Immobilization of carbonic anhydrase for CO2 capture and utilization.

Carbonic anhydrase (CA) is an excellent candidate for novel biocatalytic processes based on the capture and utilization of CO2. The setup of efficient methods for enzyme immobilization makes CA utilization in continuous bioreactors increasingly attractive and opens up new opportunities for the industrial use of CA. The development of efficient processes for CO2 capture and utilization (CCU) is one of the most challenging targets of modern chemical reaction engineering. In the general frame of CCU processes, the interest in the utilization of immobilized CA as a biocatalyst for augmentation of CO2 reactive absorption has grown consistently over the last decade. The present mini-review surveys and discusses key methodologies for CA immobilization aimed at the development of heterogeneous biocatalysts for CCU. Advantages and drawbacks of covalent attachment on fine granular solids, immobilization as cross-linked enzyme aggregates, and "in vivo" immobilization methods are presented. In particular, criteria for optimal selection of CA-biocatalyst and design of CO2 absorption units are presented and discussed to highlight the most effective solutions. Perspectives on biocatalytic CCU processes that can include the use of CA in an enzymatic reactive CO2 absorption step are eventually presented with a special focus on two examples of CO2 fixation pathways: hybrid enzyme-microalgae process and enzyme cascade for the production of carboxylic acids. KEY POINTS: • Covalent immobilization techniques applied to CA are effective for CO2 ERA. • Biocatalyst type and morphology must be selected considering CO2 ERA conditions. • Immobilized CA can offer novel routes to CO2 capture and direct utilization.

Combined antioxidant-biofuel production from coffee silverskin.

Biorefinery concept asks for an integrated processing approach to exploit all biomass components. The self-sustainability target may be approached if molecules characterized by high added value and fermentable sugars are produced simultaneously. In the present study, sequential (i) mild hydrothermal pretreatment to produce antioxidants and (ii) NaOH pretreatment to produce a fermentable sugar solution were carried out on coffee silverskin. Twenty-minute treatment and biomass to liquid ratio 1:30 (g mL-1) were identified as optimal operating conditions to extract bioactive compounds characterized by antioxidant activity (22.2 mgGAE/gCSS; 13.9 mgTE/gCSS). Twenty-minutes and biomass to liquid ratio of 1:20 (g mL-1) were identified as optimal operating conditions to maximize sugar recovery and ABE production (solvent yield YABE/Sugars and ABE productivity of 0.21 g g-1 and 0.12 g L-1 h-1 were obtained, respectively). The study marks the highly economic potential of the process aimed to exploit the CSS as feesdstock for antioxidant and biofuel production.

Deep Eutectic Solvents pretreatment of agro-industrial food waste.

BACKGROUND: Waste biomass from agro-food industries are a reliable and readily exploitable resource. From the circular economy point of view, direct residues from these industries exploited for production of fuel/chemicals is a winning issue, because it reduces the environmental/cost impact and improves the eco-sustainability of productions. RESULTS: The present paper reports recent results of deep eutectic solvent (DES) pretreatment on a selected group of the agro-industrial food wastes (AFWs) produced in Europe. In particular, apple residues, potato peels, coffee silverskin, and brewer's spent grains were pretreated with two DESs, (choline chloride-glycerol and choline chloride-ethylene glycol) for fermentable sugar production. Pretreated biomass was enzymatic digested by commercial enzymes to produce fermentable sugars. Operating conditions of the DES pretreatment were changed in wide intervals. The solid to solvent ratio ranged between 1:8 and 1:32, and the temperature between 60 and 150 °C. The DES reaction time was set at 3 h. Optimal operating conditions were: 3 h pretreatment with choline chloride-glycerol at 1:16 biomass to solvent ratio and 115 °C. Moreover, to assess the expected European amount of fermentable sugars from the investigated AFWs, a market analysis was carried out. The overall sugar production was about 217 kt yr-1, whose main fraction was from the hydrolysis of BSGs pretreated with choline chloride-glycerol DES at the optimal conditions. CONCLUSIONS: The reported results boost deep investigation on lignocellulosic biomass using DES. This investigated new class of solvents is easy to prepare, biodegradable and cheaper than ionic liquid. Moreover, they reported good results in terms of sugars' release at mild operating conditions (time, temperature and pressure).

Low-energy biomass pretreatment with deep eutectic solvents for bio-butanol production.

Waste lettuce leaves - from the "fresh cut vegetable" industry - were pretreated with the deep eutectic solvent (DES) made of choline chloride - glycerol. Reaction time (3-16h) and the operation temperature (80-150°C) were investigated. Enzymatic glucose and xylose yields of 94.9% and 75.0%, respectively were obtained when the biomass was pretreated at 150°C for 16h. Sugars contained in the biomass hydrolysate were fermented in batch cultures of Clostridium acetobutylicum DSMZ 792. The energy consumption and the energy efficiency related to the DES pretreatment were calculated and compared to the most common lignocellulosic pretreatment processes reported in the literature. The DES pretreatment process was characterized by lower energy required (about 28% decrease and 72% decrease) than the NAOH pretreatment and steam explosion process respectively. The Net Energy Ratio (NER) value related to butanol production via DES biomass pretreatment was assessed.

Renewable feedstocks for biobutanol production by fermentation.

This paper reports a study of potential feedstock for butanol production via the biotechnological route. Several waste(water) streams rich in sugars and lignocellulosic biomass were studied: cheese-whey, leftovers of high sugar-content beverages, food lost or wasted, agriculture residues. The maximum butanol production rate from each type of feedstock was assessed according to the parameters indicated in the literature: feedstock availability rate, feedstock average composition and butanol yield. In Europe the potential biotechnological production of butanol from the feedstock studied was assessed to be about 39 Mt yr-1, which would be enough to meet the current European demand of biofuels. The potential butanol production at local level was also assessed taking into account the concentration of feedstock suppliers in the Campania region.

Stabilization of Candida antarctica Lipase B (CALB) Immobilized on Octyl Agarose by Treatment with Polyethyleneimine (PEI).

Lipase B from Candida antarctica (CALB) was immobilized on octyl agarose (OC) and physically modified with polyethyleneimine (PEI) in order to confer a strong ion exchange character to the enzyme and thus enable the immobilization of other enzymes on its surface. The enzyme activity was fully maintained during the coating and the thermal stability was marginally improved. The enzyme release from the support by incubation in the non-ionic detergent Triton X-100 was more difficult after the PEI-coating, suggesting that some intermolecular physical crosslinking had occurred, making this desorption more difficult. Thermal stability was marginally improved, but the stability of the OCCALB-PEI was significantly better than that of OCCALB during inactivation in mixtures of aqueous buffer and organic cosolvents. SDS-PAGE analysis of the inactivated biocatalyst showed the OCCALB released some enzyme to the medium during inactivation, and this was partially prevented by coating with PEI. This effect was obtained without preventing the possibility of reuse of the support by incubation in 2% ionic detergents. That way, this modified CALB not only has a strong anion exchange nature, while maintaining the activity, but it also shows improved stability under diverse reaction conditions without affecting the reversibility of the immobilization.

Continuous xylose fermentation by Clostridium acetobutylicum--Assessment of solventogenic kinetics.

This work deals with the specific butanol production rate of Clostridium acetobutylicum using xylose--a relevant fraction of lignocellulosic feedstock for biofuel production--as carbon source. The tests were carried out in a CSTR equipped with a microfiltration unit. The dilution rate (D) ranged between 0.02 and 0.22 h(-1) and the ratio R between the permeate stream rate and the stream fed to the reactor ranged between 14% and 88%. The biomass present in the broth was identified as a heterogeneous cell population consisting of: acidogenic cells, solventogenic cells and spores. The results were processed to assess the concentration of acidogenic cells, solventogenic cells and spores. The specific butanol production rate was also assessed. The max butanol productivity was 1.3 g L(-1) h(-1) at D = 0.17 h(-1) and R = 30%. A comparison between the results reported in a previous work carried out with lactose was made.

Continuous lactose fermentation by Clostridium acetobutylicum--assessment of solventogenic kinetics.

This work reports the results of a series of tests on the specific butanol production rate by Clostridium acetobutylicum continuous cultures. The tests were carried out using lactose as carbon source to mimic cheese-whey. A continuous stirred tank reactor equipped with a microfiltration unit was used. The dilution rate (D) ranged between 0.02 and 0.15h(-1) and the ratio R of the permeate stream rate to the stream fed to the reactor ranged between 14% and 95%. For each set of D and R values, the continuous cultures were characterized in terms of concentration of cells, acids and solvents. Results were processed to assess the concentration of acidogenic cells, solventogenic cells, spores and the specific butanol production rate. The max butanol productivity was 0.5gL(-1)h(-1) at D=0.1h(-1) and R=95%. The butanol productivity referred to solventogenic cells was expressed as a function of concentration of lactose, acids and butanol.

Immobilization of a Pleurotus ostreatus laccase mixture on perlite and its application to dye decolourisation.

In the present study, a crude laccase preparation from Pleurotus ostreatus was successfully immobilized on perlite, a cheap porous silica material, and tested for Remazol Brilliant Blue R (RBBR) decolourisation in a fluidized bed recycle reactor. Results showed that RBBR decolourisation is mainly due to enzyme action despite the occurrence of dye adsorption-related enzyme inhibition. Fine tuning of immobilization conditions allowed balancing the immobilization yield and the resulting rate of decolourisation, with the adsorption capacity of the solid biocatalyst. In the continuous lab scale reactor, a maximum conversion degree of 56.1% was achieved at reactor space-time of 4.2 h. Stability and catalytic parameters of the immobilized laccases were also assessed in comparison with the soluble counterparts, revealing an increase in stability, despite a reduction of the catalytic performances. Both effects are most likely ascribable to the occurrence of multipoint attachment phenomena.

Continuous xylose fermentation by Clostridium acetobutylicum--kinetics and energetics issues under acidogenesis conditions.

The paper reports the assessment of the growth kinetics of Clostridium acetobutylicum DSM 792 adopting xylose as carbon source. Xylose is the fundamental component of hemicellulose hydrolysis, a relevant fraction of lignocellulosic feedstocks for biofuel production. Tests were carried out in a CSTR operated under controlled pH. The effects of acids (acetic and butyric) and solvents (acetone, ethanol and butanol) on the fermentation were investigated. The conversion process was characterized under steady-state conditions in terms of concentration of xylose, cells, acids, and pH. The growth kinetics was expressed by means of a multiple product inhibition and it was able to predict microorganism growth rate under a broad interval of operating conditions, even those typical of solvents production. The mass fractional yield of biomass and products were expressed as a function of the specific growth rate taking into account the Pirt model.

Kinetic study of a novel thermo-stable α-carbonic anhydrase for biomimetic CO2 capture.

Biomimetic CO2 capture includes environmentally friendly solutions based on carbonic anhydrase (CA), an enzyme that increases CO2 absorption rate in conventional acid-gas scrubbing processes. The present contribution reports the characterization of a new recombinant carbonic anhydrase, SspCA, isolated from the thermophile bacterium Sulphurhydrogenibium yellowstonense sp. YO3AOP1. The kinetics of SspCA was characterized in terms of first order CO2 hydration rate according to a procedure based on CO2 absorption tests in a stirred cell apparatus. The first order kinetic constant at 25°C was 9.16 × 10(6) L/(mols). An appropriate investigation on SspCA stability was carried out to assess its long-term resistance to high temperatures as in all capture processes based on absorption/vacuum-desorption cycles. Its half-life was 53 and 8 days at 40 °C and 70 °C, respectively.

Continuous lactose fermentation by Clostridium acetobutylicum--assessment of energetics and product yields of the acidogenesis.

An assessment of both the growth and the metabolism of acidogenic cells Clostridium acetobutylicum DSM 792 is reported in the paper. Tests were carried out in a CSTR under controlled pH conditions. Cultures were carried out using a semi-synthetic medium supplemented with lactose as carbon source. Acids and solvents, that represent products of the ABE process, have been purposely added in controlled amounts to the culture medium to investigate their effects on the product yields. The mass fractional yield of biomass and products were expressed as a function of the specific growth rate taking into account the Pirt model. The maximum ATP yield and the maintenance resulted 29.1 g(DM)/mol(ATP) and 0.012 mol(ATP)/g(DM)h, respectively. Quantitative features of the C. acetobutylicum growth model were in good agreement with experimental results. The model proposes as a tool to estimate the mass fractional yield even for fermentations carried out under conditions typical of the solventogenesis.

Strategies for dephenolization of raw olive mill wastewater by means of Pleurotus ostreatus.

The reduction of polyphenols content in olive mill wastewater (OMW) is a major issue in olive oil manufacturing. Although researchers have pointed out the potential of white-rot fungus in dephenolizing OMW, the results available in the literature mainly concern pretreated (sterilized) OMW. This paper deals with the reduction of polyphenols content in untreated OMW by means of a white-rot fungus, Pleurotus ostreatus. Dephenolization was performed both in an airlift bioreactor and in aerated flasks. The process was carried out under controlled non-sterile conditions, with different operating configurations (batch, continuous, biomass recycling) representative of potential industrial operations. Total organic carbon, polyphenols concentration, phenol oxidase activity, dissolved oxygen concentration, oxygen consumption rate, and pH were measured during every run. Tests were carried out with or without added nutrients (potato starch and potato dextrose) and laccases inducers (i.e., CuSO4). OMW endogenous microorganisms were competing with P. ostreatus for oxygen during simultaneous fermentation. Dephenolization of raw OMW by P. ostreatus under single batch was as large as 70%. Dephenolization was still extensive even when biomass was recycled up to six times. OMW pre-aeration had to be provided under continuous operation to avoid oxygen consumption by endogenous microorganisms that might spoil the process. The role of laccases in the dephenolization process has been discussed. Dephenolization under batch conditions with biomass recycling and added nutrients proved to be the most effective configuration for OMW polyphenols reduction in industrial plants (42-68% for five cycles).

Unstable steady state operations of substrate inhibited cultures by dissolved oxygen control.

Microorganism kinetic growth characterized by substrate inhibition was investigated by means of a continuous stirred tank reactor equipped with a feedback controller of the medium feeding flow rate. The aerobic growth of Pseudomonas sp. OX1 with phenol as carbon/energy source was adopted as a case study to test a new control strategy using dissolved oxygen concentration as a state variable. The controller was successful in steadily operating bioconversion under intrinsically unstable conditions. A simple model of the controlled system was proposed to set the feedback controller. The specific growth rate of Pseudomonas sp. OX1 was successfully described by means of the Haldane model. The regression of the experimental data yielded µ(M)=0.26 h(-1), K(Ph)=5×10(-3)g/L and K(I)=0.2g/L. The biomass-to-substrate fractional yield as a function of the specific growth rate did not change moving from substrate-inhibited to substrate-deficient state. The data was modelled according to the Pirt model: m=1.7×10(-2)g/(gh), Y(X/Ph)(Th)=1.3g/g. The specific growth rates calculated for batch and continuous growth were compared.

Modeling of an aerobic biofilm reactor with double-limiting substrate kinetics: bifurcational and dynamical analysis.

A mathematical model of an aerobic biofilm reactor is presented to investigate the bifurcational patterns and the dynamical behavior of the reactor as a function of different key operating parameters. Suspended cells and biofilm are assumed to grow according to double limiting kinetics with phenol inhibition (carbon source) and oxygen limitation. The model presented by Russo et al. is extended to embody key features of the phenomenology of the granular-supported biofilm: biofilm growth and detachment, gas-liquid oxygen transport, phenol, and oxygen uptake by both suspended and immobilized cells, and substrate diffusion into the biofilm. Steady-state conditions and stability, and local dynamic behavior have been characterized. The multiplicity of steady states and their stability depend on key operating parameter values (dilution rate, gas-liquid mass transfer coefficient, biofilm detachment rate, and inlet substrate concentration). Small changes in the operating conditions may be coupled with a drastic change of the steady-state scenario with transcritical and saddle-node bifurcations. The relevance of concentration profiles establishing within the biofilm is also addressed. When the oxygen level in the liquid phase is <10% of the saturation level, the biofilm undergoes oxygen starvation and the active biofilm fraction becomes independent of the dilution rate. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011.

Continuous lactose fermentation by Clostridium acetobutylicum--assessment of acidogenesis kinetics.

An assessment of the growth kinetics of acidogenic cells of Clostridium acetobutylicum DSM 792 is reported in the paper. Tests were carried out in a continuous stirred tank reactor under controlled conditions adopting a complex medium supplemented with lactose as carbon source to mimic cheese whey. The effects of acids (acetic and butyric), solvents (acetone, ethanol and butanol) and pH on the growth rate of acidogenic cells were assessed. The conversion process was characterized under steady-state conditions in terms of concentration of lactose, cells, acids, total organic carbon and pH. The growth kinetics was expressed by means of a multiple product inhibition and interacting model including a novel formulation to account for the role of pH. The model has the potential to predict microorganism growth rate under a broad interval of operating conditions, even those typical of solvents production.

Butanol production by Clostridium acetobutylicum in a continuous packed bed reactor.

In this study, we report on a butanol production process by immobilized Clostridium acetobutylicum in a continuous packed bed reactor (PBR) using Tygon rings as a carrier. The medium was a solution of lactose (15-30 g/L) and yeast extract (3 g/L) to emulate the cheese whey, an abundant lactose-rich wastewater. The reactor was operated under controlled conditions with respect to the pH and to the dilution rate. The pH and the dilution rate ranged between 4 and 5, the dilution rate between 0.54 and 2.4 h(-1) (2.5 times the maximum specific growth rate assessed for suspended cells). The optimal performance of the reactor was recorded at a dilution rate of 0.97 h(-1): the butanol productivity was 4.4 g/Lh and the selectivity of solvent in butanol was 88%(w).