Indian Patents. 226490:COMPUTER-BASED METHOD FOR ASSESSING COMPETENCE OF AN ORGANIZATION

Title of Invention	COMPUTER-BASED METHOD FOR ASSESSING COMPETENCE OF AN ORGANIZATION
Abstract	N/A

Full Text	FORM 2 THE PATENTS ACT, 1970 (39 Of 1970) & THE PATENTS RULES, 2003 PROVISIONAL SPECIFICATION (See section 10, rule 13) 'PROCESS FOR OVER-PRODUCTION OF HYDROGEN' TAPAN CHAKRAVARTI, MANUKONDA SURESH KUMAR, ATUL NARAYANRAO VAIDYA, SANDEEP NARAYAN MUDLIAR AND SUKUMAR DEVOTTA of National Environment Engineering Research Institute, Nehru Marg, Nagpur-440 020, Maharashtra, India and BANIBRATA PANDEY AND PIDAPARTI SESHASADRI SASTRY of Nagarjuna Fertilizers and Chemicals Limited, 82, Nagarjuna Hills, Panjagutta, Hyderabad-500 082 The following specification describes the invention. Field of the present invention The present invention relates to a method for trapping excess charged particle from an anaerobic fermentor. More particularly, the method relates to capture protons generated in an anaerobic bio-chemical reaction thereby increase in the rate of fermentation and production. More particularly, the invention pertains a electro biochemical process for the production of hydrogen by trapping protons released during the acidogenic phase of fermentation/digestion of various substrates like hexose sugar, pentose sugar, polymer of carbohydrates, Sugar juices (like sugarcane juice, sorghum juice etc), any type of biomass, any type of acids (like acetic acid, butyric acid etc) as well as carbonaceous wastewaters from food and distillery industry. However this list of substrates is not restrictive. Background and Prior Art The excessive burning of fossil fuels which results in the generation of CO2, Sox, and Nox is one of the primary causes of global warming and acid rain, which have started to affect the earth's climate, weather, vegetation and aquatic ecosystems. Hydrogen is the cleanest energy source, producing water as its only combustion product. Hydrogen can be produced from renewable raw materials such as biomass and water. Therefore, hydrogen is a potential clean energy substitute for fossil fuels. Despite the "green" nature of hydrogen as a fuel, it is still primarily produced from nonrenewable sources such as natural gas and petroleum based hydrocarbons via steam reforming, and only 4% is generated from water using electrolysis. However these processes are highly energy-intensive and not always environmentally benign. Given these 2 perspectives, biological hydrogen production assumes paramount importance as an alternative energy source. Biological processes aided by enzymes are carried out largely at ambient temperatures and pressures, and hence are less energy intensive than chemical or electrochemical ones. A large number of microbial species, including significantly different taxonomic and physiological types, can produce hydrogen. Among the various processes for the biological production of hydrogen, direct and indirect biophotolysis, fermentation, photosynthetic production and also in vitro enzymatic conversion of biomass have assumed considerable importance (Woodward et al. 1996). Biophotolysis involves light-driven decomposition of water in the presence of micro-algae or cyanobacteria. Biophotolysis may go simultaneously with the direct splitting of water to generate hydrogen by solar radiation (Benemann 1996). This water splitting can be achieved either in photochemical cells where, for example, TiO2 is employed as the catalyst, or by applying photovoltaics, which indirectly utilize solar radiation for the electrolysis of water into hydrogen and oxygen (Rao and Hall 1996). Despite its relatively lower yields of hydrogen, the fermentative route is another promising method of bio-hydrogen production due to its higher rate of hydrogen evolution in the absence of any light source as well as its versatility of using a variety of the substrates. Moreover fermentative organisms have high growth rate and do not suffer much from inhibitory effects of oxygen in the system, as the process is anoxygenic. Fermentation of glucose by all known microbiological routes (primarily by Clostridia) can produce theoretically up to 4 mol of hydrogen per mol of glucose. Woodward et al. (2000) achieved 96.7% conversion efficiency 3 (based on 4 moles of H2/mol Glucose) only by using enzymes, not bacteria as auto inhibition of hydrogen producing bacteria by hydrogen does not arise in an enzyme bioreactor. The main challenge to fermentative production of hydrogen is that only 15% of the energy from the organic source can typically be obtained in the form of hydrogen. While a conversion efficiency of 33% is theoretically possible for hydrogen production from glucose (based on maximum four moles hydrogen per mole glucose), only half of this is usually obtained under batch and continuous fermentation conditions (Van Ginkel et al. 2002; Logan et al. 2002; Fang and Liu 2002). Four moles of hydrogen could only be obtained from glucose if two moles of acetate are produced, however only two moles of hydrogen are produced when butyrate is the main fermentation product. Typically, 60-70% of the aqueous product during sugar fermentation is butyrate (Liu and Fang 2002). This is because high H2 pressure inside the reactor results in the inhibition of pyruvate ferrodoxin oxidoreductase and pyruvate formate lyase, the two enzymes responsible for conversion of pyruvate to acetate (Thauer, Jungermann & Decker). Thus a low hydrogen pressure of around 10-3 atm is necessary for achieving high conversion efficiency. A thermophilic organism has recently been reported that may be able to achieve higher conversion efficiencies (US Department of Energy; Ootegehem, W. International Patent WO 02/06503 A2, 2002). However, its biochemical route of hydrogen production is unknown, and claims of high hydrogen production conversion have not been independently verified or shown to be economical. Genetic engineering of bacteria could increase hydrogen recovery. However, even if biochemical pathways that are used by bacteria such as 4 Clostridia are successfully modified to increase hydrogen production by optimizing the production of acetate, the maximum conversion efficiency will still remain below 33% (Logan 2004). As evident from the above-refereed literature on fermentative hydrogen production, it may be noted that the yield of hydrogen is low and higher yield requires maintaining of low partial pressure of hydrogen to make the reaction thermodynamically favourable towards conversion of pyruvate to acetate and not to other reduced end products such as butyrate. Also the protons formed during fermentation lower the pH of the fermentation broth, thereby reducing the rate of hydrogen production. Various strategies (e.g. nitrogen sparging) have been reported for hydrogen removal and the advantages & disadvantages of the same are summarized in Table 2. Most of these approaches further require separation of hydrogen from the stripping inert gas thereby increasing the hydrogen production cost. Therefore, fermentative hydrogen production appears to be the process of choice in an electro-biochemical reactor designed for proton capture. In the present invention, the above-referred drawbacks are overcome by capturing the protons formed during fermentation in an electro-biochemical reactor by applying electrical potential. The protons generated in the broth will be converted to hydrogen at the negatively charged electrode and, if simultaneously removed, will enable the system in maintaining low partial pressure of hydrogen and constant pH. This in turn enhances the rate of hydrogen production as a result of low hydrogen partial pressure by activating two hydrogen repressed enzymes - pyruvate-ferredoxin oxidoreductase and pyruvate-formate lyase which convert pyruvate to acetate, an essential pre-requisite for generating four moles of hydrogen per mole of glucose. 5 Electrically reduced neutral red (NR) served as the sole source of reducing power for growth and metabolism of pure and mixed cultures of H2 consuming bacteria in a novel electrochemical bioreactor system. NR was continuously reduced by the cathodic potential (-1.5 V) generated from an electric current (0.3 to 1.0 mA), and it was subsequently oxidized by Actinobacillus succinogenes or by mixed methanogenic cultures. The A.succinogenes mutant strain FZ-6 did not grow on fumarate alone unless electrically reduced NR or hydrogen was present as the electron donor for succinate production The mutant strain, unlike the wild type , lacked the formate dehydrogenase and formate lyase. Electrically reduced NR also replaced hydrogen as the sole electron donor source for growth and production of methane from CO2. These results show that pure and mixed cultures can function as electrochemical devices when electrically generated reduced power can be used to drive metabolism (Park et al 1999). The present invention suggests a system, whereby the proton generated during acidogenic phase in an anaerobic process can be converted to hydrogen and thereby increases the yield of hydrogen in heterotrophic fermentation. Therefore the yield of hydrogen will be higher than the stoihometically possible maximum yield. Heterotrophic fermentation (HF): C6H12O6+4H2O=2CH3COO" + +4 + 4HCO-3+4H2 The above reaction in an anaerobic fermentor clearly indicates that 4 moles of molecular hydrogen can be obtained from 1 moles of glucose. The method of the present invention traps the excess proton (4H+) and converts them into molecular hydrogen there by increasing the yield. 6 Object of Invention The main objective of the present invention is to provide a novel electro biochemical system for enhanced hydrogen production by capturing the protons released during anaerobic fermentation/ digestion and simultaneous removal of hydrogen from the system with a view to maintaining a low hydrogen pressure of around 10 -3 atm which obviates the drawbacks of the hitherto known prior art as detailed above. Another object of the present invention is to provide a methodology to capture protons generated in anaerobic fermentation of carbohydrates. Still in another object of the present invention is to inhibit the growth of methanogens and lithotrophic methanogenesis in the fermentor. Further, in another object of the present invention is to provide a methodology to ensure the fermentative production of acetic acid from glucose, by maintaining a low hydrogen pressure of hydrogen (around 10-3 atm). Still in another object of the present invention is to provide a methodology to ensure the Photoheterotropic fermentative production of hydrogen from glucose by anaerobic pure cultures or mixed culture of microorganisms using Photoheterotropic organism, which will result in enhanced hydrogen production. In one more object of the present invention is to provide a methodology to ensure the Photoheterotropic fermentative production of hydrogen from sugar juice like Cane sugar juice, Sorghum juice etc. by anaerobic pure cultures or mixed culture of microorganisms using Photoheterotropic organism, which will result in enhanced hydrogen production. 7 Yet another object of the present invention is to provide a methodology to ensure the Photoheterotropic fermentative production of hydrogen from acetic acid by anaerobic pure cultures or mixed culture of microorganisms. Summary of the Invention The present invention relates to develop an electro-biochemical system which enhances the hydrogen production and to maintain a low atmospheric hydrogen pressure in anaerobic fermentation. The process uses an electro-biochemical system where protons generated in anaerobic biochemical reaction are scavenged at cathode as hydrogen. Brief description of drawings Figure 1 is a schematic representation of the electro biochemical reactor which enhances the hydrogen production by capturing the protons released during anaerobic fermentation. Detailed description of the invention The present invention relates to a novel process of trapping the protons generated during acidogenic phase of anaerobic fermentation at cathode to produce hydrogen. Said process accumulates hydrogen as metal hydride and utilize and make the electrode rechargeable by means of gaseous hydrogen. The novel process has an advantage that it maintains the low atmospheric pressure of hydrogen during the anaerobic fermentation by simultaneous removal of hydrogen which in turn helps the microorganism to activate pyruvate ferrodoxin oxidoreductase and pyruvate formate lyase. Accordingly the present invention relates to a process for over-production of hydrogen in a heterotrophic fermentation process, said process comprising the steps: 8 a) culturing anaerobically fermenting microorganism in a nutrient medium such as herein described at a temperature in the range of 25 to 40°C for a period of 36 to 72 hours in a fermentor, the fermentor comprising a charged electrode, and b) capturing protons generated during acidogenic phase of fermentation by applying an electric charge to the electrode and selectively attracting the protons to the electrode to produce molecular hydrogen and collecting the same along with the hydrogen produced by the bacteria during fermentation. In another embodiment of the present invention, the temperature is 37°C. Still in another embodiment of the present invention, the medium is selected from a group comprising sugar and fermentable organic acids. Yet in another embodiment of the present invention the sugar is selected from a group comprising hexose, pentose. The invention further relates to a bio-reactor used for heterotrophic fermentation process, said bioreactor comprising: c) a vessel for fermentation, d) an electrode, the electrode adapted to selectively capture desired charged particle when potentialized, e) an outlet to collect the gas, and f) optionally comprising a means to store produced hydrogen. In one more embodiment of the present invention is related to a method of trapping excess charged particles from a fermentor produced during bio-chemical reaction in a fermentor, said method comprising introducing into the fermentor an electrode, capturing charged particle by applying an electric charge to the electrode and selectively attracting the desired 9 charged particles to the electrode and trapping the same from the encapsulated electrode. Further, in another embodiment of the present invention, the electrode can optionally be encapsulated by gas permeable membrane. Fig 1 shows a electro-biochemical reactor A for enhanced hydrogen production by capturing the protons released during anaerobic fermentation/ digestion and simultaneous removal of hydrogen from the system with a view to maintain low H2 pressure which comprises of a fermentor containing two electrodes El & E2 connected to electric potential B ( in DC) for proton capture at the negatively charged electrode or cathode, and a gas collector F for collection of hydrogen generated at negatively charged electrode. C represents the feed pump inlet, while D represents the outlet. Examples: The following examples are given by way of illustration of the working of the invention in actual practice and therefore should not be construed to limit the scope of the present invention. Example I: Hydrogen production from anaerobic fermentation of glucose by pure culture (Clostridium species) One liter of sterilized media containing 20 g/l glucose with necessary nutrients & inoculated with pure culture (Clostridium species) was subjected to anaerobic fermentation in a 2 liter fermentor at constant temperature of 30°C. The total fermentation time was 48 hrs and the total gas produced was collected in a conventional gas collection system based on liquid displacement technique. Gas was analyzed for hydrogen content using Gas chromatograph (electron capture detector) on parapak Q SS 10 column. The yield of hydrogen was 1.2 mole/ mole substrate. This shows that H2 can be produced by anaerobic fermentation of glucose by pure culture of Clostridium specie. Example 2: Hydrogen production from anaerobic fermentation of glucose by mixed culture One litre of unsterilized media containing 20 g/l glucose with necessary nutrients & seeded with mixed culture was subjected to anaerobic fermentation in a 2 litre fermentor at constant temperature of 30°C. The total fermentation time was 48 hrs and the total gas produced was collected in a conventional gas collection system based liquid displacement technique. Gas was analyzed for hydrogen content using Gas chromatograph (electron capture detector) on parapak Q SS column. The yield of hydrogen was 0.9 mole/ mole substrate. This shows that H2 can be produced by anaerobic fermentation of glucose by mixed anaerobic culture. Example 3: Hydrogen production from anaerobic fermentation of carbonaceous wastewater by mixed culture of microorganisms. One litre of unsterilized carbonaceous wastewater containing 1 % reducing sugars with necessary nutrients & seeded with mixed culture was subjected to anaerobic fermentation in a 2 litre fermentor at constant temperature of 30°C. The total fermentation time was 48 hrs and the total gas produced was collected in a conventional gas collection system based liquid displacement technique. Gas was analyzed for hydrogen content using Gas chromatograph (electron capture detector) on parapak Q SS column. The yield of hydrogen was 0.5 mole/ mole substrate. This 11 shows that H2 can be produced by anaerobic fermentation of carboneous wastewater by mixed culture. Example 4: Hydrogen production from anaerobic fermentation of glucose by pure culture (Clostridium species) in a electro biochemical reactor One litre of sterilized media containing 20 g/l glucose with necessary nutrients & inoculated with pure culture (Clostridium specie) was subjected to anaerobic fermentation in a 2 litre electro biochemical reactor at constant temperature of 30°C. The applied cathode potential was between 2.0 and 4 V, while the current density was 0.3 and 3.0 mA. The total fermentation time was 48 hrs and the total gas produced was collected in a conventional gas collection system based liquid displacement technique. Gas was analyzed for hydrogen content using Gas chromatograph (electron capture detector) on parapak Q SS column. The yield of hydrogen was 2.1 mole/ mole substrate. This shows that H2 production can be enhanced in a electro biochemical reactor by about 75%. Example 5: Hydrogen production from anaerobic fermentation of carbonaceous wastewater by mixed culture in electrobiochemical reactor. One litre of unsterilized carbonaceous wastewater containing 1 % reducible sugars with necessary nutrients, seeded with mixed culture was subjected to anaerobic fermentation in a 2 litre electro biochemical reactor at constant temperature of 30°C. The applied cathode potential was between 2.0 and 4 V, while the current density was 0.3 and 3.0 mA. The total fermentation time was 48 hrs and the total gas produced was collected in a conventional gas collection system based liquid displacement 12 technique. Gas was analyzed for hydrogen content using Gas chromatograph (electron capture detector) on parapak Q SS column. The yield of hydrogen was 2.3 mole/ mole substrate. This shows that H2 production can be enhanced in electro biochemical reactor by about 91 %. From the above examples it can be noted that the electrobiochemical system can be used for enhanced production of hydrogen by capturing proton released during anaerobic fermentation/digestion of various substrates under low hydrogen pressure of around 10'3 atm. Proton capture at cathode will play a duel role; the capture will enhance hydrogen production and maintain the pH at near neutral (around 7.0) condition. The novelty lies in the use of charged electrodes for the capture of protons generated during anaerobic fermentation/ digestion of various substrates for the enhanced production of hydrogen using mutated cultures where enzymes converting pyruvate to acetate are insensitive to hydrogen as compared to conventional fermentative hydrogen production, which is limited due to lowering of pH and accumulation of hydrogen. Also the purity of hydrogen gas obtained from electro biochemical reactor is high as compared to that produced from conventional anaerobic fermentation. To assess the efficacy of the system of the present invention hydrogen was produced by: 1. culturing the micro-organisms in the medium of the present invention, 2. using the potentialized electrodes in the medium of the present invention, and 3. using potentialized electrodes in fermentation process, wherein in the fermentation process same medium and micro-organisms are used. 13 The yields of the hydrogen have been tabulated in the following table. Only media with electrodes gave 0.040 mol hydrogen, this value is subtracted from the actual yield of hydrogen achieved with experimental set. The results clearly indicate towards the substantial increase in the yield of the hydrogen. Sugarconsumed(mol) Hydrogen produced Experiment Stoichiometric yield of H2 (mol) Actual yield ofH2 achieved(mol) (mol.H2/ mol. sugar) Media+Culture 0.019 0.08 0.0017 0.089 Media+Culture+EI ectrodes 0.027 0.11 0.0894 3.31 Media+Electrodes Nil - 0.04 - The voltage applied for the experimental system is 4.0 v Advantages: The main advantages of the present invention are: 1. Enhanced hydrogen production compared to conventional anaerobic fermentative processes due to capture the protons generated during anaerobic digestion of various substrates & maintenance of pH at around 7.0 that prevents excessive acidity in fermentation broth. 2. Capture of protons generated from the fermentation broth will thus help in maintaining the pH without addition of alkali and also results in increase in the rate of the reaction. 3. The electrobiochemical reactor maintained at a low hydrogen pressure of around 10"3 atm can be used for enhanced hydrogen production via proton capture during anaerobic fermentation as well as anaerobic digestion of various substrates. Use of mixed consortium of microorganisms makes the process easy to operate and there is no need of sterilization of the substrate as compared to pure fermentative microorganisms 4. High purity of hydrogen can be achieved, by use of electro biochemical reactor. 14 We claim: 1. A process for over-production of hydrogen in a heterotrophic fermentation process, said process comprising the steps: g) culturing anaerobically fermenting microorganism in a nutrient medium such as herein described at a temperature in the range of 25 to 40°C for a period of 36 to 72 hours in a fermentor, the fermentor comprising a charged electrode, and h) capturing protons generated during acidogenic phase of fermentation by applying an electric charge to the electrode and selectively attracting the protons to the electrode to produce molecular hydrogen and collecting the same along with the hydrogen produced by the bacteria during fermentation. 2. A process as claimed in claim 1, wherein in step (a) the temperature is 37°C. 3. A process as claimed in claim 1, wherein the medium is selected from a group comprising sugar and fermentable organic acids. 4. A process as claimed in claim 3, wherein the sugar is selected from a group comprising hexose, pentose. 5. A bio-reactor used for heterotrophic fermentation process, said bioreactor comprising: a) a vessel for fermentation, b) an electrode, the electrode adapted to selectively capture desired charged particle when potentialized, c) an outlet to collect the gas, and d) optionally comprising a means to store produced hydrogen. 6. A method of trapping excess charged particles from a fermentor produced during bio-chemical reaction in a fermentor, said method 15 comprising introducing into the fermentor an electrode, capturing charged particle by applying an electric charge to the electrode and selectively attracting the desired charged particles to the electrode and trapping the same from the encapsulated electrode. 16

Full Text

FORM 2
THE PATENTS ACT, 1970
(39 Of 1970)
&
THE PATENTS RULES, 2003
PROVISIONAL SPECIFICATION
(See section 10, rule 13)
'PROCESS FOR OVER-PRODUCTION OF HYDROGEN'
TAPAN CHAKRAVARTI, MANUKONDA SURESH KUMAR, ATUL NARAYANRAO VAIDYA, SANDEEP NARAYAN MUDLIAR AND SUKUMAR DEVOTTA of National Environment Engineering Research Institute, Nehru Marg, Nagpur-440 020, Maharashtra, India and
BANIBRATA PANDEY AND PIDAPARTI SESHASADRI SASTRY of
Nagarjuna Fertilizers and Chemicals Limited, 82, Nagarjuna Hills, Panjagutta, Hyderabad-500 082
The following specification describes the invention.

Field of the present invention
The present invention relates to a method for trapping excess charged particle from an anaerobic fermentor. More particularly, the method
relates to capture protons generated in an anaerobic bio-chemical reaction thereby increase in the rate of fermentation and production.
More particularly, the invention pertains a electro biochemical process for the production of hydrogen by trapping protons released during the acidogenic phase of fermentation/digestion of various substrates like hexose sugar, pentose sugar, polymer of carbohydrates, Sugar juices (like sugarcane juice, sorghum juice etc), any type of biomass, any type of acids (like acetic acid, butyric acid etc) as well as carbonaceous wastewaters from food and distillery industry. However this list of substrates is not restrictive.
Background and Prior Art
The excessive burning of fossil fuels which results in the generation of CO2, Sox, and Nox is one of the primary causes of global warming and acid rain, which have started to affect the earth's climate, weather, vegetation and aquatic ecosystems. Hydrogen is the cleanest energy source, producing water as its only combustion product. Hydrogen can be produced from renewable raw materials such as biomass and water. Therefore, hydrogen is a potential clean energy substitute for fossil fuels. Despite the "green" nature of hydrogen as a fuel, it is still primarily produced from nonrenewable sources such as natural gas and petroleum based hydrocarbons via steam reforming, and only 4% is generated from water using electrolysis. However these processes are highly energy-intensive and not always environmentally benign. Given these
2

perspectives, biological hydrogen production assumes paramount importance as an alternative energy source.
Biological processes aided by enzymes are carried out largely at ambient
temperatures and pressures, and hence are less energy intensive than chemical or electrochemical ones. A large number of microbial species, including significantly different taxonomic and physiological types, can produce hydrogen. Among the various processes for the biological production of hydrogen, direct and indirect biophotolysis, fermentation, photosynthetic production and also in vitro enzymatic conversion of biomass have assumed considerable importance (Woodward et al. 1996). Biophotolysis involves light-driven decomposition of water in the presence of micro-algae or cyanobacteria. Biophotolysis may go simultaneously with the direct splitting of water to generate hydrogen by solar radiation (Benemann 1996). This water splitting can be achieved either in photochemical cells where, for example, TiO2 is employed as the catalyst, or by applying photovoltaics, which indirectly utilize solar radiation for the electrolysis of water into hydrogen and oxygen (Rao and Hall 1996).
Despite its relatively lower yields of hydrogen, the fermentative route is another promising method of bio-hydrogen production due to its higher rate of hydrogen evolution in the absence of any light source as well as its versatility of using a variety of the substrates. Moreover fermentative organisms have high growth rate and do not suffer much from inhibitory effects of oxygen in the system, as the process is anoxygenic.
Fermentation of glucose by all known microbiological routes (primarily by Clostridia) can produce theoretically up to 4 mol of hydrogen per mol of
glucose. Woodward et al. (2000) achieved 96.7% conversion efficiency
3

(based on 4 moles of H2/mol Glucose) only by using enzymes, not bacteria as auto inhibition of hydrogen producing bacteria by hydrogen does not arise in an enzyme bioreactor.
The main challenge to fermentative production of hydrogen is that only 15% of the energy from the organic source can typically be obtained in the form of hydrogen. While a conversion efficiency of 33% is theoretically possible for hydrogen production from glucose (based on maximum four moles hydrogen per mole glucose), only half of this is usually obtained under batch and continuous fermentation conditions (Van Ginkel et al. 2002; Logan et al. 2002; Fang and Liu 2002). Four moles of hydrogen could only be obtained from glucose if two moles of acetate are produced, however only two moles of hydrogen are produced when butyrate is the main fermentation product. Typically, 60-70% of the aqueous product during sugar fermentation is butyrate (Liu and Fang 2002). This is because high H2 pressure inside the reactor results in the inhibition of pyruvate ferrodoxin oxidoreductase and pyruvate formate lyase, the two
enzymes responsible for conversion of pyruvate to acetate (Thauer, Jungermann & Decker). Thus a low hydrogen pressure of around 10-3 atm is necessary for achieving high conversion efficiency.
A thermophilic organism has recently been reported that may be able to achieve higher conversion efficiencies (US Department of Energy; Ootegehem, W. International Patent WO 02/06503 A2, 2002). However, its biochemical route of hydrogen production is unknown, and claims of high hydrogen production conversion have not been independently verified or shown to be economical.
Genetic engineering of bacteria could increase hydrogen recovery. However, even if biochemical pathways that are used by bacteria such as
4

Clostridia are successfully modified to increase hydrogen production by optimizing the production of acetate, the maximum conversion efficiency will still remain below 33% (Logan 2004).
As evident from the above-refereed literature on fermentative hydrogen production, it may be noted that the yield of hydrogen is low and higher yield requires maintaining of low partial pressure of hydrogen to make the
reaction thermodynamically favourable towards conversion of pyruvate to
acetate and not to other reduced end products such as butyrate. Also the protons formed during fermentation lower the pH of the fermentation broth, thereby reducing the rate of hydrogen production. Various strategies (e.g. nitrogen sparging) have been reported for hydrogen removal and the advantages & disadvantages of the same are summarized in Table 2. Most of these approaches further require separation of hydrogen from the stripping inert gas thereby increasing the hydrogen production cost. Therefore, fermentative hydrogen production appears to be the process of choice in an electro-biochemical reactor designed for proton capture. In the present invention, the above-referred drawbacks are overcome by capturing the protons formed during fermentation in an electro-biochemical reactor by applying electrical potential. The protons generated in the broth will be converted to hydrogen at the negatively charged electrode and, if simultaneously removed, will enable the system in maintaining low partial pressure of hydrogen and constant pH. This in turn enhances the rate of hydrogen production as a result of low hydrogen partial pressure by activating two hydrogen repressed enzymes - pyruvate-ferredoxin oxidoreductase and pyruvate-formate lyase which convert pyruvate to acetate, an essential pre-requisite for generating four moles of hydrogen per mole of glucose.
5

Electrically reduced neutral red (NR) served as the sole source of reducing power for growth and metabolism of pure and mixed cultures of H2 consuming bacteria in a novel electrochemical bioreactor system. NR was continuously reduced by the cathodic potential (-1.5 V) generated from an electric current (0.3 to 1.0 mA), and it was subsequently oxidized by Actinobacillus succinogenes or by mixed methanogenic cultures. The A.succinogenes mutant strain FZ-6 did not grow on fumarate alone unless electrically reduced NR or hydrogen was present as the electron donor for succinate production The mutant strain, unlike the wild type , lacked the formate dehydrogenase and formate lyase. Electrically reduced NR also replaced hydrogen as the sole electron donor source for growth and production of methane from CO2. These results show that pure and mixed cultures can function as electrochemical devices when electrically generated reduced power can be used to drive metabolism (Park et al 1999).
The present invention suggests a system, whereby the proton generated during acidogenic phase in an anaerobic process can be converted to hydrogen and thereby increases the yield of hydrogen in heterotrophic fermentation. Therefore the yield of hydrogen will be higher than the stoihometically possible maximum yield.
Heterotrophic fermentation (HF):

C6H12O6+4H2O=2CH3COO" + +4 + 4HCO-3+4H2
The above reaction in an anaerobic fermentor clearly indicates that 4 moles of molecular hydrogen can be obtained from 1 moles of glucose. The method of the present invention traps the excess proton (4H+) and converts them into molecular hydrogen there by increasing the yield.
6

Object of Invention
The main objective of the present invention is to provide a novel electro biochemical system for enhanced hydrogen production by capturing the
protons released during anaerobic fermentation/ digestion and simultaneous removal of hydrogen from the system with a view to maintaining a low hydrogen pressure of around 10 -3 atm which obviates the drawbacks of the hitherto known prior art as detailed above.
Another object of the present invention is to provide a methodology to capture protons generated in anaerobic fermentation of carbohydrates.
Still in another object of the present invention is to inhibit the growth of methanogens and lithotrophic methanogenesis in the fermentor.
Further, in another object of the present invention is to provide a methodology to ensure the fermentative production of acetic acid from glucose, by maintaining a low hydrogen pressure of hydrogen (around 10-3 atm).
Still in another object of the present invention is to provide a methodology to ensure the Photoheterotropic fermentative production of hydrogen from glucose by anaerobic pure cultures or mixed culture of microorganisms using Photoheterotropic organism, which will result in enhanced hydrogen production.
In one more object of the present invention is to provide a methodology to ensure the Photoheterotropic fermentative production of hydrogen from sugar juice like Cane sugar juice, Sorghum juice etc. by anaerobic pure cultures or mixed culture of microorganisms using Photoheterotropic organism, which will result in enhanced hydrogen production.
7

Yet another object of the present invention is to provide a methodology to ensure the Photoheterotropic fermentative production of hydrogen from acetic acid by anaerobic pure cultures or mixed culture of microorganisms.
Summary of the Invention
The present invention relates to develop an electro-biochemical system which enhances the hydrogen production and to maintain a low atmospheric hydrogen pressure in anaerobic fermentation. The process uses an electro-biochemical system where protons generated in anaerobic biochemical reaction are scavenged at cathode as hydrogen.
Brief description of drawings
Figure 1 is a schematic representation of the electro biochemical reactor which enhances the hydrogen production by capturing the protons released during anaerobic fermentation.
Detailed description of the invention
The present invention relates to a novel process of trapping the protons generated during acidogenic phase of anaerobic fermentation at cathode to produce hydrogen. Said process accumulates hydrogen as metal hydride and utilize and make the electrode rechargeable by means of gaseous hydrogen.
The novel process has an advantage that it maintains the low atmospheric pressure of hydrogen during the anaerobic fermentation by simultaneous removal of hydrogen which in turn helps the microorganism to activate pyruvate ferrodoxin oxidoreductase and pyruvate formate lyase. Accordingly the present invention relates to a process for over-production of hydrogen in a heterotrophic fermentation process, said process comprising the steps:
8

a) culturing anaerobically fermenting microorganism in a nutrient medium such as herein described at a temperature in the range of 25 to 40°C for a period of 36 to 72 hours in a fermentor, the fermentor comprising a charged electrode, and
b) capturing protons generated during acidogenic phase of fermentation by applying an electric charge to the electrode and selectively attracting the protons to the electrode to produce molecular hydrogen and collecting the same along with the hydrogen produced by the bacteria during fermentation.
In another embodiment of the present invention, the temperature is 37°C. Still in another embodiment of the present invention, the medium is selected from a group comprising sugar and fermentable organic acids. Yet in another embodiment of the present invention the sugar is selected from a group comprising hexose, pentose.
The invention further relates to a bio-reactor used for heterotrophic fermentation process, said bioreactor comprising:
c) a vessel for fermentation,
d) an electrode, the electrode adapted to selectively capture desired charged particle when potentialized,

e) an outlet to collect the gas, and
f) optionally comprising a means to store produced hydrogen.
In one more embodiment of the present invention is related to a method of trapping excess charged particles from a fermentor produced during bio-chemical reaction in a fermentor, said method comprising introducing into the fermentor an electrode, capturing charged particle by applying an electric charge to the electrode and selectively attracting the desired
9

charged particles to the electrode and trapping the same from the encapsulated electrode.
Further, in another embodiment of the present invention, the electrode
can optionally be encapsulated by gas permeable membrane. Fig 1 shows a electro-biochemical reactor A for enhanced hydrogen production by capturing the protons released during anaerobic fermentation/ digestion and simultaneous removal of hydrogen from the system with a view to maintain low H2 pressure which comprises of a fermentor containing two electrodes El & E2 connected to electric potential B ( in DC) for proton capture at the negatively charged electrode
or cathode, and a gas collector F for collection of hydrogen generated at
negatively charged electrode. C represents the feed pump inlet, while D
represents the outlet.
Examples:
The following examples are given by way of illustration of the working of
the invention in actual practice and therefore should not be construed to
limit the scope of the present invention.
Example I:
Hydrogen production from anaerobic fermentation of glucose by pure
culture (Clostridium species)
One liter of sterilized media containing 20 g/l glucose with necessary nutrients & inoculated with pure culture (Clostridium species) was subjected to anaerobic fermentation in a 2 liter fermentor at constant temperature of 30°C. The total fermentation time was 48 hrs and the total gas produced was collected in a conventional gas collection system based on liquid displacement technique. Gas was analyzed for hydrogen content using Gas chromatograph (electron capture detector) on parapak Q SS
10

column. The yield of hydrogen was 1.2 mole/ mole substrate. This shows that H2 can be produced by anaerobic fermentation of glucose by pure culture of Clostridium specie.
Example 2:
Hydrogen production from anaerobic fermentation of glucose by mixed culture
One litre of unsterilized media containing 20 g/l glucose with necessary nutrients & seeded with mixed culture was subjected to anaerobic fermentation in a 2 litre fermentor at constant temperature of 30°C. The total fermentation time was 48 hrs and the total gas produced was collected in a conventional gas collection system based liquid displacement technique. Gas was analyzed for hydrogen content using Gas chromatograph (electron capture detector) on parapak Q SS column. The yield of hydrogen was 0.9 mole/ mole substrate. This shows that H2 can be produced by anaerobic fermentation of glucose by mixed anaerobic culture.
Example 3:
Hydrogen production from anaerobic fermentation of carbonaceous wastewater by mixed culture of microorganisms.
One litre of unsterilized carbonaceous wastewater containing 1 % reducing sugars with necessary nutrients & seeded with mixed culture was subjected to anaerobic fermentation in a 2 litre fermentor at constant temperature of 30°C. The total fermentation time was 48 hrs and the total gas produced was collected in a conventional gas collection system based liquid displacement technique. Gas was analyzed for hydrogen content using Gas chromatograph (electron capture detector) on parapak Q SS column. The yield of hydrogen was 0.5 mole/ mole substrate. This
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shows that H2 can be produced by anaerobic fermentation of carboneous wastewater by mixed culture.
Example 4:
Hydrogen production from anaerobic fermentation of glucose by pure culture (Clostridium species) in a electro biochemical reactor
One litre of sterilized media containing 20 g/l glucose with necessary
nutrients & inoculated with pure culture (Clostridium specie) was subjected
to anaerobic fermentation in a 2 litre electro biochemical reactor at
constant temperature of 30°C. The applied cathode potential was between
2.0 and 4 V, while the current density was 0.3 and 3.0 mA. The total
fermentation time was 48 hrs and the total gas produced was collected in
a conventional gas collection system based liquid displacement
technique. Gas was analyzed for hydrogen content using Gas chromatograph (electron capture detector) on parapak Q SS column. The yield of hydrogen was 2.1 mole/ mole substrate. This shows that H2 production can be enhanced in a electro biochemical reactor by about 75%.
Example 5:
Hydrogen production from anaerobic fermentation of carbonaceous wastewater by mixed culture in electrobiochemical reactor.
One litre of unsterilized carbonaceous wastewater containing 1 % reducible sugars with necessary nutrients, seeded with mixed culture was subjected to anaerobic fermentation in a 2 litre electro biochemical reactor at constant temperature of 30°C. The applied cathode potential was between 2.0 and 4 V, while the current density was 0.3 and 3.0 mA. The total fermentation time was 48 hrs and the total gas produced was collected in a conventional gas collection system based liquid displacement
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technique. Gas was analyzed for hydrogen content using Gas chromatograph (electron capture detector) on parapak Q SS column. The yield of hydrogen was 2.3 mole/ mole substrate. This shows that H2 production can be enhanced in electro biochemical reactor by about 91 %.
From the above examples it can be noted that the electrobiochemical system can be used for enhanced production of hydrogen by capturing proton released during anaerobic fermentation/digestion of various substrates under low hydrogen pressure of around 10'3 atm. Proton capture at cathode will play a duel role; the capture will enhance hydrogen production and maintain the pH at near neutral (around 7.0) condition. The novelty lies in the use of charged electrodes for the capture of protons generated during anaerobic fermentation/ digestion of various substrates for the enhanced production of hydrogen using mutated cultures where enzymes converting pyruvate to acetate are insensitive to hydrogen as compared to conventional fermentative hydrogen production, which is limited due to lowering of pH and accumulation of hydrogen. Also the purity of hydrogen gas obtained from electro biochemical reactor is high as compared to that produced from conventional anaerobic fermentation.
To assess the efficacy of the system of the present invention hydrogen
was produced by:
1. culturing the micro-organisms in the medium of the present invention,
2. using the potentialized electrodes in the medium of the present invention, and
3. using potentialized electrodes in fermentation process, wherein in the fermentation process same medium and micro-organisms are used.
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The yields of the hydrogen have been tabulated in the following table. Only media with electrodes gave 0.040 mol hydrogen, this value is
subtracted from the actual yield of hydrogen achieved with experimental
set. The results clearly indicate towards the substantial increase in the yield of the hydrogen.

Sugarconsumed(mol) Hydrogen produced
Experiment Stoichiometric yield of H2 (mol) Actual yield ofH2 achieved(mol) (mol.H2/ mol. sugar)
Media+Culture 0.019 0.08 0.0017 0.089
Media+Culture+EI ectrodes 0.027 0.11 0.0894 3.31
Media+Electrodes Nil - 0.04 -
The voltage applied for the experimental system is 4.0 v Advantages:
The main advantages of the present invention are:
1. Enhanced hydrogen production compared to conventional anaerobic fermentative processes due to capture the protons generated during anaerobic digestion of various substrates & maintenance of pH at around 7.0 that prevents excessive acidity in fermentation broth.
2. Capture of protons generated from the fermentation broth will thus help in maintaining the pH without addition of alkali and also results in increase in the rate of the reaction.
3. The electrobiochemical reactor maintained at a low hydrogen pressure of around 10"3 atm can be used for enhanced hydrogen production via proton capture during anaerobic fermentation as well as anaerobic digestion of various substrates. Use of mixed consortium of microorganisms makes the process easy to operate and there is no need of sterilization of the substrate as compared to pure fermentative microorganisms
4. High purity of hydrogen can be achieved, by use of electro biochemical reactor.
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We claim:
1. A process for over-production of hydrogen in a heterotrophic
fermentation process, said process comprising the steps:
g) culturing anaerobically fermenting microorganism in a nutrient medium such as herein described at a temperature in the
range of 25 to 40°C for a period of 36 to 72 hours in a fermentor, the fermentor comprising a charged electrode, and
h) capturing protons generated during acidogenic phase of fermentation by applying an electric charge to the electrode and selectively attracting the protons to the electrode to produce molecular hydrogen and collecting the same along with the hydrogen produced by the bacteria during fermentation.
2. A process as claimed in claim 1, wherein in step (a) the temperature is 37°C.
3. A process as claimed in claim 1, wherein the medium is selected from a group comprising sugar and fermentable organic acids.
4. A process as claimed in claim 3, wherein the sugar is selected from a group comprising hexose, pentose.
5. A bio-reactor used for heterotrophic fermentation process, said bioreactor comprising:

a) a vessel for fermentation,
b) an electrode, the electrode adapted to selectively capture desired charged particle when potentialized,
c) an outlet to collect the gas, and
d) optionally comprising a means to store produced hydrogen.
6. A method of trapping excess charged particles from a fermentor
produced during bio-chemical reaction in a fermentor, said method
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comprising introducing into the fermentor an electrode, capturing charged particle by applying an electric charge to the electrode and
selectively attracting the desired charged particles to the electrode
and trapping the same from the encapsulated electrode.

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COMPUTER-BASED METHOD FOR ASSESSING COMPETENCE OF AN ORGANIZATION

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