Acidophilic heterotrophs: basic aspects and technological applications.

Acidophiles comprise a group of microorganisms adapted to live in acidic environments. Despite acidophiles are usually associated with an autotrophic metabolism, more than 80 microorganisms capable of utilizing organic matter have been isolated from natural and man-made environments. The ability to reduce soluble and insoluble iron compounds has been described for many of these species and may be harnessed to develop new or improved mining processes when oxidative bioleaching is ineffective. Similarly, as these microorganisms grow in highly acidic media and the chances of contamination are reduced by the low pH, they may be employed to implement robust fermentation processes. By conducting an extensive literature review, this work presents an updated view of basic aspects and technological applications in biomining, bioremediation, fermentation processes aimed at biopolymers production, microbial electrochemical systems, and the potential use of extremozymes.

Production of poly (3-hydroxybutyrate) and extracellular polymeric substances from glycerol by the acidophile Acidiphilium cryptum.

Acidiphilium cryptum is an acidophilic, heterotrophic, and metallotolerant bacteria able to use dissolved oxygen or Fe(III) as an electron sink. The ability of this extremophile to accumulate poly(3-hydroxybutyrate) (PHB) and secrete extracellular polymeric substances (EPS) has also been reported. Hence, the aim of this work is to characterize the production of PHB and EPS by the wild strain DSM2389 using glycerol in shaken flasks and bioreactor. Results showed that maximum PHB accumulation (37-42% w/w) was obtained using glycerol concentrations of 9 and 15 g L-1, where maximum dry cell weight titers reached 3.6 and 3.9 g L-1, respectively. The culture in the bioreactor showed that PHB accumulation takes place under oxygen limitation, while the redox potential of the culture medium could be used for online monitoring of the PHB production. Recovered EPS was analyzed by Fourier-transform infrared spectroscopy and subjected to gas chromatography-mass spectrometry after cleavage and derivatization steps. These analyses showed the presence of sugars which were identified as mannose, rhamnose and glucose, in a proportion near to 3.2:2.3:1, respectively. Since glycerol had not been used in previous works, these findings suggest the potential of A. cryptum to produce biopolymers from this compound at a large scale with a low risk of microbial contamination due to the low pH of the fermentation process.

Production of (R)-3-hydroxybutyric acid from methane by in vivo depolymerization of polyhydroxybutyrate in Methylocystis parvus OBBP.

Methylocystis parvus OBBP accumulates polyhydroxybutyrate (PHB) using methane as the sole carbon and energy source. In this work, the feasibility of producing (R)-3-hydroxybutyric acid (R3HBA) via intracellularly accumulated PHB through depolymerization (in-vivo) was investigated. Results showed that a PHB to R3HBA conversion of 77.2 ± 0.9% (R3HBA titer of 0.153 ± 0.002 g L-1) can be attained in a mineral medium containing 1 g L-1 KNO3 at 30 °C with shaking at 200 rpm and a constant pH of 11 for 72 h. Nitrogen deprivation and neutral or acidic pHs strongly reduced the excreted R3HBA concentration. Reduced oxygen availability negatively affected the R3HBA yield, which decreased to 73.6 ± 4.9% (titer of 0.139 ± 0.01 g L-1) under microaerobic conditions. Likewise, the presence of increasing concentrations of R3HBA in the medium before the onset of PHB depolymerization reduced the initial R3HBA release rate and R3HBA yield.

Antarctic Rahnella inusitata: A Producer of Cold-Stable ß-Galactosidase Enzymes.

There has been a recent increase in the exploration of cold-active ß-galactosidases, as it offers new alternatives for the dairy industry, mainly in response to the current needs of lactose-intolerant consumers. Since extremophilic microbial compounds might have unique physical and chemical properties, this research aimed to study the capacity of Antarctic bacterial strains to produce cold-active ß-galactosidases. A screening revealed 81 out of 304 strains with ß-galactosidase activity. The strain Se8.10.12 showed the highest enzymatic activity. Morphological, biochemical, and molecular characterization based on whole-genome sequencing confirmed it as the first Rahnella inusitata isolate from the Antarctic, which retained 41-62% of its ß-galactosidase activity in the cold (4 °C-15 °C). Three ß-galactosidases genes were found in the R. inusitata genome, which belong to the glycoside hydrolase families GH2 (LacZ and EbgA) and GH42 (BglY). Based on molecular docking, some of these enzymes exhibited higher lactose predicted affinity than the commercial control enzyme from Aspergillus oryzae. Hence, this work reports a new Rahnella inusitata strain from the Antarctic continent as a prominent cold-active ß-galactosidase producer.

Beyond Intracellular Accumulation of Polyhydroxyalkanoates: Chiral Hydroxyalkanoic Acids and Polymer Secretion.

Polyhydroxyalkanoates (PHAs) are ubiquitous prokaryotic storage compounds of carbon and energy, acting as sinks for reducing power during periods of surplus of carbon source relative to other nutrients. With close to 150 different hydroxyalkanoate monomers identified, the structure and properties of these polyesters can be adjusted to serve applications ranging from food packaging to biomedical uses. Despite its versatility and the intensive research in the area over the last three decades, the market share of PHAs is still low. While considerable rich literature has accumulated concerning biochemical, physiological, and genetic aspects of PHAs intracellular accumulation, the costs of substrates and processing costs, including the extraction of the polymer accumulated in intracellular granules, still hampers a more widespread use of this family of polymers. This review presents a comprehensive survey and critical analysis of the process engineering and metabolic engineering strategies reported in literature aimed at the production of chiral (R)-hydroxycarboxylic acids (RHAs), either from the accumulated polymer or by bypassing the accumulation of PHAs using metabolically engineered bacteria, and the strategies developed to recover the accumulated polymer without using conventional downstream separations processes. Each of these topics, that have received less attention compared to PHAs accumulation, could potentially improve the economy of PHAs production and use. (R)-hydroxycarboxylic acids can be used as chiral precursors, thanks to its easily modifiable functional groups, and can be either produced de-novo or be obtained from recycled PHA products. On the other hand, efficient mechanisms of PHAs release from bacterial cells, including controlled cell lysis and PHA excretion, could reduce downstream costs and simplify the polymer recovery process.

Mechanistic Description of Convective Gas-Liquid Mass Transfer in Biotrickling Filters Using CFD Modeling.

The gas-liquid mass transfer coefficient is a key parameter to the design and operation of biotrickling filters that governs the transport rate of contaminants and oxygen from the gas phase to the liquid phase, where pollutant biodegradation occurs. Mass transfer coefficients are typically estimated via experimental procedures to produce empirical correlations, which are only valid for the bioreactor configuration and range of operational conditions under investigation. In this work, a new method for the estimation of the gas-liquid mass transfer coefficient in biotrickling filters is presented. This novel methodology couples a realistic description of the packing media (polyurethane foam without a biofilm) obtained using microtomography with computational fluid dynamics. The two-dimensional analysis reported in this study allowed capturing the mechanisms of the complex processes involved in the creeping porous air and water flow in the presence of capillary effects in biotrickling filters. Model predictions matched the experimental mass transfer coefficients (±30%) under a wide range of operational conditions.

Computational tomography and CFD simulation of a biofilter treating a toluene, formaldehyde and benzo[α]pyrene vapor mixture.

In this work, a 3D computational tomography (CT) of the packing material of a laboratory column biofilter is used to model airflow containing three contaminants. The degradation equations for toluene, formaldehyde and benzo[α]pyrene (BaP), were one-way coupled to the CFD model. Physical validation of the model was attained by comparing pressure drops with experimental measurement, while experimental elimination capacities for the pollutants were used to validate the biodegradation kinetics. The validated model was used to assess the existence of channeling and to predict the impact of the three-dimensional porous geometry on the mass transfer of the contaminants in the gas phase. Our results indicate that a physically meaningful simulation can be obtained using the techniques and approach presented in this work, without the need of performing experiments to obtain macroscopic parameters such as gas-phase axial and radial dispersion coefficients and porosities.

Methane biodegradation and enhanced methane solubilization by the filamentous fungi Fusarium solani.

Methane is one of the most important greenhouse gases emitted from natural and human activities. It is scarcely soluble in water; thus, it has a low bioavailability for microorganisms able to degrade it. In this work, the capacity of the fungus Fusarium solani to improve the solubility of methane in water and to biodegrade methane was assayed. Experiments were performed in microcosms with vermiculite as solid support and mineral media, at temperatures between 20 and 35 °C and water activities between 0.9 and 0.95, using pure cultures of F. solani and a methanotrophic consortium (Methylomicrobium album and Methylocystis sp) as a control. Methane was the only carbon and energy source. Results indicate that using thermally inactivated biomass of F. solani, decreases the partition coefficient of methane in water up to two orders of magnitude. Moreover, F. solani can degrade methane, in fact at 35 °C and the highest water activity, the methane degradation rate attained by F. solani was 300 mg m-3 h-1, identical to the biodegradation rate achieved by the consortium of methanotrophic bacteria.

Biofiltration of volatile organic compounds using fungi and its conceptual and mathematical modeling.

Volatile organic compounds (VOCs) are ubiquitous contaminants that can be found both in outdoor and indoor air, posing risks to human health and the ecosystems. The treatment of air contaminated with VOCs in low concentrations can be effectively performed using biofiltration, especially when VOCs are hydrophilic. However, the performance of biofilters inoculated with bacteria has been found to be low with sparsely water soluble molecules when compared to biofilters where fungi develop. Using conceptual and mathematical models, this review presents an overview of the physical, chemical and biological mechanisms that explain the differences in the performance of fungal and bacterial biofilters. Moreover, future research needs are proposed, with an emphasis on integrated models describing the biological and chemical reactions with the mass transfer using high-resolution descriptions of the packing material.

Biodegradation of benzo[α]pyrene, toluene, and formaldehyde from the gas phase by a consortium of Rhodococcus erythropolis and Fusarium solani.

Polycyclic aromatic hydrocarbons (PAHs) and volatile organic compounds (VOCs) are important indoor contaminants. Their hydrophobic nature hinders the possibility of biological abatement using biofiltration. Our aim was to establish whether the use of a consortium of Fusarium solani and Rhodococcus erythropolis shows an improved performance (in terms of mineralization rate and extent) towards the degradation of formaldehyde, as a slightly polar VOC; toluene, as hydrophobic VOC; and benzo[α]pyrene (BaP) as PAH at low concentrations compared to a single-species biofilm in serum bottles with vermiculite as solid support to mimic a biofilter and to relate the possible improvements with the surface hydrophobicity and partition coefficient of the biomass at three different temperatures. Results showed that the hydrophobicity of the surface of the biofilms was affected by the hydrophobicity of the carbon source in F. solani but it did not change in R. erythropolis. Similarly, the partition coefficients of toluene and BaP in F. solani biomass (both as pure culture and consortium) show a reduction of up to 38 times compared to its value in water, whereas this reduction was only 1.5 times in presence of R. erythropolis. Despite that increments in the accumulated CO2 and its production rate were found when F. solani or the consortium was used, the mineralization extent of toluene was below 25%. Regarding BaP degradation, the higher CO2 production rates and percent yields were obtained when a consortium of F. solani and R. erythropolis was used, despite a pure culture of R. erythropolis exhibits poor mineralization of BaP.

A generalized disjunctive programming framework for the optimal synthesis and analysis of processes for ethanol production from corn stover.

The aim of this study is to analyze the techno-economic performance of process configurations for ethanol production involving solid-liquid separators and reactors in the saccharification and fermentation stage, a family of process configurations where few alternatives have been proposed. Since including these process alternatives creates a large number of possible process configurations, a framework for process synthesis and optimization is proposed. This approach is supported on kinetic models fed with experimental data and a plant-wide techno-economic model. Among 150 process configurations, 40 show an improved MESP compared to a well-documented base case (BC), almost all include solid separators and some show energy retrieved in products 32% higher compared to the BC. Moreover, 16 of them also show a lower capital investment per unit of ethanol produced per year. Several of the process configurations found in this work have not been reported in the literature.

Elucidating the key role of the fungal mycelium on the biodegradation of n-pentane as a model hydrophobic VOC.

The role of the aerial mycelium of the fungus Fusarium solani in the biodegradation of n-pentane was evaluated in a continuous fungal bioreactor (FB) to determine the contribution of the aerial (hyphae) and non-aerial (monolayer) fungal biomass. The experimental results showed that although the aerial biomass fraction represented only 25.9(±3)% on a dry weight basis, it was responsible for 71.6(±4)% of n-pentane removal. The FB attained a maximum elimination capacity (ECmax) of 680(±30) g m(-3) h(-1) in the presence of fungal hyphae (which supported an interfacial area of 5.5(±1.5) × 10(6) m(2) m(-3)). In addition, a mathematical model capable of describing n-pentane biodegradation by the filamentous fungus was also developed and validated against the experimental data. This model successfully predicted the influence of the aerial biomass fraction and its partition coefficient on the n-pentane removal, with EC decreasing from 680(±30) g m(-3) h(-1) to values of 200(±14) g m(-3) h(-1) when the dimensionless partition coefficient increased from 0.21(±0.09) with aerial biomass to 0.88(±0.06) without aerial biomass.

Corn stover semi-mechanistic enzymatic hydrolysis model with tight parameter confidence intervals for model-based process design and optimization.

Uncertainty associated to the estimated values of the parameters in a model is a key piece of information for decision makers and model users. However, this information is typically not reported or the confidence intervals are too large to be useful. A semi-mechanistic model for the enzymatic saccharification of dilute acid pretreated corn stover is proposed in this work, the model is a modification of an existing one providing a statistically significant improved fit towards a set of experimental data that includes varying initial solid loadings (10-25% w/w) and the use of the pretreatment liquor and washed solids with or without supplementation of key inhibitors. A subset of 8 out of 17 parameters was identified, showing sufficiently tight confidence intervals to be used in uncertainty propagation and model analysis, without requiring interval truncation via expert judgment.

Attainable region analysis for continuous production of second generation bioethanol.

BACKGROUND: Despite its semi-commercial status, ethanol production from lignocellulosics presents many complexities not yet fully solved. Since the pretreatment stage has been recognized as a complex and yield-determining step, it has been extensively studied. However, economic success of the production process also requires optimization of the biochemical conversion stage. This work addresses the search of bioreactor configurations with improved residence times for continuous enzymatic saccharification and fermentation operations. Instead of analyzing each possible configuration through simulation, we apply graphical methods to optimize the residence time of reactor networks composed of steady-state reactors. Although this can be easily made for processes described by a single kinetic expression, reactions under analysis do not exhibit this feature. Hence, the attainable region method, able to handle multiple species and its reactions, was applied for continuous reactors. Additionally, the effects of the sugars contained in the pretreatment liquor over the enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) were assessed. RESULTS: We obtained candidate attainable regions for separate enzymatic hydrolysis and fermentation (SHF) and SSF operations, both fed with pretreated corn stover. Results show that, despite the complexity of the reaction networks and underlying kinetics, the reactor networks that minimize the residence time can be constructed by using plug flow reactors and continuous stirred tank reactors. Regarding the effect of soluble solids in the feed stream to the reactor network, for SHF higher glucose concentration and yield are achieved for enzymatic hydrolysis with washed solids. Similarly, for SSF, higher yields and bioethanol titers are obtained using this substrate. CONCLUSIONS: In this work, we demonstrated the capabilities of the attainable region analysis as a tool to assess the optimal reactor network with minimum residence time applied to the SHF and SSF operations for lignocellulosic ethanol production. The methodology can be readily modified to evaluate other kinetic models of different substrates, enzymes and microorganisms when available. From the obtained results, the most suitable reactor configuration considering residence time and rheological aspects is a continuous stirred tank reactor followed by a plug flow reactor (both in SSF mode) using washed solids as substrate.

Selection of process alternatives for lignocellulosic bioethanol production using a MILP approach.

This work proposes a decision-making framework for the selection of processes and unit operations for lignocellulosic bioethanol production. Process alternatives are described by its capital and operating expenditures, its contribution to process yield and technological availability information. A case study in second generation ethanol production using Eucalyptus globulus as raw material is presented to test the developed process synthesis tool. Results indicate that production cost does not necessarily decrease when yield increases. Hence, optimal processes can be found at the inflexion point of total costs and yield. The developed process synthesis tool provides results with an affordable computational cost, existing optimization tools and an easy-to-upgrade description of the process alternatives. These features made this tool suitable for process screening when incomplete information regarding process alternatives is available.

Process design and sustainability in the production of bioethanol from lignocellulosic materials

Background: Bioethanol is produced mainly from sugar cane and corn. In the last years it has been subject of debate due to the effects in food prices and land use change. The use of lignocellulosic materials for bioethanol production, such as agroindustry, forestry and municipal residues, wood or dendroenergetic species, has been proposed as a sustainable way for producing this biofuel. The design of a sustainable process for producing bioethanol requires a methodological approach whereby economical, environmental and social criteria are systematically integrated from the early stages of process design. Results: Until now a methodology for guiding the design of a sustainable process for bioethanol production is not available, and there are just a few studies on this subject. Moreover, with the recent global concerns on climate change, developed technologies have been confronted with additional requirements to validate their sustainability. In this sense, the inclusion of sustainability criteria on process design becomes necessary for defining a systematic methodology to select the most appropriate operations in the process stages to achieve a sustainable bioethanol production. Conclusions: A description of the stages for the production of bioethanol from lignocellulosic materials is provided in this review and the main findings in relation to the more important sustainability indicators are presented.