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.

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 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.

Improving exploitation of chicken manure via two-stage anaerobic digestion with an intermediate membrane contactor to extract ammonia.

This study describes a modified process of ammonia release through pre-hydrolysis - ammonia removal via membrane contactor - methanization for counteracting ammonia inhibition in anaerobic digestion of chicken manure. In the pre-hydrolysis step, ammonia was rapidly released within the first 3-5 days. 78%-83% of the total nitrogen was finally converted into total ammonia/ammonium (TAN) with volatile fatty acids concentration of approximately 300 g/kg·VS. In the ammonia removal process, diluting the hydrolyzed chicken manure to 1:2, the TAN could be reduced to 2 g/kg in 21 h when pH was increased to 9. The final BMP test of chicken manure verified that lower TAN concentration (decreased to 2 g/kg) significantly reduced inhibitory effects, obtaining a high methane yield of 437.0 mL/g·VS. The investigations underlined several advantages of this modified 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%.

Characterization of polyhydroxyalkanoates produced by Synechocystis salina from digestate supernatant.

The current commercial production of polyhydroxyalkanoates (PHA) is based on heterotrophic bacteria, using organic carbon sources from crops. To avoid the competition with food and feed production, cyanobacteria, metabolising PHA from carbon dioxide can be used. This research focuses on the investigation of the thermal and rheological properties of PHA polymers accumulated by Synechocystis salina, which had been cultivated in digestate supernatant and a mineral medium. The dried bacterial cells had a polymer content of 5.5-6.6%. The relevance of the derived PHA polymers for the common melt polymer processing was correlated with their molecular mass distribution as well as with their thermal and rheological properties. The determined thermal and rheological properties showed that PHA polymers accumulated by S. salina on digestate supernatant or mineral medium are comparable with the commercial available poly(3-hydroxybutyrate). However, the results showed that PHA polymers in general require modification before melt processing to increase their stability in the molten state.

The ManureEcoMine pilot installation: advanced integration of technologies for the management of organics and nutrients in livestock waste.

Manure represents an exquisite mining opportunity for nutrient recovery (nitrogen and phosphorus), and for their reuse as renewable fertilisers. The ManureEcoMine proposes an integrated approach of technologies, operated in a pilot-scale installation treating swine manure (83.7%) and Ecofrit® (16.3%), a mix of vegetable residues. Thermophilic anaerobic digestion was performed for 150 days, the final organic loading rate was 4.6 kgCOD m-3 d-1, with a biogas production rate of 1.4 Nm3 m-3 d-1. The digester was coupled to an ammonia side-stream stripping column and a scrubbing unit for free ammonia inhibition reduction in the digester, and nitrogen recovery as ammonium sulphate. The stripped digestate was recirculated daily in the digester for 15 days (68% of the digester volume), increasing the gas production rate by 27%. Following a decanter centrifuge, the digestate liquid fraction was treated with an ultrafiltration membrane. The filtrate was fed into a struvite reactor, with a phosphorus recovery efficiency of 83% (as orthophosphate). Acidification of digestate could increment the soluble orthophosphate concentration up to four times, enhancing phosphorus enrichment in the liquid fraction and its recovery via struvite. A synergistic combination of manure processing steps was demonstrated to be technologically feasible to upgrade livestock waste into refined, concentrated fertilisers.

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.

Direct fermentation of potato starch and potato residues to lactic acid by Geobacillus stearothermophilus under non-sterile conditions.

BACKGROUND: Lactic acid is an important biorefinery platform chemical. The use of thermophilic amylolytic microorganisms to produce lactic acid by fermentation constitutes an efficient strategy to reduce operating costs, including raw materials and sterilization costs. RESULTS: A process for the thermophilic production of lactic acid by Geobacillus stearothermophilus directly from potato starch was characterized and optimized. Geobacillus stearothermophilus DSM 494 was selected out of 12 strains screened for amylolytic activity and the ability to form lactic acid as the major product of the anaerobic metabolism. In total more than 30 batches at 3-l scale were run at 60 °C under non-sterile conditions. The process developed produced 37 g L-1 optically pure (98%) L-lactic acid in 20 h from 50 g L-1 raw potato starch. As co-metabolites smaller amounts (<7% w/v) of acetate, formate and ethanol were formed. Yields of lactic acid increased from 66% to 81% when potato residues from food processing were used as a starchy substrate in place of raw potato starch. CONCLUSIONS: Potato starch and residues were successfully converted to lactic acid by G. stearothermophilus. The process described in this study provides major benefits in industrial applications and for the valorization of starch-rich waste streams. © 2015 The Authors.Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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.

Influence of different mesh filter module configurations on effluent quality and long-term filtration performance.

Recently, a new type of wastewater treatment system became the focus of scientific research: the mesh filter activated sludge system. It is a modification of the membrane bioreactor (MBR), in which a membrane filtration process serves for sludge separation. The main difference is that a mesh filter is used instead of the membrane. The effluent is not of the same excellent quality as with membrane bioreactors due to the much lager pore sizes of the mesh. Nevertheless, it still resembles the quality of currently used standard treatment system, the activated sludge process. The new process shows high future potential as an alternative where a small footprint of these plants is required (3 times lower footprint than conventional activated sludge systems because of neglecting the secondary clarifier and reducing the biological stage). However, so far only limited information on this innovative process is available. In this study, the effect of different pore sizes and different mesh module configurations on the effluent quality was investigated varying the parameters cross-flow velocity (CFV) and flux rate. Furthermore the long-term filtration performance was studied in a pilot reactor system and results were compared to the full-scale conventional activated sludge process established at the same site. The results demonstrate that the configuration of the filter module has little impact on effluent quality and is only of importance with regard to engineering aspects. Most important for a successful operation are the hydrodynamic conditions within the filter module. The statement "the higher the pore size the higher the effluent turbidity" was verified. Excellent effluent quality with suspended solids between 5 and 15 mg L(-1) and high biological elimination rates (chemical oxygen demand (COD) 90-95%, biological oxygen demand (BOD5) 94-98%, total nitrogen (TN) 70-80%, and ammonium nitrogen (NH(4)-N) 95-99%) were achieved and also compared to those of conventional activated sludge systems. Regarding the air requirement for module aeration, which is the main cost factor in MBR technology, an astonishing optimization could be achieved. During the long-term filtration experiments only 4 N m(3)/m(3) was necessary to keep a stable filtration process for more than 3 weeks (MBR 20-50 N m(3)/m(3)).

Potential of combined fungal and bacterial treatment for color removal in textile wastewater.

Low efficiency of dye removal by mixed bacterial communities and high rates of dye decolorization by white-rot fungi suggest a combination of both processes to be an option of treatment of textile wastewaters containing dyes and high concentrations of organics. Bacteria were able to remove mono-azo dye but not other chemically different dyes whereas decolorization rates using Irpex lacteus mostly exceeded 90% within less than one week irrespective of dye structure. Decolorization rates for industrial textile wastewaters containing 2-3 different dyes by fungal trickling filters (FTF) attained 91%, 86%, 35% within 5-12 d. Sequential two-step application of FTF and bacterial reactors resulted in efficient decolorization in 1st step (various single dyes, 94-99% within 5 d; wastewater I, 90% within 7 d) and TOC reduction of 95-97% in the two steps. Large potential of combined use of white-rot fungi and traditional bacterial treatment systems for bioremediation of textile wastewaters was demonstrated.

The European NEAT program: an integrated approach using acamprosate and psychosocial support for the prevention of relapse in alcohol-dependent patients with a statistical modeling of therapy success prediction.

BACKGROUND: A multicenter, prospective study was conducted in five European countries to observe outcome in alcohol misusers treated for 24 weeks with acamprosate and various psychosocial support techniques, within the setting of standard patient care. METHODS: Patients diagnosed as alcohol dependent using DSM-III-R criteria were treated, for 24 weeks, with acamprosate and appropriate psychosocial support. Potential predictor variables were recorded at inclusion. Drinking behavior was monitored throughout; the proportion of cumulative abstinence days was the principal outcome measure. The influence of baseline clinical and demographic variables on outcome was assessed using multiple regression analysis. Adverse events were recorded systematically. RESULTS: A total of 1289 patients were recruited; 1230 took at least one dose of the drug and provided at least one set of follow-up data; 543 (42.1%)patients were observed for the full 24-week period. The overall proportion of cumulative abstinence days was 0.48. Multiple physical and psychiatric comorbidities and a history of drug addiction were negatively correlated with outcome, as were, to a lesser extent, multiple previous episodes of detoxification, unemployment, and living alone. Older age and stable employment were positively associated with outcome. The difference in the unadjusted proportion of cumulative abstinence days between countries was significant ( < 0.001) but less so when adjusted for the predictive factors identified in the multivariate model ( < 0.019). Overall, outcome was not influenced by the nature of the psychosocial support provided. Adverse events were generally mild, with gastrointestinal disorders, which occurred in 21.5% of patients, being the most frequent. CONCLUSIONS: This open-label study confirms the efficacy and safety of acamprosate in the treatment of alcohol dependence in the setting of standard patient care. Treatment benefit was observed irrespective of the nature of the psychosocial support provided. Predictors of the response to treatment were identified; their heterogeneous distribution within the study population explained, at least in part, the differences in outcome between countries.