Current status of the metabolic engineering of microorganisms for biohydrogen production

The improvement of H₂ production capabilities of hydrogen (H₂)-producing microorganisms is a challenging issue. Microorganisms have evolved for fast growth and substrate utilization rather than H₂ production. To develop good H₂-producing biocatalysts, many studies have focused on the redirection and/or reconstruction of cellular metabolisms. These studies included the elimination of enzymes and carbon pathways interfering or competing with H₂ production, the incorporation of non-native metabolic pathways leading to H₂ production, the utilization of various carbon substrates, the rectification of H₂-producting enzymes (nitrogenase and hydrogenase) and photophosphorylation systems, and in silico pathway flux analysis, among others. Owing to these studies, significant improvements in the yield and rate of H₂ production, and in the stability of H₂ production activity, were reached. This review presents and discusses the recent developments in biohydrogen production, with a focus on metabolic pathway engineering.     

Fermentative biohydrogen production systems integration

Acidogenic fermentation can be used to produce hydrogen from a range of biomass sources. The effluent from this process can be utilised in a number of biological processes enabling further recovery of energy from the biomass. In this review a number of candidate technologies are assessed including conventional methanogenic anaerobic digestion, dark fermentative hydrogen production, photo-fermentation, and bioelectrochemical systems. The principles, benefits and challenges associated with integrating these technologies are discussed, with particular emphasis on integration with fermentative hydrogen production, and the current state of integrative development is presented. The various system configurations for potential integrations presented here may simultaneously permit an increase in the conversion efficiency of biomass to energy, improved adaptability to varying operating conditions, and improved stability. Such integration, while increasing system complexity, may mean that these bioprocesses could be deployed in a wider range of scenarios and be used with a greater range of substrates.     

Bioreactor and process design for biohydrogen production

Biohydrogen is regarded as an attractive future clean energy carrier due to its high energy content and environmental-friendly conversion. It has the potential for renewable biofuel to replace current hydrogen production which rely heavily on fossil fuels. While biohydrogen production is still in the early stage of development, there have been a variety of laboratory- and pilot-scale systems developed with promising potential. This work presents a review of advances in bioreactor and bioprocess design for biohydrogen production. The state-of-the art of biohydrogen production is discussed emphasizing on production pathways, factors affecting biohydrogen production, as well as bioreactor configuration and operation. Challenges and prospects of biohydrogen production are also outlined.     

Metabolic engineering of Cupriavidus necator for heterotrophic and autotrophic alka(e)ne production

Alkanes of defined carbon chain lengths can serve as alternatives to petroleum-based fuels. Recently, microbial pathways of alkane biosynthesis have been identified and enabled the production of alkanes in non-native producing microorganisms using metabolic engineering strategies. The chemoautotrophic bacterium Cupriavidus necator has great potential for producing chemicals from CO₂: it is known to have one of the highest growth rate among natural autotrophic bacteria and under nutrient imbalance it directs most of its carbon flux to the synthesis of the acetyl-CoA derived polymer, polyhydroxybutyrate (PHB), (up to 80% of intracellular content). Alkane synthesis pathway from Synechococcus elongatus (2 genes coding an acyl-ACP reductase and an aldehyde deformylating oxygenase) was heterologously expressed in a C. necator mutant strain deficient in the PHB synthesis pathway. Under heterotrophic condition on fructose we showed that under nitrogen limitation, in presence of an organic phase (decane), the strain produced up to 670 mg/L total hydrocarbons containing 435 mg/l of alkanes consisting of 286 mg/l of pentadecane, 131 mg/l of heptadecene, 18 mg/l of heptadecane, and 236 mg/l of hexadecanal. We report here the highest level of alka(e)nes production by an engineered C. necator to date. We also demonstrated the first reported alka(e)nes production by a non-native alkane producer from CO₂ as the sole carbon source.     

Semi-continuous biohydrogen production as an approach to generate electricity

In this work, a semi-continuous biological system was established to produce hydrogen and generate electricity by coupling the bioreactor to a fuel cell. Heat and acid pretreatments (at 35 and 55 °C) of a seed sludge used as inoculum were performed in order to increase hydrogen producers. Different initial glucose concentrations (IGC) were tested for heat pretreated inoculum at 35 °C to determine the optimum concentration of glucose that supported the highest hydrogen production. Results showed that the heat pretreated inoculums (35 °C) reached the highest hydrogen molar yield of 2.85 mol H₂/mol glucose (0.014 L/h), which corresponds to the acetic acid pathway. At the optimum IGC (10 g/L, 35 °C) the hydrogen molar yield was 3.6 mol H₂/mol glucose (0.023 L/h). The coupled bioreactor-fuel cell system yielded an output voltage of 1.06 V, power of 0.1 W (25 °C) and a current of 68 mA. The overall results suggest that high hydrogen molar yields can be obtained through the acetic acid pathway and that is feasible to generate electricity using hydrogen from the semi- continuous bioreactor.     

Dark fermentation on biohydrogen production: Pure culture

Biohydrogen is regarded as an attractive future clean energy carrier due to its high energy content and environmental-friendly conversion. While biohydrogen production is still in the early stage of development, there have been a variety of laboratory- and pilot-scale systems developed with promising potential. This work presents a review of literature reports on the pure hydrogen-producers under anaerobic environment. Challenges and perspective of biohydrogen production with pure cultures are also outlined.     

Hydrolysates of lignocellulosic materials for biohydrogen production

Lignocellulosic materials are commonly used in bio-H₂ production for the sustainable energy resource development as they are abundant, cheap, renewable and highly biodegradable. In the process of the bio-H₂ production, the pretreated lignocellulosic materials are firstly converted to monosaccharides by enzymolysis and then to H₂ by fermentation. Since the structures of lignocellulosic materials are rather complex, the hydrolysates vary with the used materials. Even using the same lignocellulosic materials, the hydrolysates also change with different pretreatment methods. It has been shown that the appropriate hydrolysate compositions can dramatically improve the biological activities and bio-H₂ production performances. Over the past decades, hydrolysis with respect to different lignocellulosic materials and pretreatments has been widely investigated. Besides, effects of the hydrolysates on the biohydrogen yields have also been examined. In this review, recent studies on hydrolysis as well as their effects on the biohydrogen production performance are summarized. [BMB Reports 2013; 46(5): 244-251]     

Modeling and optimization of fermentative hydrogen production

Biohydrogen is a sustainable energy resource due to its potentially higher efficiency of conversion to usable power, non-polluting nature and high energy density. The purpose of modeling and optimization is to improve, analyze and predict biohydrogen production. Biohydrogen production depends on a number of variables, including pH, temperature, substrate concentration and nutrient availability, among others. Mathematical modeling of several distinct processes such as kinetics of microbial growth and products formation, steady state behavior of organic substrate along with its utilization and inhibition have been presented. Present paper summarizes the experimental design methods used to investigate effects of various factors on fermentative hydrogen production, including one-factor-at-a-time design, full factorial and fractional factorial designs. Each design method is briefly outlined, followed by the introduction of its analysis. In addition, the applications of artificial neural network, genetic algorithm, principal component analysis and optimization process using desirability function have also been highlighted.     

Microbial cell immobilization in biohydrogen production: a short overview

The high dependence on fossil fuels has escalated the challenges of greenhouse gas emissions and energy security. Biohydrogen is projected as a future alternative energy as a result of its non-polluting characteristics, high energy content (122?kJ/g), and economic feasibility. However, its industrial production has been hampered by several constraints such as low process yields and the formation of biohydrogen-competing reactions. This necessitates the search for other novel strategies to overcome this problem. Cell immobilization technology has been in existence for many decades and is widely used in various processes such as wastewater treatment, food technology, and pharmaceutical industry. In recent years, this technology has caught the attention of many researchers within the biohydrogen production field owing to its merits such as enhanced process yields, reduced microbial contamination, and improved homogeneity. In addition, the use of immobilization in biohydrogen production prevents washout of microbes, stabilizes the pH of the medium, and extends microbial activity during continuous processes. In this short review, an insight into the potential of cell immobilization is presented. A few immobilization techniques such as entrapment, adsorption, encapsulation, and synthetic polymers are discussed. In addition, the effects of process conditions on the performance of immobilized microbial cells during biohydrogen production are discussed. Finally, the review concludes with suggestions on improvement of cell immobilization technologies in biohydrogen production.     

Fermentative biohydrogen production: trends and perspectives

Biologically produced hydrogen (biohydrogen) is a valuable gas that is seen as a future energy carrier, since its utilization via combustion or fuel cells produces pure water. Heterotrophic fermentations for biohydrogen production are driven by a wide variety of microorganisms such as strict anaerobes, facultative anaerobes and aerobes kept under anoxic conditions. Substrates such as simple sugars, starch, cellulose, as well as diverse organic waste materials can be used for biohydrogen production. Various bioreactor types have been used and operated under batch and continuous conditions; substantial increases in hydrogen yields have been achieved through optimum design of the bioreactor and fermentation conditions. This review explores the research work carried out in fermentative hydrogen production using organic compounds as substrates. The review also presents the state of the art in novel molecular strategies to improve the hydrogen production.     

Feasibility of biohydrogen production from industrial wastes using defined microbial co-culture

Background

The development of clean or novel alternative energy has become a global trend that will shape the future of energy. In the present study, 3 microbial strains with different oxygen requirements, including Clostridium acetobutylicum ATCC 824, Enterobacter cloacae ATCC 13047 and Kluyveromyces marxianus 15D, were used to construct a hydrogen production system that was composed of a mixed aerobic-facultative anaerobic-anaerobic consortium. The effects of metal ions, organic acids and carbohydrate substrates on this system were analyzed and compared using electrochemical and kinetic assays. It was then tested using small-scale experiments to evaluate its ability to convert starch in 5 L of organic wastewater into hydrogen. For the one-step biohydrogen production experiment, H1 medium (nutrient broth and potato dextrose broth) was mixed directly with GAM broth to generate H2 medium (H1 medium and GAM broth). Finally, Clostridium acetobutylicum ATCC 824, Enterobacter cloacae ATCC 13047 and Kluyveromyces marxianus 15D of three species microbial co-culture to produce hydrogen under anaerobic conditions. For the two-step biohydrogen production experiment, the H1 medium, after cultured the microbial strains Enterobacter cloacae ATCC 13047 and Kluyveromyces marxianus 15D, was centrifuged to remove the microbial cells and then mixed with GAM broth (H2 medium). Afterward, the bacterial strain Clostridium acetobutylicum ATCC 824 was inoculated into the H2 medium to produce hydrogen by anaerobic fermentation.

Results

The experimental results demonstrated that the optimum conditions for the small-scale fermentative hydrogen production system were at pH 7.0, 35°C, a mixed medium, including H1 medium and H2 medium with 0.50 mol/L ferrous chloride, 0.50 mol/L magnesium sulfate, 0.50 mol/L potassium chloride, 1% w/v citric acid, 5% w/v fructose and 5% w/v glucose. The overall hydrogen production efficiency in the shake flask fermentation group was 33.7 mL/h^-1.L^-1, and those the two-step and the one-step processes of the small-scale fermentative hydrogen production system were 41.2 mL/h^-1.L^-1 and 35.1 mL/h^-1.L^-1, respectively.

Conclusion

Therefore, the results indicate that the hydrogen production efficiency of the two-step process is higher than that of the one-step process.     

Development of net energy ratio and emission factor for biohydrogen production pathways

This study investigates the energy and environmental aspects of producing biohydrogen for bitumen upgrading from a life cycle perspective. Three technologies are studied for biohydrogen production; these include the Battelle Columbus Laboratory (BCL) gasifier, the Gas Technology Institute (GTI) gasifier, and fast pyrolysis. Three different biomass feedstocks are considered including forest residue (FR), whole forest (WF), and agricultural residue (AR). The fast pyrolysis pathway includes two cases: truck transport of bio-oil and pipeline transport of bio-oil. The net energy ratios (NERs) for nine biohydrogen pathways lie in the range of 1.3-9.3. The maximum NER (9.3) is for the FR-based pathway using GTI technology. The GHG emissions lie in the range of 1.20-8.1 kg CO? eq/kg H?. The lowest limit corresponds to the FR-based biohydrogen production pathway using GTI technology. This study also analyzes the intensities for acid rain precursor and ground level ozone precursor.     

Metabolic engineering of Thermoanaerobacterium saccharolyticum for n-butanol production

The thermophilic anaerobe Thermoanaerobacterium saccharolyticum JW/SL-YS485 was investigated as a host for n-butanol production. A systematic approach was taken to demonstrate functionality of heterologous components of the clostridial n-butanol pathway via gene expression and enzymatic activity assays in this organism. Subsequently, integration of the entire pathway in the wild-type strain resulted in n-butanol production of 0.85 g/L from 10 g/L xylose, corresponding to 21% of the theoretical maximum yield. We were unable to integrate the n-butanol pathway in strains lacking the ability to produce acetate, despite the theoretical overall redox neutrality of n-butanol formation. However, integration of the n-butanol pathway in lactate deficient strains resulted in n-butanol production of 1.05 g/L from 10 g/L xylose, corresponding to 26% of the theoretical maximum.     

Isolation of cellulose-hydrolytic bacteria and applications of the cellulolytic enzymes for cellulosic biohydrogen production

Nine cellulolytic bacterial strains were isolated from soil sample taken in southern Taiwan. Through 16S rRNA sequence matching; eight of those isolates belong to Cellulomonas sp., while the other one belongs to Cellulosimicrobium cellulans. The activity of cellulolytic enzymes (cellulases and xylanase) produced from those strains was mainly present extracellularly and the enzyme production was dependent on cellulosic substrates (xylan, rice husk and rice straw) used for growth. HPLC analysis confirmed the bacterial hydrolysis of these cellulosic substrates for soluble sugars production. The efficiency of fermentative H₂ production from the enzymatically hydrolyzed rice husk was examined with seven H₂-producing pure bacterial isolates. With an initial reducing sugar concentration of 0.36 g l⁻¹, only Clostridium butyricum CGS5 exhibited efficient H₂ production from the rice husk hydrolysates with a cumulative H₂ production and H₂ yield of 88.1 ml l⁻¹ and 19.15 mmol H₂ (g reducing sugar)⁻¹ (or 17.24 mmol H₂ (g cellulose)⁻¹), respectively.     

A thermodynamic analysis of electron production during syngas fermentation

Currently, syngas fermentation is being developed as one option towards the production of biofuels from biomass. This process utilizes the acetyl-CoA (Wood-Ljungdahl) metabolic pathway. Along the pathway, CO and CO₂ are used as carbon sources. Electrons required for the metabolic process are generated from H₂ and/or from CO. This study showed that electron production from CO is always more thermodynamically favorable compared to electron production from H₂ and this finding is independent of pH, ionic strength, gas partial pressure, and electron carrier pairs. Additionally, electron production from H₂ may be thermodynamically unfavorable in some experimental conditions. Thus, it is unlikely that H₂ can be utilized for electron production in favor of CO when both species are present. Therefore, CO conversion efficiency will be sacrificed during syngas fermentation since some of the CO will provide electrons at the expense of product and cell mass formation.     

The role of biochemical engineering in the production of biofuels from microalgae

Environmental changes that have occurred due to the use of fossil fuels have driven the search for alternative sources that have a lower environmental impact. First-generation biofuels were derived from crops such as sugar cane, corn and soybean, which contribute to water scarcity and deforestation. Second-generation biofuels originated from lignocellulose agriculture and forest residues, however these needed large areas of land that could be used for food production. Based on technology projections, the third generation of biofuels will be derived from microalgae. Microalgae are considered to be an alternative energy source without the drawbacks of the first- and second-generation biofuels. Depending upon the growing conditions, microalgae can produce biocompounds that are easily converted into biofuels. The biofuels from microalgae are an alternative that can keep the development of human activity in harmony with the environment. This study aimed to present the main biofuels that can be derived from microalgae.     

Theoretical Characterization of Slurry Fermentation

This is an extensive study of the three classical fermentation systems (emerged, submerged and solid‐state fermentations), thus uncovering, a system that is an intrinsically concomitant overlap of submerged and solid‐state fermentations – slurry fermentation. It is a convenient method of fermentation with hardly any theoretical characterization. This paper details the specifics of this system and its potential fields of application.     

Thermophilic biohydrogen production by an anaerobic heat treated-hot spring culture

Batch experiments were conducted to investigate the thermophilic biohydrogen production using an enrichment culture from a Turkish hot spring. Following the enrichment, the culture was heat treated at 100 °C for 10 min to select for spore-forming bacteria. H₂ production was accompanied by production of acetate, butyrate, lactate and ethanol. H₂ production was associated by acetate–butyrate type fermentation while accumulation of lactate and ethanol negatively affected the H₂ yield. H₂ production was highest in the temperature range from 49.6 to 54.8 °C and optimum values for initial pH and concentrations of iron, yeast extract and glucose were 6.5, 40 mg/l, 4–13.5 g/l, respectively. PCR–DGGE profiling showed that the heat treated culture consisted of species closely affiliated to genus Thermoanaerobacterium.     

Assessment of optimum dilution ratio for biohydrogen production by anaerobic co-digestion of press mud with sewage and water

Anaerobic co-digestion of press mud with water or sewage at ratios of 1:7.5, 1:10 and 1:12.5 were performed in continuously fed UASB reactors for hydrogen production. At a constant hydraulic retention time of 30 h, the specific hydrogen production rate was 187 mL/g volatile solids (VS) reduced during maximum biohydrogen production of 7960 mL/day at a 1:10 ratio of press mud to sewage. Chemical oxygen demand (COD) and VS reductions of 61% and 59% were noted on peak biohydrogen yield. A pH range of 5-6 was suitable at ambient temperature for entire process; a lower pH was inhibitory. Co-digestion of acidic press mud with sewage controlled pH for fermentation. Hence press mud can be exploited for biohydrogen production.