malic acid concentrations
Optimization of malic acid concentrations
Malic acid (g/L) Replicate 1 (CFU/ml) Replicate 2 (CFU/ml) Replicate 3 (CFU/ml) Average SD
2 2.25E+08 2.60E+08 2.35E+08 2.40E+08 2E+07
4 3.46E+08 3.13E+08 3.31E+08 3.30E+08 2E+07
6 4.53E+08 4.16E+08 3.91E+08 4.20E+08 3E+07
8 5.78E+08 5.39E+08 5.63E+08 5.60E+08 2E+07
10 2.91E+09 2.66E+09 2.83E+09 2.80E+09 1E+08
12 2.23E+09 1.94E+09 2.13E+09 2.10E+09 1E+08
14 9.83E+08 8.42E+08 9.06E+08 9.10E+08 7E+07

The table presents the growth performance of Rhodospirillum rubrum in terms of colony-forming units per milliliter (CFU/ml) at varying malic acid concentrations ranging from 2 g/L to 14 g/L. The data are recorded across three replicates for each concentration, with calculated averages and standard deviations (SD) reflecting consistency and reliability of the results.

Observations:
Malic Acid Concentration (g/L) Average CFU/ml (in scientific notation) Growth Trend
2 g/L 2.40 × 10⁸ Moderate growth observed.
4 g/L 3.30 × 10⁸ Slight increase in growth.
6 g/L 4.20 × 10⁸ Gradual increase in CFU/ml.
8 g/L 5.60 × 10⁸ Significant increase in growth.
10 g/L 2.80 × 10⁹ Peak growth observed; optimal concentration for maximum biomass.
12 g/L 2.10 × 10⁹ Slight decline in CFU/ml, indicating inhibitory effects at higher concentrations.
14 g/L 9.10 × 10⁸ Further decrease in growth, confirming inhibitory effects at higher malic acid levels.

Trend Analysis:
• Growth increased progressively as the concentration of malic acid increased from 2 g/L to 10 g/L.
• Maximum biomass yield was observed at 10 g/L malic acid concentration, with an average CFU/ml of approximately 2.80 × 10⁹.
• Beyond 10 g/L, there was a decline in CFU/ml, indicating possible substrate inhibition or osmotic stress at higher concentrations (12 g/L and 14 g/L).
• The standard deviation remained relatively low across replicates, indicating reproducibility of the experimental results.
Conclusion:
The data indicates that 10 g/L malic acid is the optimal concentration for achieving maximum biomass production of Rhodospirillum rubrum in the tested conditions. Concentrations above 10 g/L show inhibitory effects on bacterial growth, possibly due to metabolic burden or osmotic imbalance.

Figure 2. Optimization of malic acid concentration for cost effective mass production of R.rubrum.
The graph illustrates the effect of different concentrations of a carbon source (likely malic acid) on the biomass yield of Rhodospirillum rubrum, measured as CFU/ml.
• The x-axis represents the concentration of the carbon source in g/L, ranging from 2 to 14 g/L.
• The y-axis represents the biomass yield in CFU/ml, ranging from 0 to 3.5 × 10⁹ CFU/ml.
• The data points are connected by a green line with circular markers indicating each measurement.
Key Observations:
1. At lower concentrations (2–8 g/L), the biomass yield increases gradually, showing a slight upward trend.
2. A sharp increase in CFU/ml is observed at 10 g/L, reaching a peak of approximately 2.8 × 10⁹ CFU/ml. This indicates the optimal concentration for maximum biomass production.
3. At 12 g/L, the CFU/ml decreases to around 2.1 × 10⁹, suggesting that higher concentrations reduce the biomass yield.
4. At 14 g/L, the CFU/ml further decreases to approximately 9 × 10⁸, showing a significant decline in biomass yield at very high concentrations.
Conclusion:
The graph demonstrates that biomass yield is concentration-dependent, with an optimal concentration at 10 g/L. Increasing concentrations beyond this point leads to a decline in CFU/ml, indicating potential inhibitory effects or substrate inhibition at higher concentrations.
Selection of nitrogen sources: Selection of nitrogen sources were done based on the research literature.
Table 3: Selection of nitrogen sources
Selection of nitrogen sources
Nitrogen Source Replicate 1 (CFU/ml) Replicate 2 (CFU/ml) Replicate 3 (CFU/ml) Average SD
Ammonium sulphate @ 1.3 g/L 1.67E+08 1.96E+08 1.77E+08 1.80E+08 1E+07
Yeast extract @ 1.29 g/L 5.14E+08 4.72E+08 4.84E+08 4.90E+08 2E+07
Glutamic acid @ 0.5 g/L 2.38E+08 2.11E+08 2.11E+08 2.20E+08 2E+07
Ammonium chloride @ 0.4 g/L 1.52E+08 1.71E+08 1.57E+08 1.60E+08 1E+07

This table presents the growth performance of Rhodospirillum rubrum using different nitrogen sources, measured in colony-forming units per milliliter (CFU/ml). The data includes three biological replicates for each condition, the calculated average CFU/ml, and the standard deviation (SD) as a measure of variability.
Nitrogen Source Replicate 1 (CFU/ml) Replicate 2 (CFU/ml) Replicate 3 (CFU/ml) Average (CFU/ml) SD
Ammonium sulphate @ 1.3 g/L 1.67 × 10⁸ 1.96 × 10⁸ 1.77 × 10⁸ 1.80 × 10⁸ 1 × 10⁷
Yeast extract @ 1.29 g/L 5.14 × 10⁸ 4.72 × 10⁸ 4.84 × 10⁸ 4.90 × 10⁸ 2 × 10⁷
Glutamic acid @ 0.5 g/L 2.38 × 10⁸ 2.11 × 10⁸ 2.11 × 10⁸ 2.20 × 10⁸ 2 × 10⁷
Ammonium chloride @ 0.4 g/L 1.52 × 10⁸ 1.71 × 10⁸ 1.57 × 10⁸ 1.60 × 10⁸ 1 × 10⁷

Key Insights:
• Yeast extract @ 1.29 g/L demonstrated the highest average CFU/ml at approximately 4.90 × 10⁸, highlighting its superior potential as a nitrogen source for R. rubrum cultivation.
• Glutamic acid @ 0.5 g/L resulted in moderate growth (2.20 × 10⁸ CFU/ml), followed by Ammonium sulphate @ 1.3 g/L (1.80 × 10⁸ CFU/ml).
• Ammonium chloride @ 0.4 g/L showed the lowest bacterial growth (1.60 × 10⁸ CFU/ml).
• The standard deviations indicate consistent replicates with minimal variability across the experiments.
Conclusion:
The data suggests that organic nitrogen sources, especially yeast extract, significantly enhance the growth of R. rubrum compared to inorganic sources. This finding supports the use of yeast extract in optimizing media formulations for cost-effective cultivation of R. rubrum at a larger scale.

Figure 3. Graph showing the total microbial count in various nitrogen sources
The bar graph illustrates the comparative growth performance of Rhodospirillum rubrum using four different nitrogen sources, based on average colony-forming units per milliliter (CFU/ml). The error bars represent the standard deviation across triplicate experiments, indicating the consistency and reproducibility of the results.
Observations:
• Yeast extract @ 1.29 g/L exhibited the highest growth among the tested nitrogen sources, with an average CFU/ml of approximately 4.90 × 10⁸. This indicates the superior nutritional value of yeast extract in supporting the growth of R. rubrum.
• Glutamic acid @ 0.5 g/L resulted in moderate growth levels, with an average CFU/ml of around 2.20 × 10⁸.
• Ammonium sulphate @ 1.3 g/L showed lower growth potential compared to organic nitrogen sources, with an average CFU/ml of approximately 1.80 × 10⁸.
• Ammonium chloride @ 0.4 g/L supported the least growth, with an average CFU/ml of around 1.60 × 10⁸.
Trend Analysis:
• The trend indicates that organic nitrogen sources (yeast extract, glutamic acid) significantly outperform inorganic nitrogen sources (ammonium sulphate, ammonium chloride) in supporting R. rubrum growth.
• The data also reveals that yeast extract is the most effective nitrogen source, suggesting its inclusion in cost-effective, high-yield media formulations for R. rubrum cultivation.
Conclusion:
The graph highlights the importance of selecting an appropriate nitrogen source for optimizing the cultivation of R. rubrum. Specifically, yeast extract emerged as the most effective nitrogen source for achieving maximum cell density, whereas inorganic sources like ammonium sulphate and ammonium chloride were less effective.
Optimization of yeast extract (nitrogen source):
Optimization of yeast extract (nitrogen source)
Yeast Extract (g/L) Replicate 1 (CFU/ml) Replicate 2 (CFU/ml) Replicate 3 (CFU/ml) Average SD
1 1.47E+08 1.22E+08 1.21E+08 1.30E+08 1E+07
2 1.57E+08 2.01E+08 1.82E+08 1.80E+08 2E+07
3 2.63E+08 2.29E+08 2.28E+08 2.40E+08 2E+07
4 3.78E+08 3.53E+08 3.49E+08 3.60E+08 2E+07
5 5.87E+08 5.54E+08 5.97E+08 5.79E+08 2E+07
6 5.07E+08 4.71E+08 4.92E+08 4.90E+08 2E+07
7 3.70E+08 3.49E+08 3.61E+08 3.60E+08 1E+07

This dataset presents the effect of Yeast Extract concentration (in g/L) on the biomass yield of a microorganism (measured in CFU/ml). The concentrations of Yeast Extract vary from 1 g/L to 7 g/L.

Key Observations:

Yeast Extract (g/L) Average CFU/ml Standard Deviation (SD) Trend
1 1.30 × 10⁸ 1 × 10⁷ Lowest biomass yield
2 1.80 × 10⁸ 2 × 10⁷ Biomass increases
3 2.40 × 10⁸ 2 × 10⁷ Biomass increases
4 3.60 × 10⁸ 2 × 10⁷ Biomass increases further
5 5.79 × 10⁸ 2 × 10⁷ Peak biomass yield
6 4.90 × 10⁸ 2 × 10⁷ Biomass decreases
7 3.60 × 10⁸ 1 × 10⁷ Biomass decreases significantly

Overall Trend:
• Biomass yield increases with increasing Yeast Extract concentration, peaking at 5 g/L (5.79 × 10⁸ CFU/ml).
• Beyond 5 g/L, the biomass yield decreases, suggesting inhibitory effects or substrate inhibition at higher concentrations.
Conclusion:
• Optimal Yeast Extract concentration for maximum biomass yield is 5 g/L.
• Concentrations above 5 g/L lead to a decrease in CFU/ml, indicating possible nutrient excess stress or osmotic effects affecting microbial growth.

Description of the Graph (Effect of Yeast Extract on Biomass Yield):
Graph Overview:
• The graph shows the relationship between yeast extract concentration (g/L) on the X-axis and biomass yield (CFU/ml) on the Y-axis.
• The data points represent average CFU/ml values from multiple replicates, with error bars indicating standard deviation (SD).
Trend and Key Observations:
Yeast Extract (g/L) Average CFU/ml (approximate) Trend
1 ~1.3 × 10⁸ Low biomass
2 ~1.8 × 10⁸ Slight increase in biomass
3 ~2.4 × 10⁸ Continued growth
4 ~3.6 × 10⁸ Noticeable increase
5 ~5.8 × 10⁸ Peak biomass yield
6 ~4.9 × 10⁸ Decline in biomass after peak
7 ~3.6 × 10⁸ Further decline

Pattern Interpretation:
• Growth Phase: Biomass yield increases steadily from 1 g/L to 5 g/L, indicating that increasing yeast extract concentration up to 5 g/L supports higher biomass production.
• Optimal Concentration: Maximum biomass yield is achieved at 5 g/L yeast extract.
• Decline Phase: Beyond 5 g/L, biomass yield decreases, suggesting substrate inhibition or toxic effects at higher concentrations.
• Error Bars: The small error bars across concentrations indicate consistent replicate results and reliable data.
Conclusion:
• 5 g/L of yeast extract is the optimal concentration for maximum biomass production.
• Higher concentrations negatively affect growth, possibly due to osmotic stress, nutrient imbalance, or toxic accumulation.
Optimization of Inoculum Percentage:
Table 4. Optimization of inoculum percentage
Inoculum (%) Replicate 1 (CFU/ml) Replicate 2 (CFU/ml) Replicate 3 (CFU/ml) Average SD
5 (standard) 5.99E+08 6.05E+08 5.33E+08 5.79E+08 4E+07
10 3.48E+09 3.74E+09 3.59E+09 3.60E+09 1E+08
15 3.41E+09 3.13E+09 3.36E+09 3.30E+09 1E+08
20 3.50E+09 3.23E+09 3.46E+09 3.40E+09 1E+08

Based on the results, the optimal parameters were determined to be 10 g/L malic acid, 5 g/L yeast extract, and 10% inoculum.

Graph Overview:
• The graph depicts the effect of different percentage concentrations (%) on biomass yield (CFU/ml).
• The X-axis represents percentage concentration (%) (approx. 5%, 10%, 15%, and 20%).
• The Y-axis represents biomass yield (CFU/ml), measured in scientific notation (e.g., 5E+08 = 5 × 10⁸ CFU/ml).
• Error bars indicate standard deviation (SD), reflecting variability across replicates.
Key Observations:
Percentage (%) CFU/ml (approximate) Trend
5% ~5.0 × 10⁸ Low biomass yield
10% ~3.5 × 10⁹ Sharp increase, peak biomass yield
15% ~3.2 × 10⁹ Slight decline from peak
20% ~3.3 × 10⁹ Slight increase, stable compared to 15%

Trend Summary:
• Sharp Increase: Biomass yield increases dramatically from 5% to 10%.
• Peak Biomass: 10% concentration shows the highest biomass yield (~3.5 × 10⁹ CFU/ml).
• Slight Decrease and Stability: Biomass yield slightly decreases at 15% but stabilizes around 3.2–3.3 × 10⁹ CFU/ml up to 20%.
• Error Bars: Small error bars indicate low variability and consistent replicates.
Interpretation:
• Optimal Concentration: 10% is the optimal concentration for maximum biomass yield.
• Stabilization: Beyond 10%, the system reaches a plateau, suggesting saturation or limitation by other factors.
• The initial sharp increase suggests that below 10%, the concentration is limiting growth, while concentrations above 10% provide sufficient nutrients for maximum biomass.
3. Large-Scale Cultivation in HDPE Containers
A cost-effective cultivation process was developed using HDPE water bubble cans with a capacity of 20 L.
Process Steps:
1. HDPE water bubble cans were sterilized with 70% isopropyl alcohol.
2. The optimized nutrient medium (10 g/L malic acid, 5 g/L yeast extract) was prepared in 18 L of water.
3. The nutrient solution was sterilized at 121°C for 15 min at 15 psi.
4. 10% Rhodospirillum rubrum inoculum was added to each can.
5. The cans were filled to the brim with the sterilized nutrient solution.
6. The cans were sealed with plastic stretch film to maintain anaerobic conditions.
7. The cans were exposed to direct sunlight for 15 days to facilitate bacterial growth.
Batch Trial Results:
A batch trial using the optimized parameters resulted in a bacterial CFU count of 3.8 x 10⁹/ml, demonstrating the efficiency of the proposed method. Whereas the mass production of R.rubrum using photobioreactors achieved the CFU count of 1.1 x 10⁹/ml.
4. Results and Industrial Applications: The batch trial using the optimized parameters resulted in a final CFU of 3.8 × 10⁹/ml, demonstrating the effectiveness of the method. This cost-effective process significantly reduces production costs compared to conventional photobioreactors.
Applications of the produced R. rubrum include:
• Biohydrogen production
• Nitrogen fixation in agriculture
• Coenzyme Q10 biosynthesis
• Single-cell protein production.
The process for cultivating Rhodospirillum rubrum has been optimized to reduce costs and simplify large-scale production. The key steps are as follows:
1. Utilization of Polypropylene Clear Bubble Cans: Instead of expensive photobioreactors, clear polypropylene bubble cans are employed. These containers are first cleaned with 70% isopropyl alcohol to ensure sterility.
2. Preparation of Nutrient Solution: For each 20 L container, nutrients are measured to achieve concentrations of 10 g/L malic acid and 5 g/L yeast extract, dissolved in 18 L of water.
3. Sterilization: The nutrient solution is sterilized by autoclaving at 121°C for 15 minutes at 15 psi to eliminate any contaminants.
4. Inoculation: Each container receives an inoculum of R. rubrum amounting to 10% of the total volume.
5. Filling and Sealing: The containers are filled to the brim with the sterilized nutrient solution, capped, and sealed with plastic stretch film to maintain anaerobic conditions.
6. Incubation: The sealed containers are placed in direct sunlight for a period of 15 days to facilitate growth.
This method leverages cost-effective materials and natural sunlight, providing an efficient alternative to traditional photobioreactor systems for the mass cultivation of R. rubrum.
In another embodiment, A system for cultivating Rhodospirillum rubrum, comprising:
a) at least one transparent, sterilized polypropylene container;
b) a nutrient solution containing malic acid at a concentration of approximately 10 g/L and yeast extract at a concentration of approximately 5 g/L;
c) an inoculum of Rhodospirillum rubrum added to the nutrient solution in an amount of approximately 10% of the total volume;
d) a sealing mechanism configured to maintain anaerobic conditions, said mechanism comprising a cap and plastic stretch film; and
e) an incubation system wherein the sealed container is exposed to natural sunlight for a period of approximately 15 days under anaerobic and phototrophic conditions. The container has a volume capacity of 20 liters. The container is pre-cleaned using 70% isopropyl alcohol to ensure sterility before inoculation. The nutrient solution is sterilized by autoclaving at a temperature of 121°C and pressure of 15 psi for 15 minutes. The incubation system relies exclusively on ambient sunlight without artificial lighting. The polypropylene container is reusable after sterilization.
ADVANTAGES OF THE INVENTION
• Cost Reduction: The use of HDPE water bubble cans eliminates the need for expensive photobioreactors.
• Scalability: The process is easily scalable for industrial applications.
• High Yield: The optimized medium formulation and cultivation conditions maximize bacterial growth.
• Ease of Implementation: The method requires minimal technical expertise and infrastructure.

, Claims:CLAIMS:
We claim:
1. A cost-effective method for cultivating Rhodospirillum rubrum at scale comprising the steps of:
a. providing a transparent container made of clear polypropylene material;
b) sterilizing said container with a 70% isopropyl alcohol solution;
c) preparing a nutrient solution by dissolving malic acid at a concentration of 10 g/L and yeast extract at 5 g/L in water to a total volume of 18 liters per container;
d) sterilizing the nutrient solution by autoclaving at a temperature of 121°C and pressure of 15 psi for 15 minutes;
e) inoculating the sterilized nutrient solution with Rhodospirillum rubrum at a concentration equivalent to 10% of the final culture volume;
f) filling the container to the brim with the inoculated nutrient solution;
g) sealing the container with a cap and wrapping with plastic stretch film to establish anaerobic conditions; and
h) incubating the sealed container in direct sunlight for a period of approximately 15 days to allow microbial growth.
2. The method as claimed in claim 1, wherein the optimized medium enhances bacterial CFU count to 3.8 x 10⁹/ml.
3. The method as claimed in claim 1, wherein the process eliminates the need for traditional photobioreactors, reducing production costs.
4. The method as claimed in claim 1, wherein the container used is a clear polypropylene bubble can with a capacity of 20 liters.
5. The method as claimed in claim 1, wherein the step of sterilizing the nutrient solution is performed before inoculation to eliminate microbial contaminants.
6. The method as claimed in claim 1, wherein sealing the container with stretch film ensures the maintenance of an anaerobic environment throughout incubation.
7. The method as claimed in claim 1, wherein incubation in direct sunlight provides the phototrophic energy source required for Rhodospirillum rubrum proliferation, eliminating the need for artificial lighting.
8. The method as claimed in claim 1, wherein the entire cultivation system is adapted for batch processing to enable scalability and low-cost operation.
9. The method as claimed in claim 1, wherein no mechanical agitation or artificial temperature control is used during incubation, leveraging ambient conditions to further reduce energy costs.
10)A system for cultivating Rhodospirillum rubrum, comprising:
a) at least one transparent, sterilized polypropylene container;
b) a nutrient solution containing malic acid at a concentration of approximately 10 g/L and yeast extract at a concentration of approximately 5 g/L;
c) an inoculum of Rhodospirillum rubrum added to the nutrient solution in an amount of approximately 10% of the total volume;
d) a sealing mechanism configured to maintain anaerobic conditions, said mechanism comprising a cap and plastic stretch film; and
e) an incubation system wherein the sealed container is exposed to natural sunlight for a period of approximately 15 days under anaerobic and phototrophic conditions.
11.The system as claimed in claim 10, wherein the container has a volume capacity of 20 liters.
12.The system as claimed in claim 10, wherein the container is pre-cleaned using 70% isopropyl alcohol to ensure sterility before inoculation.
13.The system as claimed in claim 10, wherein the nutrient solution is sterilized by autoclaving at a temperature of 121°C and pressure of 15 psi for 15 minutes.
14.The system as claimed in claim 10, wherein the incubation system relies exclusively on ambient sunlight without artificial lighting.
15.The system as claimed in claim 10, wherein the polypropylene container is reusable after sterilization.
Date: 14/06/2025 For, Biofac Inputs pvt ltd,
Agent of the applicant
Pallavi unmesh Deshmukh