Reactor microbiome enriches vegetable oil with n-caproate and n-caprylate for potential functionalized feed additive production via extractive lactate-based chain elongation

Background Biotechnological processes for efficient resource recovery from residual materials rely on complex conversions carried out by reactor microbiomes. Chain elongation microbiomes produce valuable medium-chain carboxylates (MCC) that can be used as biobased starting materials in the chemical, agriculture and food industry. In this study, sunflower oil is used as an application-compatible solvent to accumulate microbially produced MCC during extractive lactate-based chain elongation. The MCC-enriched solvent is harvested as a potential novel product for direct application without further MCC purification, e.g., direct use for animal nutrition. Sunflower oil biocompatibility, in situ extraction performance and effects on chain elongation were evaluated in batch and continuous experiments. Microbial community composition and dynamics of continuous experiments were analyzed based on 16S rRNA gene sequencing data. Potential applications of MCC-enriched solvents along with future research directions are discussed. Results Sunflower oil showed high MCC extraction specificity and similar biocompatibility to oleyl alcohol in batch extractive fermentation of lactate and food waste. Continuous chain elongation microbiomes produced the MCC n-caproate (nC6) and n-caprylate (nC8) from l-lactate and acetate at pH 5.0 standing high undissociated n-caproic acid concentrations (3 g L−1). Extractive chain elongation with sunflower oil relieved apparent toxicity of MCC and production rates and selectivities reached maximum values of 5.16 ± 0.41 g nC6 L−1 d−1 (MCC: 11.5 g COD L−1 d−1) and 84 ± 5% (e− eq MCC per e− eq products), respectively. MCC were selectively enriched in sunflower oil to concentrations up to 72 g nC6 L−1 and 3 g nC8 L−1, equivalent to 8.3 wt% in MCC-enriched sunflower oil. Fermentation at pH 7.0 produced propionate and n-butyrate instead of MCC. Sunflower oil showed stable linoleic and oleic acids composition during extractive chain elongation regardless of pH conditions. Reactor microbiomes showed reduced diversity at pH 5.0 with MCC production linked to Caproiciproducens co-occurring with Clostridiumtyrobutyricum, Clostridiumluticellarii and Lactobacillus species. Abundant taxa at pH 7.0 were Anaerotignum, Lachnospiraceae and Sporoanaerobacter. Conclusions Sunflower oil is a suitable biobased solvent to selectively concentrate MCC. Extractive reactor microbiomes produced MCC with improved selectivity and production rate, while downstream processing complexity was reduced. Potential applications of MCC-enriched solvents may include feed, food and biofuels purposes.

In situ extraction (milking) of the two promising Botryococcus braunii strains Showa and Bot22 under optimized extraction time

In situ extraction or “milking” of microalgae is a promising approach to reduce downstream costs in order to produce low-value substances such as lipids from microalgae in an economical way. Due to its ability to secrete high amounts of long-chain hydrocarbons to an extracellular matrix, the green microalga Botryococcus braunii is suitable for the process of in situ extraction as the cost intensive steps of harvesting, dewatering, and cell disruption could be omitted. Based on a previous study investigating various B. braunii strains in terms of growth, lipid accumulation, and solvent compatibility, the B. braunii strains Showa and Bot22 (both B race) were identified as potential candidates for the process of in situ extraction. In order to prove the suitability of these two strains for the process of in situ extraction, this study first determined the optimal extraction time using short-term in situ extraction over 7 days at different starting biomass concentrations of 1.5 and 2.5 g L−1. Furthermore, both strains were treated applying the optimal extraction time in long-term in situ extractions for 30 days to confirm the results from the short-term extractions. The results indicate a strain-dependent optimal extraction time of 300 min day−1 for strain Showa and 200 min day−1 for strain Bot22. During long-term in situ extraction for 30 days, hydrocarbon productivity was 16.99 mg L−1 day−1 (10.53 mg gDW−1 day−1) for strain Showa and 14.53 mg L−1 day−1 (10.48 mg gDW−1 day−1) for strain Bot22. Furthermore, a direct correlation between hydrocarbon productivity achieved by in situ extraction and the hydrocarbon concentration in the biomass of the respective strain could be established. It could be shown that the consideration of the effective extraction time and the phase boundary area is required to calculate an extraction system independent value for the comparison of different extraction setups.

Tailor-Made Workflow to Understand and Forecast H2S Production Risks in Thermal Projects

In-situ extraction of heavy sulfured oil based on steam injection comes with a high level of risk in terms of H2S production, resulting from aquathermolysis reactions. This could lead to on-site living being casualties, environment damage, surface facilities and wells corrosion. Also, there is a strong need to understand aquathermolysis reactions and to forecast acid gases generation in such context. For that aim a tailor-made workflow was developed to estimate H2S concentration at the well-head. To meet these challenges, a 3-steps approach combining laboratory studies and numerical predictions has been developed. It firstly consists in a fast preliminary assessment of the highest H2S risk areas, based on the measurements of sulfur characteristics of reservoir core samples using our Rock-Eval Sulfur set-up. Then aquathermolysis experiments from the previously selected core samples are conducted in order to calibrate the compositional chemical model of the reservoir simulator. The latter is eventually used to carry out thermal compositional reactive simulations at field scale. The reservoir simulator allows simulations of SAGD processes and handles H2S distribution over oil/water/gas phases and its migration in these phases. Simulation results show that acid gases are generated within the steam chamber, before they accumulate at the chamber edges where they dissolve in the water and oil phases. This contributes to reduce viscosity, allowing the oil to flow along the steam chamber edges before it has reached the steam temperature. Therefore the H2S produced at surface is mainly carried towards the wells by water and to a lesser extent oil. This flowing oil has not reacted with the steam: its composition is close to the initial reservoir oil but enriched with dissolved gases. The steam chamber shape, the temperature distribution and the H2S produced at surface are strongly modified when heterogeneities are introduced in the reservoir model. Synthetic cases allow a deeper understanding of the effects of heterogeneities. Vertical permeability is thus found to be a key factor of H2S production variations. When steam reaches a lower permeability lithology, a delayed rise in H2S production at wellhead is observed as aquathermolysis reactions rates increase. Finally Foam Assisted-SAGD has been considered. While the foam improves the Steam-Oil ratio, no clear improvement was observed regarding the H2S production.

Fermentative production of enantiopure (S)-linalool using a metabolically engineered Pantoea ananatis

Background Linalool, an acyclic monoterpene alcohol, is extensively used in the flavor and fragrance industries and exists as two enantiomers, (S)- and (R)-linalool, which have different odors and biological properties. Linalool extraction from natural plant tissues suffers from low product yield. Although linalool can also be chemically synthesized, its enantioselective production is difficult. Microbial production of terpenes has recently emerged as a novel, environmental-friendly alternative. Stereoselective production can also be achieved using this approach via enzymatic reactions. We previously succeeded in producing enantiopure (S)-linalool using a metabolically engineered Pantoea ananatis, a member of the Enterobacteriaceae family of bacteria, via the heterologous mevalonate pathway with the highest linalool titer ever reported from engineered microbes. Results Here, we genetically modified a previously developed P. ananatis strain expressing the (S)-linalool synthase (AaLINS) from Actinidia arguta to further improve (S)-linalool production. AaLINS was mostly expressed as an insoluble form in P. ananatis; its soluble expression level was increased by N-terminal fusion of a halophilic β-lactamase from Chromohalobacter sp. 560 with hexahistidine. Furthermore, in combination with elevation of the precursor supply via the mevalonate pathway, the (S)-linalool titer was increased approximately 1.4-fold (4.7 ± 0.3 g/L) in comparison with the original strain (3.4 ± 0.2 g/L) in test-tube cultivation with an aqueous-organic biphasic fermentation system using isopropyl myristate as the organic solvent for in situ extraction of cytotoxic and semi-volatile (S)-linalool. The most productive strain, IP04S/pBLAAaLINS-ispA*, produced 10.9 g/L of (S)-linalool in “dual-phase” fed-batch fermentation, which was divided into a growth-phase and a subsequent production-phase. Thus far, this is the highest reported titer in the production of not only linalool but also all monoterpenes using microbes. Conclusions This study demonstrates the potential of our metabolically engineered P. ananatis strain as a platform for economically feasible (S)-linalool production and provides insights into the stereoselective production of terpenes with high efficiency. This system is an environmentally friendly and economically valuable (S)-linalool production alternative. Mass production of enantiopure (S)-linalool can also lead to accurate assessment of its biological properties by providing an enantiopure substrate for study.

Occurrence of Pharmaceutical Compounds in Groundwater from the Gran Canaria Island (Spain)

The presence of pharmaceutical compounds in the whole environment is a growing concern. These compounds might be present in the effluents of wastewater treatment plants and, hence, irrigation with treated sewage may be a source of groundwater pollution. The volcanic aquifer that lies NE of Gran Canaria (Spain) was studied to address the relationship of the occurrence of pharmaceutical compounds and a golf course that has been irrigated with regenerated water since 1973. Of the 14 analyzed groundwater samples, five wells were chosen to perform annual monitoring. Irrigation water and soil leachate were also evaluated. The target analytes were atenolol, metamizole, fluoxetine, ibuprofen, nicotine, permethrin, caffeine, and their metabolite paraxanthine. The environmental risk is limited as the concentrations of the pharmaceuticals measured in the sampled wells were always below 60 ng·L−1 (lower than the detected caffeine and nicotine concentrations). Wide variations for the same wells were measured among sampling campaigns, and also among the different wells. The study points to the importance of sample conservation during transport and the need to perform analyses immediately, or to follow an in-situ extraction procedure to carry concentrated samples under better conditions.