Eight Up-Coming Biotech Tools to Combat Climate Crisis.

Biotechnology has a high potential to substantially contribute to a low-carbon society. Several green processes are already well established, utilizing the unique capacity of living cells or their instruments. Beyond that, the authors believe that there are new biotechnological procedures in the pipeline which have the momentum to add to this ongoing change in our economy. Eight promising biotechnology tools were selected by the authors as potentially impactful game changers: (i) the Wood-Ljungdahl pathway, (ii) carbonic anhydrase, (iii) cutinase, (iv) methanogens, (v) electro-microbiology, (vi) hydrogenase, (vii) cellulosome and, (viii) nitrogenase. Some of them are fairly new and are explored predominantly in science labs. Others have been around for decades, however, with new scientific groundwork that may rigorously expand their roles. In the current paper, the authors summarize the latest state of research on these eight selected tools and the status of their practical implementation. We bring forward our arguments on why we consider these processes real game changers.

Comparison of Carbonic Anhydrases for CO2 Sequestration.

Strategies for depleting carbon dioxide (CO2) from flue gases are urgently needed and carbonic anhydrases (CAs) can contribute to solving this problem. They catalyze the hydration of CO2 in aqueous solutions and therefore capture the CO2. However, the harsh conditions due to varying process temperatures are limiting factors for the application of enzymes. The current study aims to examine four recombinantly produced CAs from different organisms, namely CAs from Acetobacterium woodii (AwCA or CynT), Persephonella marina (PmCA), Methanobacterium thermoautotrophicum (MtaCA or Cab) and Sulphurihydrogenibium yellowstonense (SspCA). The highest expression yields and activities were found for AwCA (1814 WAU mg-1 AwCA) and PmCA (1748 WAU mg-1 PmCA). AwCA was highly stable in a mesophilic temperature range, whereas PmCA proved to be exceptionally thermostable. Our results indicate the potential to utilize CAs from anaerobic microorganisms to develop CO2 sequestration applications.

Design and engineering of artificial microbial consortia for biohydrogen production.

In natural microbial ecosystems the metabolic diversity of the organisms enables interaction among the community members and allows them to engage in syntrophic interactions. With regard to biotechnology, artificial microbial consortium engineering is used to improve productivities and yields of bioprocesses. However, to achieve supreme productivity or efficiency at industrial scale, defined ecosystems must be physiologically well-selected to meet eco-biotechnological demands. Here, we present an artificial microbial consortia design and engineering pipeline for developing dark fermentative biohydrogen production processes. The proposed pipeline might be considered as a blue-print for enhancing other bioprocesses that fundamentally face metabolic restrictions or kinetic limitations.

Biohydrogen production beyond the Thauer limit by precision design of artificial microbial consortia.

Dark fermentative biohydrogen (H2) production could become a key technology for providing renewable energy. Until now, the H2 yield is restricted to 4 moles of H2 per mole of glucose, referred to as the "Thauer limit". Here we show, that precision design of artificial microbial consortia increased the H2 yield to 5.6 mol mol-1 glucose, 40% higher than the Thauer limit. In addition, the volumetric H2 production rates of our defined artificial consortia are superior compared to any mono-, co- or multi-culture system reported to date. We hope this study to be a major leap forward in the engineering of artificial microbial consortia through precision design and provide a breakthrough in energy science, biotechnology and ecology. Constructing artificial consortia with this drawing-board approach could in future increase volumetric production rates and yields of other bioprocesses. Our artificial consortia engineering blueprint might pave the way for the development of a H2 production bioindustry.

Controlled enzymatic hydrolysis and synthesis of lignin cross-linked chitosan functional hydrogels.

This study presents a novel fully enzymatic process for the controlled depolymerisation of fungal and shrimp chitosan, and their subsequent use in the synthesis of lignin cross-linked chitosan (CTS) hydrogels. Cellobiosehydrolase (CBH) was used to depolymerize CTS resulting in decrease in average molecular weight (Mw) of shrimp CTS from 140 kDa and degree of deacetylation (DD %) from 91.3% to an average MW of 15 kDa and 16% DD. Similarly, fungal chitosan average molecular weight decreased from 92 kDa and the degree of deacetylation (DD) of 48.3% to 12 kDa and a DD of 13%. The depolymerized CTS were completely soluble in water and miscible with lignosulfonates without encountering the usual problem of formation of flocs. Introduction of laccase into a lignosulfonate-chitosan mixture resulted in the oxidation and generation of lignin reactive phenoxyl radicals that cross-linked with CTS-NH2 reactive groups resulting in the increase of Mw from 20 kDa to >500 kDa and viscosity from 20 mPa to >500 mPa. This resulted in the formation of stable lignin-cross-linked hydrogels with elongation at break of 111% and tensile strength of 7 mPa. The produced functional hydrogels have potential application in food and biomedical industries as e.g. as oxygen barriers in packaging or as functional wound dressing or tissue engineering platforms.

Anaerobic Digestion of Pretreated Industrial Residues and Their Energetic Process Integration.

The food and beverage industry offers a wide range of organic feedstocks for use in biogas production by means of anaerobic digestion (AD). Microorganisms convert organic compounds-solid, pasty, or liquid ones-within four steps to biogas mainly consisting of CH4 and CO2. Therefore, various conversion technologies are available with several examples worldwide to show for the successful implementation of biogas technologies on site. The food and beverage industry offer a huge potential for biogas technologies due to the sheer amount of process residues and their concurrent requirement for heat and power. The following study analyzes specific industries with respect to their implementation potential based on arising waste and heat and power demand. Due to their chemical composition, several feedstocks are resistant against microbiological degradation to a great extent. A combination of physical-, chemical-, and microbiological pretreatment are used to increase the biological availability of the feedstock. The following examples will discuss how to best implement AD technology in industrial processes. The brewery industry, dairy production, slaughterhouses, and sugar industry will serve as examples.

Formate Utilization by the Crenarchaeon Desulfurococcus amylolyticus.

Formate is one of the key compounds of the microbial carbon and/or energy metabolism. It owes a significant contribution to various anaerobic syntrophic associations, and may become one of the energy storage compounds of modern energy biotechnology. Microbial growth on formate was demonstrated for different bacteria and archaea, but not yet for species of the archaeal phylum Crenarchaeota. Here, we show that Desulfurococcus amylolyticus DSM 16532, an anaerobic and hyperthermophilic Crenarchaeon, metabolises formate without the production of molecular hydrogen. Growth, substrate uptake, and production kinetics on formate, glucose, and glucose/formate mixtures exhibited similar specific growth rates and similar final cell densities. A whole cell conversion experiment on formate revealed that D. amylolyticus converts formate into carbon dioxide, acetate, citrate, and ethanol. Using bioinformatic analysis, we examined whether one of the currently known and postulated formate utilisation pathways could be operative in D. amylolyticus. This analysis indicated the possibility that D. amylolyticus uses formaldehyde producing enzymes for the assimilation of formate. Therefore, we propose that formate might be assimilated into biomass through formaldehyde dehydrogenase and the oxidative pentose phosphate pathway. These findings shed new light on the metabolic versatility of the archaeal phylum Crenarchaeota.

The physiological effect of heavy metals and volatile fatty acids on Methanococcus maripaludis S2.

BACKGROUND: Methanogenic archaea are of importance to the global C-cycle and to biological methane (CH4) production through anaerobic digestion and pure culture. Here, the individual and combined effects of copper (Cu), zinc (Zn), acetate, and propionate on the metabolism of the autotrophic, hydrogenotrophic methanogen Methanococcus maripaludis S2 were investigated. Cu, Zn, acetate, and propionate may interfere directly and indirectly with the acetyl-CoA synthesis and biological CH4 production. Thus, these compounds can compromise or improve the performance of M. maripaludis, an organism which can be applied as biocatalyst in the carbon dioxide (CO2)-based biological CH4 production (CO2-BMP) process or of methanogenic organisms applied in anaerobic digestion. RESULTS: Here, we show that Cu concentration of 1.9 µmol L-1 reduced growth of M. maripaludis, whereas 4.4 and 6.3 µmol L-1 of Cu even further retarded biomass production. However, 1.0 mmol L-1 of Zn enhanced growth, but at Zn concentrations > 2.4 mmol L-1 no growth could be observed. When both, Cu and Zn, were supplemented to the medium, growth and CH4 production could even be observed at the highest tested concentration of Cu (6.3 µmol L-1). Hence, it seems that the addition of 1 mmol L-1 of Zn enhanced the ability of M. maripaludis to counteract the toxic effect of Cu. The physiological effect to rising concentrations of acetate (12.2, 60.9, 121.9 mmol L-1) and/or propionate (10.3, 52.0, 104.1 mmol L-1) was also investigated. When instead of acetate 10.3 mmol L-1 propionate was provided in the growth medium, M. maripaludis could grow without reduction of the specific growth rate (µ) or the specific CH4 productivity (qCH4). A combination of inorganic and/or organic compounds resulted in an increase of µ and qCH4 for Zn/Cu and Zn/acetate beyond the values that were observed if only the individual concentrations of Zn, Cu, acetate were used. CONCLUSIONS: Our study sheds light on the physiological effect of VFAs and heavy metals on M. maripaludis. Differently from µ and qCH4, MER was not influenced by the presence of these compounds. This indicated that each of these compounds directly interacted with the C-fixation machinery of M. maripaludis. Until now, the uptake of VFAs other than acetate was not considered to enhance growth and CH4 production of methanogens. The finding of propionate uptake by M. maripaludis is important for the interpretation of VFA cycling in anaerobic microenvironments. Due to the importance of methanogens in natural and artificial anaerobic environments, our results help to enhance the understanding the physiological and biotechnological importance with respect to anaerobic digestion, anaerobic wastewater treatment, and CO2-BMP. Finally, we propose a possible mechanism for acetate uptake into M. maripaludis supported by in silico analyses.

The physiology and biotechnology of dark fermentative biohydrogen production.

A CO2-neutral energy production alternative compared to conventional fossil fuel utilization is biohydrogen (H2) production. Three basic mechanisms for microbial H2 production exist: photosynthetic H2 production, photo-fermentative H2 production, and dark fermentative H2 production (DFHP). Despite surmounting reports in literature on the characterization and optimization of DFHP systems, H2 production has not yet reached an industrial scale. Here, DFHP characteristics of pure culture of microorganisms from more than one century were reviewed and analysed. Analysing pure culture DFHP has the advantage that the physiology and the biotechnological potential of a specific organism can be exploited with the aim to optimize and establish a straightforward H2 production bioprocess. Essential to this effort is the analysis of reported values across phylogenetically distinct groups of microorganisms. Therefore, an extensive review and subsequent in-depth meta-data analysis of DFHP from pure cultures was performed with the goals of providing: a comprehensive overview to their physiology, reviewing closed batch, batch, and continuous culture DFHP from an energy production perspective, and to integrate physiology and biotechnology through comprehensive meta-data analyses, statistics, and modelling. We revealed that a comparison of H2 productivity and H2 yield (Y(H2/S)) could unambiguously be performed on a carbon molar level. Clear dependencies between Y(H2/S) and the metabolic pathways of specific phylogenetic DFHP groups were found. With respect to specific H2 productivity and Y(H2/S) the superior phylogenetic group for DFHP was Thermococcaceae. Moreover, a distinct correlation between high Y(H2/S) and high H2 productivity was identified. The best substrate for H2 production was found to be formate. Statistical analysis and modelling provided the input parameter sets that could be used to optimize of H2 production of Clostridiaceae and Enterobacteriaceae. With respect to the overall goal to improve H2 production beyond reported values, we suggest to utilize Thermococcaceae, and to integrate these organisms into a H2 production set-up encompassing a cell retention system that would allow the accumulation of a high biomass density. Then both, high H2 production and Y(H2/S) might be achieved at the same time. Such an integrated system could finally render DFHP a biotechnologically useful process.

Optimisation of continuous gas fermentation by immobilisation of acetate-producing Acetobacterium woodii.

Hydrogen from water electrolysis is often suggested as a way of storing the excess energy from wind and solar power plants. However, unlike natural gas, hydrogen is difficult to store and distribute. One solution is to convert the hydrogen into other fuels or bulk chemicals. In this study we investigated fermentation in which homoacetogenic clostridia apply the Wood-Ljungdahl pathway to generate acetate from H2 and CO2. Acetate can be used as a bulk chemical or further transformed into biofuels. Autotrophic growth with CO2 as the sole carbon source is slow compared to heterotrophic growth, so the aim of this work was to improve continuous gas fermentation by immobilising the acetate-producing clostridia, thus preventing their wash out from the bioreactor. Two homoacetogenic bacterial strains (Acetobacterium woodii and Moorella thermoacetica) were tested for their acetate production potential, with A. woodii proving to be the better strain with maximum acetate concentration of 29.57 g l-1. Due to its stability during fermentation and good bacterial immobilisation, linen was chosen as immobilisation material for continuous fermentation. This study demonstrates the successful continuous fermentation of acetate from H2 and CO2 using A. woodii immobilised on a low-cost surface at high volumetric productivity of 1.21 ± 0.05 g acetate l-1 d-1. This has great industrial potential and future studies should focus on the scale-up of this process.

Characteristics of adapted hydrogenotrophic community during biomethanation.

The results presented in this study were carried out as concomitant experiments during the start-up and operation of a biomethanation unit to evaluate the effect of process parameters on carbon conversion, product formation (methane and acetate) and community composition. For that, two different samples were withdrawn from a trickle-bed reactor with immobilized enrichment culture of hydrogenotrophic methanogens adapted from sewage sludge. One sample was taken from the recirculation liquid during start-up phase while the other was withdrawn directly from the carrier material in the reactor. Elevated acid levels especially during start-up were shown to affect the overall carbon conversion. This effect was also seen during the acid tolerance testing reported here. Final acid concentrations of 1.6±0.3g/L resulted in a reduced conversion ratio of only 46%. Without acid addition complete conversion of CO2 in the headspace was achieved. However, maximum methane production of 0.55±0.02mmol after 4days of incubation was monitored at moderate initial acetate concentration of 0.4g/L. In both analyzed inoculation materials Methanobacterium species were by far the most dominant Archaea with 21.8% in the recirculation liquid during start-up and 84.8% in the enrichment culture immobilized on the carrier material. The microbial composition of the two analyzed samples is in accordance with the results obtained for the carbon conversion and product formation. With approximately 50% of Bacteroidetes and Firmicutes present during reactor start-up the acetic acid production significantly contributed to the overall carbon conversion. In contrast, methane was produced almost exclusively in trials representing continuous operation where acetogenic bacteria accounted only up to 17.5%. In summary, the acid accumulation monitored during reactor start-up of a biomethanation unit is most likely to result from the microbial composition present. Nevertheless, complete adaptation to hydrogenotrophic conditions was proven to alter the consortium and yield methane as main product alongside high carbon conversion of up to 70.5±1.8%.

Overcoming the bottlenecks of anaerobic digestion of olive mill solid waste by two-stage fermentation.

Two-stage anaerobic digestion (AD) of two-phase olive mill solid waste (OMSW) was applied for reducing the inhibiting factors by optimizing the acidification stage. Single-stage AD and co-fermentation with chicken manure were conducted coinstantaneous for direct comparison. Degradation of the polyphenols up to 61% was observed during the methanogenic stage. Nevertheless the concentration of phenolic substances was still high; the two-stage fermentation remained stable at OLR 1.5 kgVS/m³day. The buffer capacity of the system was twice as high, compared to the one-stage fermentation, without additives. The two-stage AD was a combined process - thermophilic first stage and mesophilic second stage, which pointed out to be the most profitable for AD of OMSW for the reduced hydraulic retention time (HRT) from 230 to 150 days, and three times faster than the single-stage and the co-fermentation start-up of the fermentation. The optimal HRT and incubation temperature for the first stage were determined to four days and 55°C. The performance of the two-stage AD concerning the stability of the process was followed by the co-digestion of OMSW with chicken manure as a nitrogen-rich co-substrate, which makes them viable options for waste disposal with concomitant energy recovery.

Efficient anaerobic mono-digestion of N-rich slaughterhouse waste: influence of ammonia, temperature and trace elements.

Three mono-digestion experiments treating slaughterhouse waste with high TKN concentration (∼11g/kg) were applied in lab-scale at mesophilic and psychrophilic conditions to study the impact of high ammonia concentrations and additives. Precipitation of sulphur by addition of ferrous chloride did not influence process behaviour, whereas supplementation of trace elements significantly improved process stability by reducing volatile fatty acid concentration towards zero. The limit of NH4-N concentration causing a rise of VFAs to 19,000mg/l and reduction of methane by 25% was found between 7.7 and 9.1g/kg which correspond to NH3 concentrations of 830-1060mg/l. Psychrophilic operation (25°C) lowered inhibitory NH3 concentration to 140mg/l, but process performance was stable only at low OLR of 0.4kgVS/m(3)d. Robust performance at highest possible NH4-N concentration (7.7g/kg), low VFA accumulation and satisfying methane yield of about 280Nm(3)/t COD was observed at OLR of 2.5kgVS/m(3)d at 37°C.

Reducing the risk of foaming and decreasing viscosity by two-stage anaerobic digestion of sugar beet pressed pulp.

Anaerobic digestion (AD) of sugar beet pressed pulp (SBPP) is a promising treatment concept. It produces biogas as a renewable energy source making sugar production more energy efficient and it turns SBPP from a residue into a valuable resource. In this study one- and two-stage mono fermentation at mesophilic conditions in a continuous stirred tank reactor were compared. Also the optimal incubation temperature for the pre-acidification stage was studied. The fastest pre-acidification, with a hydraulic retention time (HRT) of 4 days, occurred at a temperature of 55 °C. In the methanogenic reactor of the two-stage system stable fermentation at loading rate of 7 kg VS/m³ d was demonstrated. No artificial pH adjustment was necessary to maintain optimum levels in both the pre-acidification and the methanogenic reactor. The total HRT of the two-stage AD was 36 days which is considerably lower compared to the one-stage AD (50 days). The frequently observed problem of foaming at high loading rates was less severe in the two-stage reactor. Moreover the viscosity of digestate in the methanogenic stage of the two-stage fermentation was in average tenfold lower than in the one-stage fermentation. This decreases the energy input for the reactor stirring about 80 %. The observed advantages make the two-stage process economically attractive, despite higher investments for a two reactor system.

Changes in bacterial communities from anaerobic digesters during petroleum hydrocarbon degradation.

Anaerobic biodegradation of petroleum hydrocarbons (PHC) to methane has been recognized to occur in oil reservoirs and contaminated surface sites alike. This process could be employed efficiently for the treatment of contaminated materials, including petrochemical wastes and PHC-contaminated soil, since no external electron acceptor is required. Moreover, the controlled production of methane in digestion plants, similarly to the anaerobic digestion (AD) of energy crops or organic residues, would enable for energy recovery from these wastes. At present, little is known about the bacterial communities involved in and responsible for hydrocarbon fermentation, the initial step in PHC conversion to methane. In the present study, the fate of two different methanogenic communities derived from the AD of wastewater (WWT) and of biowaste, mixed with PHC-contaminated soil (SWT), was monitored during incubation with PHC using denaturing gradient gel electrophoresis (DGGE) of 16S rDNA genes amplified with Bacteria-specific primers. During 11 months of incubation, slight but significant degradation of PHC occurred in both sludges and distinct bacterial communities were developing. In both sludges, Bacteroidetes were found. In addition, in WWT, the bacterial community was found to be dominated by Synergistetes and Proteobacteria, while Firmicutes and unidentified members were abundant in SWT. These results indicate that bacterial communities from anaerobic digesters can adapt to and degrade petroleum hydrocarbons. The decontamination of PHC-containing waste via fermentative treatment appears possible.