Thermocaproicibacter melissae gen. nov., sp. nov., a thermophilic chain-elongating bacterium, producing n-caproate from polymeric carbohydrates.

Strain MDTJ8T is a chain-elongating thermophilic bacterium isolated from a thermophilic acidogenic anaerobic digestor treating human waste while producing the high commodity chemical n-caproate. The strain grows and produces formate, acetate, n-butyrate, n-caproate and lactate from mono-, di- and polymeric saccharides at 37-60 °C (optimum, 50-55 °C) and at pH 5.0-7.0 (optimum, pH 6.5). The organism is an obligate anaerobe, is motile and its cells form rods (0.3-0.5×1.0-3.0 µm) that stain Gram-positive and occur primarily as chains. Phylogenetic analysis of both the 16S rRNA gene and full genome sequence shows that strain MDTJ8T belongs to a group that consists of mesophylic chain-elongating bacteria within the family Oscillospiraceae, being nearest to Caproicibacter fermentans EA1T (94.8 %) and Caproiciproducens galactitolivorans BS-1T (93.7 %). Its genome (1.96 Mbp) with a G+C content of 49.6 mol% is remarkably smaller than those of other chain-elongating bacteria of the family Oscillospiraceae. Pairwise average nucleotide identity and DNA-DNA hybridization values between strain MDJT8T and its mesophilic family members are less than 70 and 35 %, respectively, while pairwise average amino acid identity values are less than 68 %. In addition, strain MDJT8T uses far less carbohydrate and non-carbohydrate substrates compared to its nearest family members. The predominant cellular fatty acids of strain MDTJ8T are C14 : 0, C14 : 0 DMA (dimethyl acetal) and C16 : 0, while its polar lipid profile shows three unidentified glycophospholipids, 11 glycolipids, 13 phospholipids and six unidentified lipids. No respiratory quinones and polyamines are detected. Based on its phylogenetic, genotypic, morphological, physiological, biochemical and chemotaxonomic characteristics, strain MDTJ8T represents a novel species and novel genus of the family Oscillospiraceae and Thermocaproicibacter melissae gen. nov., sp. nov. is proposed as its name. The type strain is MDTJ8T (=DSM 114174T=LMG 32615T=NCCB 100883T).

Isolation and characterization of a thermophilic chain elongating bacterium that produces the high commodity chemical n-caproate from polymeric carbohydrates.

A thermophilic chain elongating bacterium, strain MDTJ8, was isolated from a thermophilic acidogenic anaerobic digestor producing n-caproate from human waste, growing optimally at 50-55 °C and pH 6.5. 16S rRNA gene analysis suggests that MDTJ8 represents a new species/genus within a group currently composed of mesophilic chain elongators of the Oscillospiraceae family. Genome analysis showed that strain MDTJ8 contains homologues of genes encoding for chain elongation and energy conservation but also indicated n-caproate production from carbohydrates including polymeric substances. This was confirmed by culturing experiments in which MDTJ8 converted, at pH 6.5 and 55 °C, mono-, di- and polymeric carbohydrates (starch and hemicellulose) to n-caproate reaching concentrations up to 283 mg/L and accounting for up to 10 % of the measured fermentation products. MDTJ8 is the first axenic organism that thermophilically performs chain elongation, opening doors to understand and intensify thermophilic bioprocesses targeting anaerobic digestion towards the production of the value-added chemical n-caproate.

Limnospira indica PCC8005 growth in photobioreactor: model and simulation of the ISS and ground experiments.

The Arthrospira-B experiment is the first experiment in space ever allowing the online measurements of both oxygen production rate and growth rate of Limnospira indica PCC8005 in batch photobioreactors running on-board ISS. Four bioreactors were integrated in the ISS Biolab facility. Each reactor was composed of two chambers (gas and liquid) separated by a PTFE membrane and was run in batch conditions. Oxygen production was monitored by online measurement of the total pressure increase in the gas chamber. The experiments are composed of several successive batch cultures for each reactor, performed in parallel on ISS and on ground. In this work, a model for the growth of the cyanobacterium Limnospira indica PCC8005 (also known as Arthrospira or spirulina) in these space membrane photobioreactors was proposed and the simulation results obtained are compared to the experimental results gathered in space and on ground. The photobioreactor model was based on a light transfer limitation model, already used to describe and predict the growth and oxygen production in small to large scale ground photobioreactors. It was completed by a model for pH prediction in the liquid phase allowing assessment of the pH increase associated to the bicarbonate consumption for the biomass growth. A membrane gas-liquid transfer model is used to predict the gas pressure increase in the gas chamber. Substrate limitation is considered in the biological model. A quite satisfactory fit was achieved between experimental and simulation results when a suitable mixing of the liquid phase was maintained. The data showed that microgravity has no first order effect on the oxygen production rate of Limnospira indica PCC8005 in a photobioreactor operating in space in zero gravity conditions.

Assessment of transient effects of alternative nitrogen sources in continuous cultures of Arthrospira sp. using proteomic, modeling and biochemical tools.

The ability of cyanobacterium Arthrospira sp. to assimilate waste nitrogen sources (ammonium and urea) makes it an important candidate for wastewater management. The aim of this work was to evaluate a cultivation approach based on continuous-transitional-feeding regime (nitrate-ammonium-nitrate) in a photobioreactor to assess the effects of ammonium salts on Arthrospira sp. PCC 8005 metabolism. Using a comprehensive biochemical, proteomic and stoichiometric profiling of biomass, this study demonstrated that the proposed cultivation approach could increase the proteins and pigments yields by 20-30%, compared to the respective yields obtained from wild-type Arthrospira sp. strain A light-energy-transfer model was used to predict the biomass and oxygen productivities of Arthrospira sp. cultivated under transitional-feeding regime. 95 ±â€¯2% match was observed between the experimental and simulated productivities. This study thus opened new avenues for use of ammonium rich wastewater for commercial production of high value pigments, biofuel and bioplastics using Arthrospira sp.

Growth modelling of Nitrosomonas europaea ATCC® 19718 and Nitrobacter winogradskyi ATCC® 25391: A new online indicator of the partial nitrification.

The aim of the present work was to study the growth of two nitrifying bacteria. For modelling the nitrifying subsystem of the MELiSSA loop, Nitrosomonas europaea ATCC® 19718 and Nitrobacter winogradskyi ATCC® 25931 were grown separately and in cocultures. The kinetic parameters of a stoichiometric mass balanced Pirt model were identified: µmax=0.054h(-1), decay rate b=0.003h(-1) and maintenance rate m=0.135gN-NH4(+)·gX(-1)·h(-1) for Nitrosomonas europaea; µmax=0.024h(-1), b=0.001h(-1) and m=0.467gN-NO2(-)·gX(-1)·h(-1) for Nitrobacter winogradskyi. A predictive structured model of nitrification in co-culture was developed. The online evolution of the addition of KOH is correlated to the nitritation; the dissolved oxygen concentration is correlated to both nitritation and nitratation. The model suitably represents these two variables so that transient partial nitrification is assessed. This is a clue for avoiding partial nitrification by predictive functional control.

Improvement and modeling of culture parameters to enhance biomass and lipid production by the oleaginous yeast Cryptococcus curvatus grown on acetate.

The improvement of culture parameters for lipid production from acetate as carbon source was investigated using the oleaginous yeast Cryptococcus curvatus. A new pH regulation system dispensing acetate was developed for fed-batch culture and allowed obtaining nearly 80 g/L biomass within 60 h with a maximal growth rate of 0.28 h(-1). A biological model was developed from experimental data. The influence of three C/N ratios of 300, 500 and 900 were tested during a multi-phases process on lipid accumulation. The C/N ratio of 300 was reported to be the most suitable for lipid storage. No significant increase of lipids content was obtained with higher value. A maximal content of 60% DCW of lipid was obtained. The determination of fatty acids profiles of the microbial oils has confirmed that the valorization of acetate by microbial oils production was a promising perspective.

Cellulase activity mapping of Trichoderma reesei cultivated in sugar mixtures under fed-batch conditions.

BACKGROUND: On-site cellulase production using locally available lignocellulosic biomass (LCB) is essential for cost-effective production of 2nd-generation biofuels. Cellulolytic enzymes (cellulases and hemicellulases) must be produced in fed-batch mode in order to obtain high productivity and yield. To date, the impact of the sugar composition of LCB hydrolysates on cellulolytic enzyme secretion has not been thoroughly investigated in industrial conditions. RESULTS: The effect of sugar mixtures (glucose, xylose, inducer) on the secretion of cellulolytic enzymes by a glucose-derepressed and cellulase-hyperproducing mutant strain of Trichoderma reesei (strain CL847) was studied using a small-scale protocol representative of the industrial conditions. Since production of cellulolytic enzymes is inducible by either lactose or cellobiose, two parallel mixture designs were performed separately. No significant difference between inducers was observed on cellulase secretion performance, probably because a common induction mechanism occurred under carbon flux limitation. The characteristics of the enzymatic cocktails did not correlate with productivity, but instead were rather dependent on the substrate composition. Increasing xylose content in the feed had the strongest impact. It decreased by 2-fold cellulase, endoglucanase, and cellobiohydrolase activities and by 4-fold ß-glucosidase activity. In contrast, xylanase activity was increased 6-fold. Accordingly, simultaneous high ß-glucosidase and xylanase activities in the enzymatic cocktails seemed to be incompatible. The variations in enzymatic activity were modelled and validated with four fed-batch cultures performed in bioreactors. The overall enzyme production was maintained at its highest level when substituting up to 75% of the inducer with non-inducing sugars. CONCLUSIONS: The sugar substrate composition strongly influenced the composition of the cellulolytic cocktail secreted by T. reesei in fed-batch mode. Modelling can be used to predict cellulolytic activity based on the sugar composition of the culture-feeding solution, or to fine tune the substrate composition in order to produce a desired enzymatic cocktail.

Test of an anaerobic prototype reactor coupled with a filtration unit for production of VFAs.

The artificial ecosystem MELiSSA, supported by the European Space Agency is a closed loop system consisting of 5 compartments in which food, water and oxygen are produced out of organic waste. The first compartment is conceived as a thermophilic anaerobic membrane bioreactor liquefying organic waste into VFAs, ammonium and CO2 without methane. A 20 L reactor was assembled to demonstrate the selected design and process at prototype scale. We characterized system performance from start-up to steady state and evaluated process efficiencies with special attention drawn to the mass balances. An overall efficiency for organic matter biodegradation of 50% was achieved. The dry matter content was stabilized around 40-50 g L(-1) and VFA production around 5-6 g L(-1). The results were consistent for the considered substrate mixture and can also be considered relevant in a broader context, as a first processing step to produce building blocks for synthesis of primary energy vectors.

A new stoichiometric miniaturization strategy for screening of industrial microbial strains: application to cellulase hyper-producing Trichoderma reesei strains.

BACKGROUND: During bioprocess development, secondary screening is a key step at the boundary between laboratory and industrial conditions. To ensure an effective high-throughput screening, miniaturized laboratory conditions must mimic industrial conditions, especially for oxygen transfer, feeding capacity and pH stabilization. RESULTS: A feeding strategy has been applied to develop a simple screening procedure, in which a stoichiometric study is combined with a standard miniaturization procedure. Actually, the knowledge of all nutriments and base or acid requirements leads to a great simplification of pH stabilization issue of miniaturized fed-batch cultures. Applied to cellulase production by Trichoderma reesei, this strategy resulted in a stoichiometric mixed feed of carbon and nitrogen sources. While keeping the pH between shake flask and stirred bioreactor comparable, the developed shake flask protocol reproduced the strain behaviour under stirred bioreactor conditions. Compared to a an already existing miniaturized shake flasks protocol, the cellulase concentration was increased 5-fold, reaching about 10 g L-1. Applied to the secondary screening of several clones, the newly developed protocol succeeded in selecting a clone with a high industrial potential. CONCLUSIONS: The understanding of a bioprocess stoichiometry contributed to define a simpler and more effective miniaturization. The suggested strategy can potentially be applied to other fed-batch processes, for the screening of either strain collections or experimental conditions.

Dynamic aspects and controllability of the MELiSSA project: a bioregenerative system to provide life support in space.

Manmade ecosystems differ from their prototype biosphere by the principle of control. The Earth Biosphere is sustainable by stochastic control and very large time constants. By contrast, in a closed ecosystem such as the micro-ecological life support system alternative (MELiSSA system) developed by the European Space Agency for space exploration, a deterministic control is a prerequisite of sustainable existence. MELiSSA is an integrated sum of interconnected biological subsystems. On one hand, all unit operations in charge of the elementary functions constitutive of the entire life support system are studied until a thorough understanding and mathematical modelling. On the other hand, the systemic approach of complex, highly branched systems with feedback loops is performed. This leads to study in the same perspective, with the same degree of accuracy and with the same language, waste degradation, water recycling, atmosphere revitalisation and food production systems prior to the integration of knowledge-based control models. This paper presents the mathematical modelling of the MELiSSA system and the interface between the control strategy of the entire system and the control of the bioreactors.

Chemical and conformational study of the interactions involved in mycotoxin complexation with beta-D-glucans.

In a previous paper we reported that beta-D-glucans isolated from Saccharomyces cerevisiae could adsorb zearalenone, reduce its bioavailability in the digestive tract, and protect animals against its adverse effects. We have now investigated, in vitro, the kinetics of the interaction between other mycotoxins and beta-D-glucans from several sources at three pH values found along the digestive tract (3.0, 6.0, and 8.0). Acid and neutral conditions gave the highest affinity rates for aflatoxins B1 > deoxynivalenol > ochratoxin A and involved both the (1 --> 3)-beta-D-glucans and the (1 --> 6)-beta-D-glucans. Alkaline conditions, owing to their destructuring action on glucans, were favorable only for the adsorption of patulin. Using molecular mechanics, we found that hydroxyl, ketone, and lactone groups are involved in the formation of both hydrogen bonds and van der Waals interactions between aflatoxins B1, deoxynivalenol and patulin, and beta-D-glucans. Differences in the binding capacity of the mycotoxins are due to their specific physical and chemical characteristics.

Alkali extraction of beta-d-glucans from Saccharomyces cerevisiae cell wall and study of their adsorptive properties toward zearalenone.

The isolated cell wall of Saccharomyces cerevisiae has some capacity to adsorb zearalenone (affinity near 30%) and reduce the bioavailability of toxins in the digestive tract. The adsorption process was quantified in vitro, and the data obtained when plotted with Hill's equation indicated a cooperative process. The model showed that the adsorption capacity was related to the yeast cell wall composition. This work focused on the role of various beta-d-glucan types in the efficacy of zearalenone adsorption by yeast cell wall and sought to elucidate some of the adsorption mechanisms. Zearalenone was mixed at 37 degrees C with a constant quantity of alkali-soluble or alkali-insoluble beta-d-glucans isolated from yeast cell walls, and the amount of adsorbed zearalenone was measured. Given that the alkali solubility of beta-d-glucans is a determining factor for their three-dimensional conformation and that the alkali-insoluble fraction had a greater affinity (up to 50%) than the alkali-soluble fraction ( approximately 16%), it was concluded that the three-dimensional structure strongly influences the adsorption process. The alkali insolubility of beta-d-glucans led to the formation of single and/or triple helices, which have been identified as the most favorable structures for zearalenone adsorption efficacy. The beta(1,3)-d-glucan and beta(1,6)-d-glucan compositions of the two alkali-extracted fractions and their involvement in the adsorption process are discussed.

A novel technique to evaluate interactions between Saccharomyces cerevisiae cell wall and mycotoxins: application to zearalenone.

Three models based on sigmoidal plotting were tested for their ability to describe zearalenone adsorption on Saccharomyces cerevisiae cell walls in vitro. All three models closely fitted the experimental data, but Hill's equation gave the most accurate parameters, and provided information on the physical and chemical mechanisms involved in the adsorption of mycotoxin on yeast cell walls.