Biomining hydrogen. Hydrogen-producing microbial technology.

Autor: Georgii Feodoridi

Place: HYDITEX CORPORATION, North Cyprus

Date: October 2024

Abstract: A promising and unique direction of hydrogen fuel production is the dark fermentation of residual petroleum hydrocarbons as organic matter by anaerobic bacteria. This method could be a promising alternative to other hydrogen production methods currently in use, because depleted, decommissioned oil wells have the organic matter and temperature needed for hydrogen production, and are in locations with existing infrastructure and require less cost.

In this article, we provide information on studies to date that demonstrate that bacteria are capable of biopurifying hydrocarbons to hydrogen. The rate and production of hydrogen from hydrocarbons depends on the presence of glucose and other surfactants in the growth media. This article also provides an overview of special technologies, such as genetic engineering and nanotechnology, aimed at changing the properties of microbial communities and growing media to reduce the sensitivity of bacteria to environmental conditions, increase the bioactivity of microorganisms to increase the rate of conversion of feedstock to hydrogen. 

Key Words: Hydrogen, Biohydrogen, Gold hydrogen, Dark fermentation, crude oil, hydrogen from hydrocarbons

DOI: 10.13140/RG.2.2.29715.49441

Biomining hydrogen. Hydrogen-producing microbial technology.

Introduction

In recent years, there has been a significant increase in interest in hydrogen energy, because hydrogen is an environmentally friendly fuel, virtually the only combustion product of which is water. An important advantage of its use as an energy carrier is the elimination of greenhouse gas emissions and other pollutants in the environment and reduction of carbon dioxide emissions into the atmosphere.

Realisation of hydrogen fuel production is primarily associated with the search and development of economical and environmentally safe technology for its production. One of the promising directions in this area, which is rapidly gaining momentum, is obtaining fuel from waste (organic and industrial waste, wastewater, etc.) by processing them through microbial conversion. So-called ‘bio-hydrogen’, i.e. hydrogen produced by biological (microbiological) means. 

It is currently known that molecular hydrogen can be produced by microorganisms of different groups: green microalgae, cyanobacteria, anaerobic photosynthetic bacteria and anaerobic fermentative bacteria. Based on this, the following methods of biohydrogen production can be distinguished:

Among the above-mentioned methods, the most promising is the production of biohydrogen by dark fermentation [1, 2, 3, 4, 5], it consists in the fact that bacteria release hydrogen as a result of fermentation during growth under anaerobic conditions on various carbon-containing substrates. The rate of hydrogen generation by bacteria carrying out fermentation can reach up to 400 ml/l/hour. 

Dark fermentation is possible in mesophilic (approximately 20-40 °C) [6], thermophilic (approximately 50-60 °C) [7] and hyperthermophilic (above 80 °C) [8, 9] temperature ranges (The division into these temperature ranges is rather conventional, as the bacterial growth temperatures overlap considerably). But hydrogen production at elevated temperatures is thermodynamically more favourable [10], and thermophilic production of biohydrogen benefits from the general features of high temperature processes sn  uch as lower viscosity, better mixing and higher reaction rates [11, 12]. 

Hydrogen from hydrocarbons

Biohydrogen production requires a carbon source and high temperatures. Therefore, a new and unique way to produce biohydrogen is to inject specially engineered microorganisms and nutrients into depleted oil wells, where they will break down the residual petroleum hydrocarbons there and convert them into hydrogen and carbon dioxide. Mature oil reservoirs have the high temperatures required for hydrogen production, and they contain significant amounts of organic matter in the form of residual hydrocarbons.

This method of hydrogen production is now being used by an American company. This company does not disclose what bacteria and nutrients it uses in its project, but states that the bacteria used are not classified as genetically modified. For the project, they only enhanced the natural abilities of microorganisms, which allowed them to increase the productivity of microbes by 6 times. The company estimates that the cost of hydrogen produced using such technology will be $1 per kilogram, which could compete with other methods of fuel production [13].

The company has named its product ‘golden hydrogen’. In some sources, the term ‘golden hydrogen’ may also refer to hydrogen that is produced by natural processes in the Earth's interior and seeps through various geological media to its surface [13].

In 2020, an article [14] was published on a study conducted by Danish and Norwegian scientists. This study evaluated the ability of microorganisms to biopurify hydrocarbons to hydrogen.

Bacteria isolated from hydrocarbon reservoirs, all of the Thermotogales suborder, which can produce biohydrogen from a variety of simple and complex sugars with yields close to Tower's theoretical limit of 4 mol hydrogen/mol of glucose consumed, were selected for the study [15]. Three bacteria, Thermotoga petrophila RKU-1 (strain DSM-13995) [16], Pseudothermotoga hypogea (strain DSM-11164) [17] and Pseudothermotoga elfii (strain DSM-9442) [14, 18].

Glucose, n-hexadecane and crude oil were used as carbon sources in this study in different experiments. Two growth media were used to evaluate the production of biohydrogen: the first medium, called ‘base medium’ did not contain glucose in its composition. The second medium, had glucose and surfactant (Tween 80) as n-hexadecane and crude oil are not miscible in the aqueous phase and the addition of surfactant can stimulate hydrogen production from hydrocarbons by reducing the interfacial tension between the two phases and hence increasing the availability of hydrocarbons to microorganisms [14].

The microorganism Thermotoga petrophila RKU-1 (strain DSM-13995), failed to provide detectable hydrogen concentrations in the absence of glucose and in the presence of hydrocarbons, further the study cites results for only two bacterial strains [14].

For the bacterium Pseudothermotoga elfii (strain DSM 9442), the maximum rate of hydrogen production in the base medium was 0.72 mmol l-1-h-1, the rate increased to 0.85 mmol l-1-h-1 (when n-hexadecane was added) and to 0.99 mmol l-1-h-1 (when crude oil was added). Thus, Pseudothermotoga elfii (strain DSM 9442) can produce more hydrogen in experiments where the base medium was supplemented with crude oil or n-hexadecane [14].

For the bacterium Pseudothermotoga hypogea (strain DSM-11164), the maximum hydrogen production rate was 0.94 mmol l-1-h-1 in the base medium, the rate increased to 1.28 mmol l-1-h-1 when n-hexadecane was added and decreased to 0.88 mmol l-1-h-1 when crude oil was added. Thus, the addition of n-hexadecane can also increase hydrogen production by the bacterium Pseudothermotoga elfii (strain DSM 9442). But addition of crude oil leads to higher hydrogen production in early growth stages but the final hydrogen production is lower compared to the other two cases [14].

By subtracting the rate of hydrogen production in hydrocarbon experiments from experiments without hydrocarbons (baseline), hydrogen production from hydrocarbons can be calculated: For Pseudothermotoga elfii (strain DSM 9442), the maximum rate of hydrogen production from hydrocarbons is 0.041 mmol l-1-h-1 (for n-hexadecane) and 0.033 mmol l-1-h-1 (for crude oil). For the bacterium Pseudothermotoga hypogea (strain DSM-11164), the maximum rate of hydrogen formation from n-hexadecane is 0.0027 mmol l-1-h-1. Thus, the rate of hydrogen formation from hydrocarbons is one to two orders of magnitude lower than the rate of hydrogen formation from glucose [14].

The study shows that the addition of glucose and surfactant increased hydrogen production from hydrocarbons about 12-fold (from 0.47 to 5.7 mmol/L) for n-hexadecane and 3-fold (from 1.019 to 3.16 mmol/L) for crude oil by the bacterium Pseudothermotoga elfii (strain DSM 9442). Similarly, for the bacterium Pseudothermotoga hypogea (strain DSM-11164), addition of glucose and surfactant increased hydrogen release from hydrocarbons about 4-fold (from 0.94 to 3.35 mmol/L for n-hexadecane and from 1.1 to 4.23 mmol/L for crude oil) [14].

Determining the rate of hydrogen production as stated above, it can be concluded that the addition of glucose and surfactant (Tween 80) increased the maximum rate of hydrogen production from hydrocarbons by the bacterium Pseudothermotoga elfii (strain DSM 9442) by about 6-fold (from 0.04 to 0.28 mmol l-1-h-1 for n-hexadecane and from 0.03 to 0.17 for crude oil). For the bacterium Pseudothermotoga hypogea (strain DSM-11164), the addition of glucose and surfactant (Tween 80) increased the maximum rate of hydrogen production from n-hexadecane about 400-fold (from 0.0027 to 0.14 mmol l-1-h-1) and produced hydrogen from crude oil at a maximum rate of 0.14 mmol l-1-h-1 [14].

Changing the properties of the growth medium to increase hydrogen yield through the addition of surfactants has been described in the literature and previously, for example mechanisms such as these have been described [14]: 

The addition of small amounts of glucose and surfactant significantly stimulates hydrogen production from hydrocarbons, allowing the strains to achieve production rates that are only about 3-5 times less than hydrogen production from pure highly concentrated glucose. It is assumed that the initial supply of glucose provides the microorganisms with sufficient energy to begin the energy-consuming decomposition of the incoming hydrocarbons. In addition, the surfactant provides better availability of hydrocarbons. But it is not yet possible to conclude whether the increase in hydrogen production from hydrocarbons is due to the addition of surfactant, glucose or their combination as more research is needed [14]. 

Other hydrogen-producing bacteria isolated from hydrocarbon reservoirs, such as Acetomicrobium thermoterrenum and Acetomicrobium hydrogeniformans, obligate anaerobes of the Synergistetes type have the same ability as the previously discussed Thermotogales bacteria to produce almost 4 hydrogen molecules/molecule of glucose [22]. 

Based on this study, the main conclusion is that bioconversion of crude oil to hydrogen can be a promising alternative to other hydrogen production methods currently in use (methane vapour conversion, water electrolysis, etc.) and also cost effective, because depleted, decommissioned oil wells are in locations with existing infrastructure above and around the well to collect and transport gases. Therefore, it requires less cost to convert it for hydrogen collection and transport. This method of hydrogen production will allow oil and gas companies to ‘reuse’ old oil and gas assets by converting them into underground hydrogen biorefineries, thereby increasing the profitability of drilling projects.

Application of special technologies to increase hydrogen productivity

Biohydrogen production requires a carbon source and high temperatures. Therefore, a new and unique way to produce biohydrogen is to inject specially engineered microorganisms and nutrients into depleted oil wells, where they will break down the residual petroleum hydrocarbons there and convert them into hydrogen and carbon dioxide. Mature oil reservoirs have the high temperatures required for hydrogen production, and they contain significant amounts of organic matter in the form of residual hydrocarbons.

The future success of the industrial biological hydrogen production process strictly depends on its ability to compete economically with modern petrochemical methods. Therefore, many research developments are aimed at further improvement of biohydrogen production technologies.

Most of the studies in this field are devoted to the search for microbial communities capable of growing on cheap organic media with high hydrogen production rates, as well as to improving the hydrogen production performance by regulating external factors: light intensity [23], medium pH [24, 25], cultivation temperature [26], gas composition of the medium [23, 26, 27] and nutrients [28, 29].

In our article we would like to pay special attention to the research aimed at using special technologies to change the properties of microbial communities in order to reduce the sensitivity of bacteria to environmental conditions, to increase the bioactivity of microorganisms in order to increase the rate of conversion of feedstock into hydrogen. There are two types of special technologies: genetic engineering and nanotechnology.

Genetic engineering

They are used to make changes to the genomic material of bacterial cells, i.e. to switch off certain genes that hinder hydrogen production by a given cell and to add genomic material to that bacterium that will further increase the rate of hydrogen production. 

Let's take the example of a hydrogen-producing bacterium belonging to the genus Rhodobacter. These bacteria produce hydrogen in extremely small quantities. They need it only to get rid of unnecessary reducing equivalents in their metabolic pathways. Hydrogen production is precisely the method of such disposal. And in general, the activity of these microorganisms has nothing to do with hydrogen. Thanks to an extremely diverse metabolism, they easily adapt to radical changes in the conditions of existence. These bacteria are photosynthesising cells, but if you turn off the light, they can live on. They need oxygen, but they are comfortable in anaerobic conditions. In other words, there is a huge ‘biochemical potential’ in these cells, and one or another metabolic pathway comes into play depending on what happens to that cell. With such a variety of metabolic pathways, a change in external conditions (optimisation of the nutrient environment) does not affect the increase in hydrogen production, i.e. when conditions change, the bacteria adapt to them.

In this case, it is necessary to make changes in the gene material of bacterial cells. The first way is to switch off some genes, which leads to switching off some biochemical reactions inside the cell that prevent hydrogen production. If everything is done correctly, the bacterial cell will start to synthesise hydrogen.

The second way is to add genomic material to this bacterium that will further increase the rate of hydrogen production. In this case there are two solutions, either to make copies of already existing genes that are needed for hydrogen production, or to include in the genome of this bacterium individual genes from the genomes of other bacteria.

To date, a lot of research has been done, in so-called gene editing, to improve hydrogen production. For example, such as: precise gene editing with zinc. -finger nuclease technology and CRISPR / Cas9 technology [30, 31, 32]; miRNA gene silencing for metabolic engineering [33], the use of riboswitches, specific components of the mRNA molecule that regulate gene expression in the cyanobacterium Synechococcus elongatus and can be applied to C. reinhardtii [34], improving hydrogen photoproduction by fusing the Nac2 gene to the cycl6 promoter, which is induced under anaerobic conditions or copper deficiency [QW6], developing several artificial microRNAs (amiRNAs) to increase hydrogen yield by stimulating faster oxygen consumption or suppressing the expression of the psbA gene, which encodes the D1 protein associated with FS2 [35, 36]. and many other studies [37]. 

In this way, hydrogen production can be accelerated by the latest technological advances in genetic engineering.

Nanotechnology

The use of nanostructured materials can impart unique physical and chemical properties to microorganisms and influence the factors that determine hydrogen yield.

Let us consider this as an example of one study [38] of biohydrogen production, using colloidal gold nanoparticles of 5, 10 or 20 nm in size as a catalyst. Clostridium butyricum anaerobes were used for hydrogen production. Significantly higher percentages and hydrogen yields were obtained in all tests using gold nanoparticles. The highest improvement was found with 5nm gold particles. The maximum cumulative hydrogen yield obtained using 5 nm gold particles was 4.48 mol per mol of sucrose compared to about 2.5 mol per mol of sucrose in the absence of gold nanoparticles, corresponding to a sucrose-to-hydrogen conversion efficiency of 56%. These results show that gold nanoparticles can significantly enhance the biological activity of hydrogen-producing microbes and that the enhancement effect is strongly dependent on the size of the gold particles. This is a promising method to enhance the catalytic activity of microbial hydrogenases, with potentially great significance for biohydrogen production [38].

There is currently tremendous interest in the use of nanoparticles for biological applications including DNA transfection [39], biosensors [40, 41, 42, 43], nanochemical devices [44, 45] and drug delivery [46]. Nanoparticles, especially in the 1-10 nm range (intermediate between the size of small molecules and bulk metal size), will exhibit electronic structures reflecting the electronic zone structure of nanoparticles due to quantum mechanical rules [47]. It follows that hydrogen production can be influenced by modern nanotechnology [38].

Conclusions

The success of industrial-scale biohydrogen production depends directly on the speed and production of hydrogen by the bacteria, the availability of the required volume of organic matter, and the financial costs of the technology. Depleted hydrocarbon reserves can provide exactly the organic matter sources, volumes and temperatures required for biohydrogen production, as well as the necessary, already developed infrastructure.

Increasing the productivity of hydrocarbon-to-hydrogen conversion requires further research into different hydrogen-producing bacteria and the application of nanotechnology and genetic engineering to improve biohydrogen production technologies.

Reference