| 1. |
- Dererie, Debebe Yilma, et al.
(författare)
-
Improved bio-energy yields via sequential ethanol fermentation and biogas digestion of steam exploded oat straw
- 2011
-
Ingår i: Bioresource Technology. - : Elsevier. - 0960-8524 .- 1873-2976. ; 102:6, s. 4449-4455
-
Tidskriftsartikel (refereegranskat)abstract
- Using standard laboratory equipment, thermochemically pretreated oat straw was enzymatically saccharified and fermented to ethanol, and after removal of ethanol the remaining material was subjected to biogas digestion. A detailed mass balance calculation shows that, for steam explosion pretreatment, this combined ethanol fermentation and biogas digestion converts 85-87% of the higher heating value (HHV) of holocellulose (cellulose and hemicellulose) in the oat straw into biofuel energy. The energy (HHV) yield of the produced ethanol and methane was 9.5-9.8 MJ/(kg dry oat straw), which is 28-34% higher than direct biogas digestion that yielded 7.3-7.4 MJ/(kg dry oat straw). The rate of biogas formation from the fermentation residues was also higher than from the corresponding pretreated but unfermented oat straw, indicating that the biogas digestion could be terminated after only 24 days. This suggests that the ethanol process acts as an additional pretreatment for the biogas process.
|
|
| 2. |
- Blomqvist, Johanna, et al.
(författare)
-
Fermentation of lignocellulosic hydrolysate by the alternative industrial ethanol yeast Dekkera bruxellensis
- 2011
-
Ingår i: Letters in Applied Microbiology. - Malden, USA : Wiley-Blackwell. - 0266-8254 .- 1472-765X. ; 53:1, s. 73-78
-
Tidskriftsartikel (refereegranskat)abstract
- Aim: Testing the ability of the alternative ethanol production yeast Dekkera bruxellensis to produce ethanol from lignocellulose hydrolysate and comparing it to Saccharomyces cerevisiae.Methods and Results: Industrial isolates of D. bruxellensis and S. cerevisiae were cultivated in small-scale batch fermentations of enzymatically hydrolysed steam exploded aspen sawdust. Different dilutions of hydrolysate were tested. None of the yeasts grew in undiluted or 1 : 2 diluted hydrolysate [final glucose concentration always adjusted to 40 g l(-1) (0.22 mol l(-1))]. This was most likely due to the presence of inhibitors such as acetate or furfural. In 1 : 5 hydrolysate, S. cerevisiae grew, but not D. bruxellensis, and in 1 : 10 hydrolysate, both yeasts grew. An external vitamin source (e.g. yeast extract) was essential for growth of D. bruxellensis in this lignocellulosic hydrolysate and strongly stimulated S. cerevisiae growth and ethanol production. Ethanol yields of 0 42 +/- 0 01 g ethanol (g glucose)(-1) were observed for both yeasts in 1 : 10 hydrolysate. In small-scale continuous cultures with cell recirculation, with a gradual increase in the hydrolysate concentration, D. bruxellensis was able to grow in 1 : 5 hydrolysate. In bioreactor experiments with cell recirculation, hydrolysate contents were increased up to 1 : 2 hydrolysate, without significant losses in ethanol yields for both yeasts and only slight differences in viable cell counts, indicating an ability of both yeasts to adapt to toxic compounds in the hydrolysate.Conclusions: Dekkera bruxellensis and S. cerevisiae have a similar potential to ferment lignocellulose hydrolysate to ethanol and to adapt to fermentation inhibitors in the hydrolysate.Significance and Impact of the study: This is the first study investigating the potential of D. bruxellensis to ferment lignocellulosic hydrolysate. Its high competitiveness in industrial fermentations makes D. bruxellensis an interesting alternative for ethanol production from those substrates.
|
|
| 3. |
- Gudmundsson, Mikael, et al.
(författare)
-
Structural and Electronic Snapshots during the Transition from a Cu(II) to Cu(I) Metal Center of a Lytic Polysaccharide Monooxygenase by X-ray Photoreduction
- 2014
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 289, s. 18782-18792
-
Tidskriftsartikel (refereegranskat)abstract
- Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of enzymes that employ a copper-mediated, oxidative mechanism to cleave glycosidic bonds. The LPMO catalytic mechanism likely requires that molecular oxygen first binds to Cu(I), but the oxidation state in many reported LPMO structures is ambiguous, and the changes in the LPMO active site required to accommodate both oxidation states of copper have not been fully elucidated. Here, a diffraction data collection strategy minimizing the deposited x-ray dose was used to solve the crystal structure of a chitin-specific LPMO from Enterococcus faecalis (EfaCBM33A) in the Cu(II)-bound form. Subsequently, the crystalline protein was photoreduced in the x-ray beam, which revealed structural changes associated with the conversion from the initial Cu(II)-oxidized form with two coordinated water molecules, which adopts a trigonal bipyramidal geometry, to a reduced Cu(I) form in a T-shaped geometry with no coordinated water molecules. A comprehensive survey of Cu(II) and Cu(I) structures in the Cambridge Structural Database unambiguously shows that the geometries observed in the least and most reduced structures reflect binding of Cu(II) and Cu(I), respectively. Quantum mechanical calculations of the oxidized and reduced active sites reveal little change in the electronic structure of the active site measured by the active site partial charges. Together with a previous theoretical investigation of a fungal LPMO, this suggests significant functional plasticity in LPMO active sites. Overall, this study provides molecular snapshots along the reduction process to activate the LPMO catalytic machinery and provides a general method for solving LPMO structures in both copper oxidation states.
|
|
| 4. |
- Haataja, Topi, et al.
(författare)
-
Enzyme kinetics by GH7 cellobiohydrolases on chromogenic substrates is dictated by non-productive binding : insights from crystal structures and MD simulation
- 2023
-
Ingår i: The FEBS Journal. - : John Wiley & Sons. - 1742-464X .- 1742-4658. ; 290:2, s. 379-399
-
Tidskriftsartikel (refereegranskat)abstract
- Cellobiohydrolases (CBHs) in the glycoside hydrolase family 7 (GH7) (EC3.2.1.176) are the major cellulose degrading enzymes both in industrial settings and in the context of carbon cycling in nature. Small carbohydrate conjugates such as p-nitrophenyl-beta-d-cellobioside (pNPC), p-nitrophenyl-beta-d-lactoside (pNPL) and methylumbelliferyl-beta-d-cellobioside have commonly been used in colorimetric and fluorometric assays for analysing activity of these enzymes. Despite the similar nature of these compounds the kinetics of their enzymatic hydrolysis vary greatly between the different compounds as well as among different enzymes within the GH7 family. Through enzyme kinetics, crystallographic structure determination, molecular dynamics simulations, and fluorometric binding studies using the closely related compound o-nitrophenyl-beta-d-cellobioside (oNPC), in this work we examine the different hydrolysis characteristics of these compounds on two model enzymes of this class, TrCel7A from Trichoderma reesei and PcCel7D from Phanerochaete chrysosporium. Protein crystal structures of the E212Q mutant of TrCel7A with pNPC and pNPL, and the wildtype TrCel7A with oNPC, reveal that non-productive binding at the product site is the dominating binding mode for these compounds. Enzyme kinetics results suggest the strength of non-productive binding is a key determinant for the activity characteristics on these substrates, with PcCel7D consistently showing higher turnover rates (k(cat)) than TrCel7A, but higher Michaelis-Menten (K-M) constants as well. Furthermore, oNPC turned out to be useful as an active-site probe for fluorometric determination of the dissociation constant for cellobiose on TrCel7A but could not be utilized for the same purpose on PcCel7D, likely due to strong binding to an unknown site outside the active site.
|
|
| 5. |
- Haddad Momeni, Majid, et al.
(författare)
-
A novel starch-binding laccase from the wheat pathogen Zymoseptoria tritici highlights the functional diversity of ascomycete laccases
- 2019
-
Ingår i: BMC Biotechnology. - : Springer Science and Business Media LLC. - 1472-6750. ; 19
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Laccases are multicopper oxidases, which are assigned into auxiliary activity family 1 (AA1) in the CAZy database. These enzymes, catalyzing the oxidation of phenolic and nonphenolic substrates coupled to reduction of O2 to H2O, are increasingly attractive as eco-friendly oxidation biocatalysts. Basidiomycota laccases are well characterized due to their potential in de-lignification of lignocellulose. By contrast, insight into the biochemical diversity of Ascomycota counterparts from saprophytes and plant pathogens is scarce. Results: Here, we report the properties of the laccase from the major wheat pathogen Zymoseptoria tritici (ZtrLac1A), distinguished from common plant fungal pathogens by an apoplastic infection strategy. We demonstrate that ZtrLac1A is appended to a functional starch-binding module and displays an activity signature disfavoring relatively apolar phenolic redox mediators as compared to the related biochemically characterized laccases. By contrast, the redox potential of ZtrLac1A (370 mV vs. SHE) is similar to ascomycetes counterparts. The atypical specificity is consistent with distinctive sequence substitutions and insertions in loops flanking the T1 site and the enzyme C-terminus compared to characterized laccases. Conclusions: ZtrLac1A is the first reported modular laccase appended to a functional starch-specific carbohydrate binding module of family 20 (CBM20). The distinct specificity profile of ZtrLac1A correlates to structural differences in the active site region compared to previously described ascomycetes homologues. These differences are also highlighted by the clustering of the sequence of ZtrLac1A in a distinct clade populated predominantly by plant pathogens in the phylogenetic tree of AA1 laccases. The possible role of these laccases in vivo merits further investigations. These findings expand our toolbox of laccases for green oxidation and highlight the binding functionality of CBM-appended laccases as versatile immobilization tags.
|
|
| 6. |
- Haddad Momeni, Majid, et al.
(författare)
-
Biochemical and Structural Characterizations of Two Dictyostelium Cellobiohydrolases from the Amoebozoa Kingdom Reveal a High Level of Conservation between Distant Phylogenetic Trees of Life
- 2016
-
Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 82, s. 3395-3409
-
Tidskriftsartikel (refereegranskat)abstract
- Glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) are enzymes commonly employed in plant cell wall degradation across eukaryotic kingdoms of life, as they provide significant hydrolytic potential in cellulose turnover. To date, many fungal GH7 CBHs have been examined, yet many questions regarding structure-activity relationships in these important natural and commercial enzymes remain. Here, we present the crystal structures and a biochemical analysis of two GH7 CBHs from social amoeba: Dictyostelium discoideum Cel7A (DdiCel7A) and Dictyostelium purpureum Cel7A (DpuCel7A). DdiCel7A and DpuCel7A natively consist of a catalytic domain and do not exhibit a carbohydrate-binding module (CBM). The structures of DdiCel7A and DpuCel7A, resolved to 2.1 angstrom and 2.7 angstrom respectively, are homologous to those of other GH7 CBHs with an enclosed active-site tunnel. Two primary differences between the Dictyostelium CBHs and the archetypal model GH7 CBH, Trichoderma reesei Cel7A (TreCel7A), occur near the hydrolytic active site and the product-binding sites. To compare the activities of these enzymes with the activity of TreCel7A, the family 1 TreCel7A CBM and linker were added to the C terminus of each of the Dictyostelium enzymes, creating DdiCel7A(CBM) and DpuCel7A(CBM), which were recombinantly expressed in T. reesei. DdiCel7A(CBM) and DpuCel7A(CBM) hydrolyzed Avicel, pretreated corn stover, and phosphoric acid-swollen cellulose as efficiently as TreCel7A when hydrolysis was compared at their temperature optima. The K-i of cellobiose was significantly higher for DdiCel7A(CBM) and DpuCel7A(CBM) than for TreCel7A: 205, 130, and 29 mu M, respectively. Taken together, the present study highlights the remarkable degree of conservation of the activity of these key natural and industrial enzymes across quite distant phylogenetic trees of life.IMPORTANCEGH7 CBHs are among the most important cellulolytic enzymes both in nature and for emerging industrial applications for cellulose breakdown. Understanding the diversity of these key industrial enzymes is critical to engineering them for higher levels of activity and greater stability. The present work demonstrates that two GH7 CBHs from social amoeba are surprisingly quite similar in structure and activity to the canonical GH7 CBH from the model biomass-degrading fungus T. reesei when tested under equivalent conditions (with added CBM-linker domains) on an industrially relevant substrate.
|
|
| 7. |
- Haddad Momeni, Majid, et al.
(författare)
-
Expression, crystal structure and cellulase activity of the thermostable cellobiohydrolase Cel7A from the fungus Humicola grisea var. thermoidea
- 2014
-
Ingår i: Acta Crystallographica Section D: Biological Crystallography. - 0907-4449 .- 1399-0047. ; 70, s. 2356-2366
-
Tidskriftsartikel (refereegranskat)abstract
- Glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) play a key role in biomass recycling in nature. They are typically the most abundant enzymes expressed by potent cellulolytic fungi, and are also responsible for the majority of hydrolytic potential in enzyme cocktails for industrial processing of plant biomass. The thermostability of the enzyme is an important parameter for industrial utilization. In this study, Cel7 enzymes from different fungi were expressed in a fungal host and assayed for thermostability, including Hypocrea jecorina Cel7A as a reference. The most stable of the homologues, Humicola grisea var. thermoidea Cel7A, exhibits a 10 degrees C higher melting temperature (T-m of 72.5 degrees C) and showed a 4-5 times higher initial hydrolysis rate than H. jecorina Cel7A on phosphoric acid-swollen cellulose and showed the best performance of the tested enzymes on pretreated corn stover at elevated temperature (65 degrees C, 24 h). The enzyme shares 57% sequence identity with H. jecorina Cel7A and consists of a GH7 catalytic module connected by a linker to a C-terminal CBM1 carbohydrate-binding module. The crystal structure of the H. grisea var. thermoidea Cel7A catalytic module (1.8 angstrom resolution; R-work and R-free of 0.16 and 0.21, respectively) is similar to those of other GH7 CBHs. The deviations of several loops along the cellulose-binding path between the two molecules in the asymmetric unit indicate higher flexibility than in the less thermostable H. jecorina Cel7A.
|
|
| 8. |
- Haddad Momeni, Majid, et al.
(författare)
-
Improved bio-energy yields via sequential ethanol fermentation and biogas digestion of steam exploded oat straw
- 2011
-
Ingår i: Bioresource Technology. - : Elsevier BV. - 0960-8524 .- 1873-2976. ; 102, s. 4449-4455
-
Tidskriftsartikel (refereegranskat)abstract
- Using standard laboratory equipment, thermochemically pretreated oat straw was enzymatically saccharified and fermented to ethanol, and after removal of ethanol the remaining material was subjected to biogas digestion. A detailed mass balance calculation shows that, for steam explosion pretreatment, this combined ethanol fermentation and biogas digestion converts 85-87% of the higher heating value (HHV) of holocellulose (cellulose and hemicellulose) in the oat straw into biofuel energy. The energy (HHV) yield of the produced ethanol and methane was 9.5-9.8 MJ/(kg dry oat straw), which is 28-34% higher than direct biogas digestion that yielded 7.3-7.4 MJ/(kg dry oat straw). The rate of biogas formation from the fermentation residues was also higher than from the corresponding pretreated but unfermented oat straw, indicating that the biogas digestion could be terminated after only 24 days. This suggests that the ethanol process acts as an additional pretreatment for the biogas process. (C) 2010 Elsevier Ltd. All rights reserved.
|
|
| 9. |
- Haddad Momeni, Majid, et al.
(författare)
-
Structural, Biochemical, and Computational Characterization of the Glycoside Hydrolase Family 7 Cellobiohydrolase of the Tree-killing Fungus Heterobasidion irregulare
- 2013
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 288:8, s. 5861-5872
-
Tidskriftsartikel (refereegranskat)abstract
- Root rot fungi of the Heterobasidion annosum complex are the most damaging pathogens in temperate forests, and the recently sequenced Heterobasidion irregulare genome revealed over 280 carbohydrate-active enzymes. Here, H. irregulare was grown on biomass, and the most abundant protein in the culture filtrate was identified as the only family 7 glycoside hydrolase in the genome, which consists of a single catalytic domain, lacking a linker and carbohydrate-binding module. The enzyme, HirCel7A, was characterized biochemically to determine the optimal conditions for activity. HirCel7A was crystallized and the structure, refined at 1.7 angstrom resolution, confirms that HirCel7A is a cellobiohydrolase rather than an endoglucanase, with a cellulose-binding tunnel that is more closed than Phanerochaete chrysosporium Cel7D and more open than Hypocrea jecorina Cel7A, suggesting intermediate enzyme properties. Molecular simulations were conducted to ascertain differences in enzyme-ligand interactions, ligand solvation, and loop flexibility between the family 7 glycoside hydrolase cellobiohydrolases from H. irregulare, H. jecorina, and P. chrysosporium. The structural comparisons and simulations suggest significant differences in enzyme-ligand interactions at the tunnel entrance in the -7 to -4 binding sites and suggest that a tyrosine residue at the tunnel entrance of HirCel7A may serve as an additional ligand-binding site. Additionally, the loops over the active site in H. jecorina Cel7A are more closed than loops in the other two enzymes, which has implications for the degree of processivity, endo- initiation, and substrate dissociation. Overall, this study highlights molecular level features important to understanding this biologically and industrially important family of glycoside hydrolases.
|
|
| 10. |
- Haddad Momeni, Majid
(författare)
-
Structural insights into the catalytic mechanism, protein dynamics, inhibition and thermostability of GH7 cellobiohydrolases
- 2014
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Glycoside hydrolase family 7 cellobiohydrolases (GH7 CBH) are typically the most abundant enzymes of cellulolytic fungi and play a key role in biomass recycling in Nature, as well as in biofuel production from plant biomass. This thesis examines molecular properties of this biologically and industrially important class of enzymes. Paper I shows that HirCel7A is the most abundant protein of the serious forest pathogen Heterobasidion irregulare. The HirCel7A exhibits intermediate dynamical and structural properties between CBHs with the most closed and most open tunnels known in GH7. The results point to tunnel-enclosing loops as important for carbohydrate processivity and association-dissociation on cellulose. Paper II presents the first Michaelis complex, with cellononaose spanning 42 Å of the active site, and the first glycosyl-enzyme intermediate trapped in a GH7 CBH. QM/MM calculations determine optimal reaction coordinates, and rate constants at 11 s-1 for Step1 and 5300 s-1 for Step2, showing that the glycosylation step is rate-limiting. A product-assisted mechanism is revealed for the deglycosylation step, indicating that expulsion of the cellobiose product is not required prior to hydrolysis of the intermediate. In Paper III, HgrCel7A from Humicola grisea var. thermoidea showed 10 °C higher Tm and 75% higher yield in a biomass performance assay at 65 °C than the canonical HjeCel7A of Hypocrea jecorina. The crystal structure of HgrCel7A indicates higher flexibility in tunnel-defining loops, and structural features potentially related to thermostability and enhanced activity, including a putative conformational switch in an active-center loop not reported previously in GH7. In Paper IV, structures of HjeCel7A in complex with xylooligosaccharides of DP 3-5 show predominant binding in the beginning of the tunnel and partial occupancy for a second binding mode near the catalytic centre. Birchwood xylan displayed ~100-fold stronger inhibition based on mass, suggesting that it may penetrate further into the tunnel and occupy a longer stretch of the active site.
|
|
| 11. |
- Haddad Momeni, Majid, et al.
(författare)
-
Structural insights into the inhibition of cellobiohydrolase Cel7A by xylo-oligosaccharides
- 2015
-
Ingår i: The FEBS Journal. - : Wiley. - 1742-464X. ; 282:11, s. 2167-2177
-
Tidskriftsartikel (refereegranskat)abstract
- The filamentous fungus Hypocreajecorina (anamorph of Trichodermareesei) is the predominant source of enzymes for industrial saccharification of lignocellulose biomass. The major enzyme, cellobiohydrolase Cel7A, constitutes nearly half of the total protein in the secretome. The performance of such enzymes is susceptible to inhibition by compounds liberated by physico-chemical pre-treatment if the biomass is kept unwashed. Xylan and xylo-oligosaccharides (XOS) have been proposed to play a key role in inhibition of cellobiohydrolases of glycoside hydrolase family7. To elucidate the mechanism behind this inhibition at a molecular level, we used X-ray crystallography to determine structures of H.jecorina Cel7A in complex with XOS. Structures with xylotriose, xylotetraose and xylopentaose revealed a predominant binding mode at the entrance of the substrate-binding tunnel of the enzyme, in which each xylose residue is shifted similar to 2.4 angstrom towards the catalytic center compared with binding of cello-oligosaccharides. Furthermore, partial occupancy of two consecutive xylose residues at subsites -2 and -1 suggests an alternative binding mode for XOS in the vicinity of the catalytic center. Interestingly, the -1 xylosyl unit exhibits an open aldehyde conformation in one of the structures and a ring-closed pyranoside in another complex. Complementary inhibition studies with p-nitrophenyl lactoside as substrate indicate mixed inhibition rather than pure competitive inhibition. DatabaseThe atomic coordinates and structure factors are available in the Protein Data Bank under accession number (H. jecorina Cel7A E212Q variant, complex with xylotriose), (H. jecorina Cel7A E217Q variant, complex with xylotriose), (H. jecorina Cel7A E212Q variant, complex with xylopentaose), (H. jecorina Cel7A E217Q variant, complex with xylopentaose), (wild-type H. jecorina Cel7A, complex with xylopentaose) and (H. jecorina Cel7A E217Q variant, complex with xylotetraose).
|
|
| 12. |
- Haddad Momeni, Majid, et al.
(författare)
-
The Mechanism of Cellulose Hydrolysis by a Two-Step, Retaining Cellobiohydrolase Elucidated by Structural and Transition Path Sampling Studies
- 2014
-
Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 136, s. 321-329
-
Tidskriftsartikel (refereegranskat)abstract
- Glycoside hydrolases (GHs) cleave glycosidic linkages in carbohydrates, typically via inverting or retaining mechanisms, the latter of which proceeds via a two-step mechanism that includes formation of a glycosyl-enzyme intermediate. We present two new structures of the catalytic domain of Hypocrea jecorina GH Family 7 cellobiohydrolase Cel7A, namely a Michaelis complex with a full cellononaose ligand and a glycosyl-enzyme intermediate, that reveal details of the 'static' reaction coordinate. We also employ transition path sampling to determine the 'dynamic' reaction coordinate for the catalytic cycle. The glycosylation reaction coordinate contains components of forming and breaking bonds and a conformational change in the nucleophile. Deglycosylation proceeds via a product-assisted mechanism wherein the glycosylation product, cellobiose, positions a water molecule for nucleophilic attack on the anomeric carbon of the glycosyl-enzyme intermediate. In concert with previous structures, the present results reveal the complete hydrolytic reaction coordinate for this naturally and industrially important enzyme family.
|
|
| 13. |
- Tiukova, Ievgeniia, et al.
(författare)
-
Adaptation of Dekkera bruxellensis to lignocellulose-based substrate
- 2014
-
Ingår i: Biotechnology and applied biochemistry. - : Wiley. - 0885-4513 .- 1470-8744. ; 61:1, s. 51-57
-
Tidskriftsartikel (refereegranskat)abstract
- Adaptation of Dekkera bruxellensis to lignocellulose hydrolysate was investigated. Cells of D. bruxellensis were grown for 72 and 192H in batch and continuous culture, respectively (adapted cells). Cultivations in semisynthetic medium were run as controls (nonadapted cells). To test the adaptation, cells from these cultures were reinoculated in the lignocellulose medium, and growth and ethanol production characteristics were monitored. Cells adapted to lignocellulose hydrolysate had a shorter lag phase, grew faster, and produced a higher ethanol concentration as compared with nonadapted cells. A stability test showed that after cultivation in rich medium, cells partially lost the adapted phenotype but still showed faster growth and higher ethanol production as compared with nonadapted cells. Because alcohol dehydrogenase genes have been described to be involved in the adaptation to furfural in Saccharomyces cerevisiae, an analogous mechanism of adaptation to lignocelluloses hydrolysate of D. bruxellensis was hypothesized. However, gene expression analysis showed that genes homologous to S. cerevisiae ADH1 were not involved in the adaptation to lignocelluloses hydrolysate in D. bruxellensis.
|
|
