Real-Time Monitoring of the Yeast Intracellular State During Bioprocesses With a Toolbox of Biosensors

Industrial fermentation processes strive for high robustness to ensure optimal and consistent performance. Medium components, fermentation products, and physical perturbations may cause stress and lower performance. Cellular stress elicits a range of responses, whose extracellular manifestations have been extensively studied; whereas intracellular aspects remain poorly known due to lack of tools for real-time monitoring. Genetically encoded biosensors have emerged as promising tools and have been used to improve microbial productivity and tolerance toward industrially relevant stresses. Here, fluorescent biosensors able to sense the yeast intracellular environment (pH, ATP levels, oxidative stress, glycolytic flux, and ribosome production) were implemented into a versatile and easy-to-use toolbox. Marker-free and efficient genome integration at a conserved site on chromosome X of Saccharomyces cerevisiae strains and a commercial Saccharomyces boulardii strain was developed. Moreover, multiple biosensors were used to simultaneously monitor different intracellular parameters in a single cell. Even when combined together, the biosensors did not significantly affect key physiological parameters, such as specific growth rate and product yields. Activation and response of each biosensor and their interconnection were assessed using an advanced micro-cultivation system. Finally, the toolbox was used to screen cell behavior in a synthetic lignocellulosic hydrolysate that mimicked harsh industrial substrates, revealing differences in the oxidative stress response between laboratory (CEN.PK113-7D) and industrial (Ethanol Red) S. cerevisiae strains. In summary, the toolbox will allow both the exploration of yeast diversity and physiological responses in natural and complex industrial conditions, as well as the possibility to monitor production processes.

Eukaryotic Parasites Are Integral to a Productive Microbial Food Web in Oxygen-Depleted Waters

Oxygen-depleted water columns (ODWCs) host a diverse community of eukaryotic protists that change dramatically in composition over the oxic-anoxic gradient. In the permanently anoxic Cariaco Basin, peaks in eukaryotic diversity occurred in layers where dark microbial activity (chemoautotrophy and heterotrophy) were highest, suggesting a link between prokaryotic activity and trophic associations with protists. Using 18S rRNA gene sequencing, parasites and especially the obligate parasitic clade, Syndiniales, appear to be particularly abundant, suggesting parasitism is an important, but overlooked interaction in ODWC food webs. Syndiniales were also associated with certain prokaryotic groups that are often found in ODWCs, including Marinimicrobia and Marine Group II archaea, evocative of feedbacks between parasitic infection events, release of organic matter, and prokaryotic assimilative activity. In a network analysis that included all three domains of life, bacterial and archaeal taxa were putative bottleneck and hub species, while a large proportion of edges were connected to eukaryotic nodes. Inclusion of parasites resulted in a more complex network with longer path lengths between members. Together, these results suggest that protists, and especially protistan parasites, play an important role in maintaining microbial food web complexity, particularly in ODWCs, where protist diversity and microbial productivity are high, but energy resources are limited relative to euphotic waters.

Glacial lake outburst floods enhance benthic microbial productivity in perennially ice-covered Lake Untersee (East Antarctica)

Benthic ecosystems of perennially ice-covered lakes in Antarctica are highly sensitive to climate-driven changes. Lake Untersee has been in hydrological steady-state for several hundred years with a high pH water column and extremely low levels of dissolved inorganic carbon. Here, we show that glacial lake outburst floods can replenish carbon dioxide-depleted lakes with carbon, enhancing phototrophic activity of the benthic ecosystem. In 2019, a glacial lake outburst flood brought 17.5 million m3 of water to Lake Untersee, the most substantial reported increase for any surface lake in Antarctica. High-resolution grain-size and carbon isotope analyses of microbial mats suggest that glacial lake outburst floods have occurred periodically over the Holocene and help explain the complex patterns of carbon cycling and sequestration observed in the lake. Our findings suggest that periodic flooding events may provide biological stimuli to other carbon dioxide-depleted Antarctic ecosystems and perhaps even icy lakes on early Mars.

Upper Ordovician (Sandbian–Katian) carbonate outliers in the northern Ottawa–Bonnechere graben (central Canada): records of transgressions and sedimentation patterns in the Laurentian platform interior

Small Ordovician sedimentary outliers, including Brent Crater, within the northern Ottawa–Bonnechere graben are remnants of a once expansive Upper Ordovician sedimentary cover extending across the southern Canadian Shield. Facies successions along with updated macrofossil and conodont biostratigraphy, and isotope (C, O, Sr) chemostratigraphy provide additional insights into the terrestrial-to-marine transformation, carbonate-platform development, and oceanographic communication across the southern Laurentian platform. Four of the outliers document Sandbian shoreline-to-nearshore deposition: near Deux Rivières, Manitou Islands, the upper part of the Brent Crater sedimentary fill, and at nearby Cedar Lake. Marine transgression initially reworked local fine-grained to boulder-rich regolith within high-energy shoreface siliciclastic environments that gave way to low- to high-energy inner carbonate-ramp setting. Continued transgression resulted in more offshore rhythmic and diverse lithofacies successions defining mixed heterozoan, photozoan, and microbial productivity and marine isotope (C, Sr) signatures, but δ13C excursions suggest periods of greater mixing of terrestrial and marine carbon reservoirs. Lower Katian strata are preserved near Lake Nipissing and characterize deepening from high-energy ooid-heterozoan skeletal shoals to deeper water mid-ramp siliciclastics and skeletal carbonates, host to a Cruziana ichnofacies. An upsection decline in δ13C values through this succession may identify deposition during the post-peak decline of the global Guttenberg δ13C excursion. This lithic succession fits well with contemporary expansion of heterozoan skeletal lithofacies across the Laurentian platform, yet the presence of ooids identifies prevailing warm waters within the platform interior during early stages of transgression.

PERBAIKAN BIOPROSES UNTUK PENINGKATAN PRODUKSI BIOETANOL DARI MOLASE TEBU / Bioprocess Improvement for Enhanching Bioethanol Production of Sugarcane Molase

<p align="center">ABSTRAK </p><p>Bioetanol merupakan salah satu bahan bakar alternatif yang strategis untuk dikembangkan. Salah satu substrat yang menjanjikan untuk digunakan adalah molase. Molase merupakan hasil samping industri gula kristal tebu yang masih mengandung gula yaitu sekitar 45-54,6%. Bioetanol dari molase tebu berpotensi untuk dikembangkan karena sangat menguntungkan, pasokan cukup besar, tersedianya teknologi proses, serta tidak bersaing dengan pangan. Tulisan ini mengulas hasil-hasil penelitian dan implikasinya tentang bahan baku, proses, lingkungan yang berpengaruh serta strategi untuk meningkatkan produktivitas bioetanol dari molase tebu melalui rekayasa proses fermentasi. Pada pembuatan etanol, fermentasi merupakan proses yang memegang peranan penting. Pengaturan lingkungan fermentasi seperti suhu, pH, dan tekanan berpengaruh terhadap bioproses dalam fermentasi. Begitu pula penambahan bahan suplemen seperti gula, garam, dan ion logam menurut jenis dan konsentrasi yang tepat juga dapat mengoptimalkan proses fermentasi. Selain pengelolaan lingkungan dan penambahan bahan suplemen<span style="text-decoration: underline;">, s</span>trategi untuk peningkatan produktivitas bioetanol dari molase dapat dilakukan dengan: 1) penggunaan mikrobia selain <em>Saccharomyces cerevisiae</em>; 2) <em>pretreatment</em>; dan 3) metode fermentasi kontinyu. Penggunaan mikrobia selain <em>Saccharomyces cerevisiae</em>, seperti <em>Zymomonas mobilis</em> dapat meningkatkan produktivitas etanol hingga 55,8 g/L atau 27,9% dari total gula reduksi. Perlakuan <em>pretreatment</em> dapat meningkatkan produktivitas mikrobia dalam mengkonversi gula menjadi etanol, sedangkan penggunaan metode fermentasi secara kontinyu dapat meningkatkan produktivitas sebesar <span style="text-decoration: underline;">+</span> 4.75 g/L/jam.</p><p> </p><p align="center">ABSTRACT </p><p>Bioethanol is one of strategic alternative fuel to develop. One of substrate that promises to be used is molasses. Molasses is by-product of sugar industry which contain of sugar about 45-54,6%. Bioethanol from sugarcane molase is necessary to develope because it is very profitable, large supply, availability technology, and no-competion to food. This paper was aimed to reviews some research results and their implications on raw materials, processes, advanced environments and strategies to increas bioethanol productivity of molasses through the fermentation process engineering. In the manufacture of ethanol, fermentation is an important holding process. In ethanol production, fermentation plays an important role. Fermentation environments arragement such as temperature, pH, and pressure can effect on bioprocess of fermentation. Similarly, the addition of supplemental ingredients such as sugar, salt, and metal ions by appropriate type and concentration can also optimize the fermentation process. In addition to environmental arrangement and supplemental adding, strategies to improve bioethanol productivity of molasses can be accomplished by 1) the use of microbes other than Saccharomyces cerevisiae; 2) pretreatment; and 3) continuous fermentation method. The use of microbes other than Saccharomyces cerevisiae, such as Zymomonas mobilis can increase ethanol productivity up to 55.8 g / L or 27.9% of total sugar reduction. Pretreatment can increase microbial productivity in converting sugar to ethanol, while continuous use of fermentation method can increase productivity by <span style="text-decoration: underline;">+</span> 4.75 g / L / hr.</p><p> </p>

Proteomic and Transcriptomic Analyses of “Candidatus Pelagibacter ubique” Describe the First PII-Independent Response to Nitrogen Limitation in a Free-Living Alphaproteobacterium

ABSTRACTNitrogen is one of the major nutrients limiting microbial productivity in the ocean, and as a result, most marine microorganisms have evolved systems for responding to nitrogen stress. The highly abundant alphaproteobacterium “CandidatusPelagibacter ubique,” a cultured member of the orderPelagibacterales(SAR11), lacks the canonical GlnB, GlnD, GlnK, and NtrB/NtrC genes for regulating nitrogen assimilation, raising questions about how these organisms respond to nitrogen limitation. A survey of 266Alphaproteobacteriagenomes found these five regulatory genes nearly universally conserved, absent only in intracellular parasites and members of the orderPelagibacterales, including “Ca. Pelagibacter ubique.” Global differences in mRNA and protein expression between nitrogen-limited and nitrogen-replete cultures were measured to identify nitrogen stress responses in “Ca.Pelagibacter ubique” strain HTCC1062. Transporters for ammonium (AmtB), taurine (TauA), amino acids (YhdW), and opines (OccT) were all elevated in nitrogen-limited cells, indicating that they devote increased resources to the assimilation of nitrogenous organic compounds. Enzymes for assimilating amine into glutamine (GlnA), glutamate (GltBD), and glycine (AspC) were similarly upregulated. Differential regulation of the transcriptional regulator NtrX in the two-component signaling system NtrY/NtrX was also observed, implicating it in control of the nitrogen starvation response. Comparisons of the transcriptome and proteome supported previous observations of uncoupling between transcription and translation in nutrient-deprived “Ca.Pelagibacter ubique” cells. Overall, these data reveal a streamlined, PII-independent response to nitrogen stress in “Ca.Pelagibacter ubique,” and likely otherPelagibacterales, and show that they respond to nitrogen stress by allocating more resources to the assimilation of nitrogen-rich organic compounds.IMPORTANCEPelagibacteralesare extraordinarily abundant and play a pivotal role in marine geochemical cycles, as one of the major recyclers of labile dissolved organic matter. They are also models for understanding how streamlining selection can reshape chemoheterotroph metabolism. Streamlining and its broad importance to environmental microbiology are emerging slowly from studies that reveal the complete genomes of uncultured organisms. Here, we report another remarkable example of streamlined metabolism inPelagibacterales, this time in systems that control nitrogen assimilation.Pelagibacteralesare major contributors to metatranscriptomes and metaproteomes from ocean systems, where patterns of gene expression are used to gain insight into ocean conditions and geochemical cycles. The data presented here supply background that is essential to interpreting data from field studies.

Development of Four Unit Processes for Biobased PLA Manufacturing

Polylactide (PLA), which is one of the most important biocompatible polyesters that are derived from annually renewable biomass such as corn and sugar beets, has attracted much attention for automotive parts application. The manufacturing method of PLA is the ring-opening polymerization of the dimeric cyclic ester of lactic acid, lactide. For the stereocomplex PLA, we developed the four unit processes, fermentation, separation, lactide conversion, and polymerization. Fermentation of sugars to D-lactic acid is little studied, and its microbial productivity is not well known. Therefore, we investigated D-lactic acid fermentation with a view to obtaining the strains capable of producing D-lactic acid, and we got a maximum lactic acid production 60 g/L. Lactide is prepared by a two-step process: first, the lactic acid is converted into oligo(lactic acid) by a polycondensation reaction; second, the oligo(lactic acid) is thermally depolymerized to form the cyclic lactide via an unzipping mechanism. Through catalyst screening test for polycondensation and depolymerization reactions, we got a new method which shortens the whole reaction time 50% the level of the conventional method. Poly(L-lactide) was obtained from the ring-opening polymerization of L-lactide. We investigated various catalysts and polymerization conditions. Finally, we got the best catalyst system and the scale-up technology.

Organic matter content and quality in supraglacial debris across the ablation zone of the Greenland ice sheet

Quantifying the biogeochemical cycling of carbon in glacial ecosystems is of great significance for regional, and potentially global, carbon flow estimations. The concentration and quality of organic carbon (OC) is an important indicator of biogeochemical and physical processes that prevail in an ice-sheet ecosystem. Here we determine the content and quality of OC in debris from the surface of the Greenland ice sheet (GrIS) using microscopic, chromatographic, spectrophotometric and high-temperature combustion techniques. The total OC content in the debris increased with distance from the edge of the ice sheet, from virtually zero to >6% dry weight at 50 km inland, and there was a peak in the carbohydrate proportion and the microbial abundance at ∼6km inland. The highest (galactose + mannose)/(arabinose + xylose) ratios, indicating maximum autochthonous microbial production, were found at >10km inland. We propose that three key processes influence the carbon cycling on the GrIS: aeolian input of microbial inoculum and nutrients, in situ biological C transformation and the wash-away of supraglacial debris by meltwaters. We show that all these processes have significant spatial variability. While the total OC content of the debris on the ice sheet is probably controlled by the physical processes of wind transport and wash-away by meltwater, the microbial abundance and the quantity of the labile cell-contained OC within the debris is likely to be driven by the balance between the wash-away and the microbial productivity.