Green Extractants in Assisting Recovery of REEs: A Case Study.

The recycling of REEs from the end of life (EoL) products, such as nickel metal hydride batteries (NiMH), offers great opportunities for their supply in Europe. In the presented paper, the application of 'green' extractants such as citric (CA), metatartaric (TA), and ethylenediaminedisuccinic acid (EDDS) (also with H2O2 addition) for the recovery of REEs was studied. The studies were conducted considering the effects of the phase contact time, the initial concentration of CA, TA, and EDDS, as well as H2O2, pH, and temperature. It was found that the addition of TA to the CA solution meant that higher rates of metal ion binding and, thus, leaching was observed. The optimal conditions were obtained in the system: CA-TA and H2O2 for the concentration 0.6M-0.3 M-2%.

Early-stage sustainability assessment of biotechnological processes: A case study of citric acid production.

Sustainability assessment using a life-cycle approach is indispensable to contemporary bioprocess development. This assessment is particularly important for early-stage bioprocess development. As early-stage investigations of bioprocesses involve the evaluation of their ecological and socioeconomic effects, they can be adjusted more effectively and improved towards sustainability, thereby reducing environmental risk and production costs. Early-stage sustainability assessment is an important precautionary practice and, despite limited data, a unique opportunity to determine the primary impacts of bioprocess development. To this end, a simple and robust method was applied based on the standardized life-cycle sustainability assessment methodology and commercially available datasets. In our study, we elaborated on the yeast-based citric acid production process with Yarrowia lipolytica assessing 11 different substrates in different process modes. The focus of our analysis comprised both cultivation and down-stream processing. According to our results, the repeated batch raw glycerol based bioprocess alternative showed the best environmental performance. The second- and third-best options were also glycerol-based. The least sustainable processes were those using molasses, chemically produced ethanol, and soy bean oil. The aggregated results of environmental, economic, and social impacts display waste frying oil as the best-ranked alternative. The bioprocess with sunflower oil in the batch mode ranked second. The least favorable alternatives were the chemically produced ethanol-, soy oil-, refined glycerol-, and molasses-based citric acid production processes. The scenario analysis demonstrated that the environmental impact of nutrients and wastewater treatment is negligible, but energy demand of cultivation and down-stream processing dominated the production process. However, without energy demand the omission of neutralizers almost halves the total impact, and neglecting pasteurization also considerably decreases the environmental impact.

Novel multifunctional ion exchangers for metal ions removal in the presence of citric acid.

The present study deals with the potential application of Purolite S957 and Diphonix Resin® for the removal of rare earth elements from aqueous liquors as a result of the extraction of spent Ni-MH batteries in the presence of citric acid. The effects of the metal ion and the citric acid ratio, pH, ion exchanger dose, contact time, initial concentration and temperature were studied using the batch technique. The Langmuir and Freundlich adsorption isotherm models were used for the description of the adsorption process. The equilibrium adsorption data were fitted using the pseudo first order, pseudo second order, intraparticle diffusion, Boyd, film diffusion and Dumwald-Wagner models. The maximum adsorption capacity q0 obtained from the Langmuir isotherm was found to be 46.63 mg/g for Ni(II) and 60.75 mg/g for La(III) on Purolite S957 as well as 46.55 mg/g for Ni(II) and 60.12 mg/g for La(III) on Diphonix Resin®. The kinetics followed the pseudo second order reaction. Based on the Weber-Morris model the adsorption process proved to proceed in two stages. Based on the Boyd model the rate controlling steps were film and intraparticle diffusions. The adsorption process was spontaneous and endothermic in nature. Reusability of ion exchangers in the desorption studies was also evaluated as a sustainable approach. The physicochemical properties of Purolite S957 and Diphonix Resin® were studied using the ASAP analysis, optical and scanning electron microscopy, potentiometric titration, pHPZC and FT-IR as well as XPS analysis.

Contrasting effects of biochar on N2O emission and N uptake at different N fertilizer levels on a temperate sandy loam.

Biochar has been frequently suggested as an amendment to improve soil quality and mitigate climate change. To investigate the optimal management of nitrogen (N) fertilization, we examined the combined effect of biochar and N fertilizer on plant N uptake and N2O emissions in a cereal rotation system in a randomized two-factorial field experiment on a sandy loam soil in Brandenburg, Germany. The biochar treatment received 10Mgha-1 wood-derived biochar in September 2012. Four levels of N fertilizer, corresponding to 0, 50%, 100%, 130% of the recommended fertilizer level, were applied in winter wheat (Triticum aestivum L.)) and winter rye (Secale cereal L.) in 2013 and 2014 followed by the catch crop oil radish (Raphanus sativus L. var. oleiformis). Biomass and N uptake of winter wheat and winter rye were significantly affected by the level of N fertilizer but not by biochar. For N uptake of oil radish an interaction effect was observed for biochar and N fertilizer. Without applied fertilizer, 39% higher N uptake was found in the presence of biochar, accompanied by higher soil NH4+ content and elevated cumulative CO2 emissions. At 130% of the recommended fertilizer level, 16% lower N uptake and lower cumulative N2O emissions were found in the biochar-mediated treatment. No significant change in abundance of microbial groups and nosZ gene were observed. Our results highlight that biochar can have a greenhouse gas mitigation effect at high levels of N supply and may stimulate nutrient uptake when no N is supplied.

A Comparison of Carbon Footprint and Production Cost of Different Pasta Products Based on Whole Egg and Pea Flour.

Feed and food production are inter alia reasons for high greenhouse gas emissions. Greenhouse gas emissions could be reduced by the replacement of animal components with plant components in processed food products, such as pasta. The main components currently used for pasta are semolina, and water, as well as additional egg. The hypothesis of this paper is that the substitution of whole egg with plant-based ingredients, for example from peas, in such a product might lead to reduced greenhouse gas emissions (GHG) and thus a reduced carbon footprint at economically reasonable costs. The costs and carbon footprints of two pasta types, produced with egg or pea protein, are calculated. Plant protein-based pasta products proved to cause 0.57 kg CO2 equivalents (CO2eq) (31%) per kg pasta less greenhouse gas emissions than animal-based pasta, while the cost of production increases by 10% to 3.00 €/kg pasta.

Effects of different biochars and digestate on N2O fluxes under field conditions.

Field studies that have investigated the effects of char materials on the emission of nitrous oxide (N2O) are still scarce. Therefore, we conducted a field trial with bio- and hydrochars and measured N2O emissions for one whole year. It was hypothesised that the incorporation of chars reduces the emissions of N2O. Chars were produced by pyrolysis and hydrothermal carbonisation (HTC) using either maize silage or wood residues as feedstock. In addition, after production chars were post-treated with digestate in order to accelerate the ageing process of the chars. Chars and digestate were applied to the soil to raise the C content. Emissions of N2O were measured weekly and soil samples for inorganic nitrogen (N) and soil water-content were taken once a month. Additionally, the abundance of functional marker genes from denitrification (nosZ) was determined in October 2012 and in June 2013. The treatment with pure digestate emitted the most N2O compared to the control and char treatments. However, this was significant only in one case. There were no great differences between the char treatments due to high spatial variability and gene abundance of nosZ did not differ between treatments. Overall, emissions of N2O were relatively low. This was attributed to the heterogeneous distribution of the chars and the sandy soils that did not favour the production of N2O. To conclude, the emissions of N2O were mainly influenced by temperature and precipitation and to a minor extent by the type of char and post-treatment.

The influence of oxygen limitation for the production of succinic acid with recombinant strains of Yarrowia lipolytica.

The yeast Yarrowia lipolytica is able to produce high amounts of several organic acids such as pyruvic, citric, isocitric, alpha-ketoglutaric, and succinic acid. Here we report on the influence of the reduced activity of succinate dehydrogenase in Y. lipolytica on its ability to produce succinate. The recombinant strains Y. lipolytica H222-AZ1 and H222-AZ2 were created by exchange of the native promoter of the succinate dehydrogenase subunit 2 encoding gene by inducible promoters. During the cultivation of the strain Y. lipolytica H222-AZ1 in shaking flask experiments, it was found that the promoter exchange resulted in an increase in succinic acid (SA) production. Moreover, it was found that the production of SA depends on an additional limitation of oxygen. Fed-batch cultivations in 1-l bioreactors confirmed this fundamental finding. Y. lipolytica H222-AZ1 produced 2 g l(-1) of SA with oxygen supply and 9.2 g l(-1) under the limitation of oxygen after 165 h. By using a less active promoter in Y. lipolytica H222-AZ2, the production of SA was increased to 25 g l(-1) with a productivity of 0.152 g (l*h)(-1) and a selectivity of 67 % after 165 h. Yields of 2.39 g SA per gram biomass and 0.26 g SA per gram glycerol were found.

Engineering the α-ketoglutarate overproduction from raw glycerol by overexpression of the genes encoding NADP+-dependent isocitrate dehydrogenase and pyruvate carboxylase in Yarrowia lipolytica.

To establish and develop a biotechnological process of α-ketoglutaric acid (KGA) production by Yarrowia lipolytica, it is necessary to increase the KGA productivity and to reduce the amounts of by-products, e.g. pyruvic acid (PA) as major by-product and fumarate, malate and succinate as minor by-products. The aim of this study was the improvement of KGA overproduction with Y. lipolytica by a gene dose-dependent overexpression of genes encoding NADP(+)-dependent isocitrate dehydrogenase (IDP1) and pyruvate carboxylase (PYC1) under KGA production conditions from the renewable carbon source raw glycerol. Recombinant Y. lipolytica strains were constructed, which harbour multiple copies of the respective IDP1, PYC1 or IDP1 and PYC1 genes together. We demonstrated that a selective increase in IDP activity in IDP1 multicopy transformants changes the produced amount of KGA. Overexpression of the gene IDP1 in combination with PYC1 had the strongest effect on increasing the amount of secreted KGA. About 19% more KGA compared to strain H355 was produced in bioreactor experiments with raw glycerol as carbon source. The applied cultivation conditions with this strain significantly reduced the main by-product PA and increased the KGA selectivity to more than 95% producing up to 186 g l(-1) KGA. This proved the high potential of this multicopy transformant for developing a biotechnological KGA production process.

Microbiologically produced carboxylic acids used as building blocks in organic synthesis.

Oxo- and hydroxy-carboxylic acids are of special interest in organic synthesis. However, their introduction by chemical reactions tends to be troublesome especially with regard to stereoselectivity. We describe herein the biotechnological preparation of selected oxo- and hydroxycarboxylic acids under "green" conditions and their use as promising new building blocks. Thereby, our biotechnological goal was the development of process fundamentals regarding the variable use of renewable raw materials, the development of a multi purpose bioreactor and application of a pilot plant with standard equipment for organic acid production to minimize the technological effort. Furthermore the development of new product isolation procedures, with the aim of direct product recovery, capture of products or single step operation, was necessary. The application of robust and approved microorganisms, also genetically modified, capable of using a wide range of substrates as well as producing a large spectrum of products, was of special importance. Microbiologically produced acids, like 2-oxo-glutaric acid and 2-oxo-D-gluconic acid, are useful educts for the chemical synthesis of hydrophilic triazines, spiro-connected heterocycles, benzotriazines, and pyranoic amino acids. The chiral intermediate of the tricarboxylic acid cycle, (2R,3S)-isocitric acid, is another promising compound. For the first time our process provides large quantities of enantiopure trimethyl (2R,3S)-isocitrate which was used in subsequent chemical transformations to provide new chiral entities for further usage in total synthesis and pharmaceutical research.Oxo- and hydroxy-carboxylic acids are of special interest in organic synthesis. However, their introduction by chemical reactions tends to be troublesome especially with regard to stereoselectivity. We describe herein the biotechnological preparation of selected oxo- and hydroxycarboxylic acids under "green" conditions and their use as promising new building blocks. Thereby, our biotechnological goal was the development of process fundamentals regarding the variable use of renewable raw materials, the development of a multi purpose bioreactor and application of a pilot plant with standard equipment for organic acid production to minimize the technological effort. Furthermore the development of new product isolation procedures, with the aim of direct product recovery, capture of products or single step operation, was necessary. The application of robust and approved microorganisms, also genetically modified, capable of using a wide range of substrates as well as producing a large spectrum of products, was of special importance. Microbiologically produced acids, like 2-oxo-glutaric acid and 2-oxo-D-gluconic acid, are useful educts for the chemical synthesis of hydrophilic triazines, spiro-connected heterocycles, benzotriazines, and pyranoic amino acids. The chiral intermediate of the tricarboxylic acid cycle, (2R,3S)-isocitric acid, is another promising compound. For the first time our process provides large quantities of enantiopure trimethyl (2R,3S)-isocitrate which was used in subsequent chemical transformations to provide new chiral entities for further usage in total synthesis and pharmaceutical research.

Variation of the by-product spectrum during α-ketoglutaric acid production from raw glycerol by overexpression of fumarase and pyruvate carboxylase genes in Yarrowia lipolytica.

The yeast Yarrowia lipolytica secretes high amounts of various organic acids, like citric, isocitric, pyruvic (PA), and α-ketoglutaric (KGA) acids, triggered by growth limitation and excess of carbon source. This is leading to an increased interest in this non-conventional yeast for biotechnological applications. To improve the KGA production by Y. lipolytica for an industrial application, it is necessary to reduce the amounts of by-products, e.g., fumarate (FU) and PA, because production of by-products is a main disadvantage of the KGA production by this yeast. We have examined whether the concentration of secreted organic acids (main product KGA and PA as major by-product and FU, malate (MA), and succinate (SU) as minor by-products) can be influenced by a gene-dose-dependent overexpression of fumarase (FUM) or pyruvate carboxylase (PYC) genes under KGA production conditions. Recombinant Y. lipolytica strains were constructed, which harbor multiple copies of the respective FUM1, PYC1 or FUM1, and PYC1 genes. Overexpression of the genes FUM1 and PYC1 resulted in strongly increased specific enzyme activities during cultivation of these strains on raw glycerol as carbon source in bioreactors. The recombinant Y. lipolytica strains showed different product selectivity of the secreted organic acids KGA, PA, FU, MA, and SU. Concentrations of the by-products FU, MA, SU, and PA decreased significantly at overproduction of FUM and increased at overproduction of PYC and also of FUM and PYC simultaneously. In contrast, the production of KGA with the multicopy strains H355A(FUM1) and H355A(FUM1-PYC1) was comparable with the wild-type strain H355 or slightly lower in case of H355(PYC1). KGA productivity was not changed significantly compared with strain H355 whereas product selectivity of the main product KGA was increased in H355A(FUM1).

Substrate utilization by recombinant Yarrowia lipolytica growing on sucrose.

We report the study of the dynamics of substrate utilization by the genetic modified strain Yarrowia lipolytica H222-S4(p67ICL1) T5. In contrast to its wild-type equivalent, this recombinant strain is able to excrete the sucrose cleaving enzyme invertase. Both the sucrose degradation rate and the glucose and fructose consumption rate have been investigated. In all experiments, satisfied amounts of invertase were produced so that all sucrose was cleaved into its monomers. While glucose and fructose as sole carbon sources were consumed with the same uptake rate, a clear preference for glucose uptake was detected in cultivations with sucrose as sole carbon source or mixed substrates when compared with fructose. Nevertheless, no real diauxie could be observed because of partly simultaneous consumption of both monosaccharides. Fructose being present in the cultivation medium at the beginning of the fermentation led to the retardation of glucose uptake. This effect was observed for various fructose starting concentrations in the range of 5-85 g/l.

Repeated fed-batch fermentation using biosensor online control for citric acid production by Yarrowia lipolytica.

Biosensor-controlled substrate feeding was used in a citric acid production process with the yeast strain Yarrowia lipolytica H222 with glucose as the carbon source. The application of an online glucose biosensor measurement facilitated the performance of long-time repeated fed-batch process with automated bioprocess control. Ten cycles of repeated fed-batch fermentation were carried out in order to validate both the stability of the microorganism for citric acid production and the robustness of the glucose biosensor in a long-time experiment. In the course of this fermentation with a duration of 553 h, a slight loss of productivity from 1.4 g/(L×h) to 1.1 g/(L×h) and of selectivity for citric acid from 91% to 88% was observed. The glucose biosensor provided 6,227 measurements without any loss of activity.

Overexpression of alpha-ketoglutarate dehydrogenase in Yarrowia lipolytica and its effect on production of organic acids.

The yeast Yarrowia lipolytica is one of the most intensively studied "non-conventional" yeast species. Its ability to secrete various organic acids, like pyruvic (PA), citric, isocitric, and alpha-ketoglutaric (KGA) acid, in large amounts is of interest for biotechnological applications. We have studied the effect of the alpha-ketoglutarate dehydrogenase (KGDH) complex on the production process of KGA. Being well studied in Saccharomyces cerevisiae this enzyme complex consists of three subunits: alpha-ketoglutarate dehydrogenase, dihydrolipoyl transsuccinylase, and lipoamide dehydrogenase. Here we report the effect of overexpression of these subunits encoding genes and resulting increase of specific KGDH activity on organic acid production under several conditions of growth limitation and an excess of carbon source in Y. lipolytica. The constructed strain containing multiple copies of all three KGDH genes showed a reduced production of KGA and an elevated production of PA under conditions of KGA production. However, an increased activity of the KGDH complex had no influence on organic acid production under citric acid production conditions.

Citric acid production from sucrose using a recombinant strain of the yeast Yarrowia lipolytica.

The yeast Yarrowia lipolytica is able to secrete high amounts of several organic acids under conditions of growth limitation and carbon source excess. Here we report the production of citric acid (CA) in a fed-batch cultivation process on sucrose using the recombinant Y. lipolytica strain H222-S4(p67ICL1) T5, harbouring the invertase encoding ScSUC2 gene of Saccharomyces cerevisiae under the inducible XPR2 promoter control and multiple ICL1 copies (10-15). The pH-dependent expression of invertase was low at pH 5.0 and was identified as limiting factor of the CA-production bioprocess. The invertase expression was sufficiently enhanced at pH 6.0-6.8 and resulted in production of 127-140 g l(-1) CA with a yield Y (CA) of 0.75-0.82 g g(-1), whereas at pH 5.0, 87 g l (-1) with a yield Y (CA) of 0.51 g g(-1) were produced. The CA-productivity Q (CA) increased from 0.40 g l (-1) h(-1) at pH 5.0 up to 0.73 g l (-1) h(-1) at pH 6.8. Accumulation of glucose and fructose at high invertase expression level at pH 6.8 indicated a limitation of CA production by sugar uptake. The strain H222-S4(p67ICL1) T5 also exhibited a gene-dose-dependent high isocitrate lyase expression resulting in strong reduction (<5%) of isocitric acid, a by-product during CA production.

Effect of sildenafil (Viagra) on cerebral blood flow velocity: a pilot study.

Sildenafil (Viagra) is a selective inhibitor of the cyclic guanosine monophosphate (cGMP) specific phosphodiesterase 5 (PDE5) used for the oral treatment of male erectile dysfunction due to vasodilation. However, assessment of direct effects of sildenafil on cerebral arteries is unknown. The aim of this present experiment is to measure the possible changes in blood flow velocity of the middle cerebral artery after the administration of sildenafil and a placebo. Blood-flow velocity changes of the middle cerebral artery were measured before and 1 to 2 h after the administration of the drug. Neither the intake of sildenafil nor the application of placebo resulted in any significant changes in blood flow velocity of the right middle cerebral artery. The next step for future experiments will be in vitro measurement of the diameters of the cerebral arteries under the influence of sildenafil and in vivo measurement of carbon dioxide and cerebral blood flow velocity during sexual stimulation after sildenafil intake instead of under resting conditions.