Bioconversion of Agave tequilana fructans by exo-inulinases from indigenous Aspergillus niger CH-A-2010 enhances ethanol production from raw Agave tequilana juice

Agave tequilana fructans are the source of fermentable sugars for the production of tequila. Fructans are processed by acid hydrolysis or by cooking in ovens at high temperature. Enzymatic hydrolysis is considered an alternative for the bioconversion of fructans. We previously described the isolation of Aspergillus niger CH-A-2010, an indigenous strain that produces extracellular inulinases. Here we evaluated the potential application of A. niger CH-A-2010 inulinases for the bioconversion of A. tequilana fructans, and its impact on the production of ethanol. Inulinases were analyzed by Western blotting and thin layer chromatography. Optimal pH and temperature conditions for inulinase activity were determined. The efficiency of A. niger CH-A-2010 inulinases was compared with commercial enzymes and with acid hydrolysis. The hydrolysates obtained were subsequently fermented by Saccharomyces cerevisiae to determine the efficiency of ethanol production. Results indicate that A. niger CH-A-2010 predominantly produces an exo-inulinase activity. Optimal inulinase activity occurred at pH 5.0 and 50 °C. Hydrolysis of raw agave juice by CH-A-2010 inulinases yielded 33.5 g/l reducing sugars, compared with 27.3 g/l by Fructozyme^® (Novozymes Corp, Bagsværd, Denmark) and 29.4 g/l by acid hydrolysis. After fermentation of hydrolysates, we observed that the conversion efficiency of sugars into ethanol was 97.5 % of the theoretical ethanol yield for enzymatically degraded agave juice, compared to 83.8 % for acid-hydrolyzed juice. These observations indicate that fructans from raw Agave tequilana juice can be efficiently hydrolyzed by using A. niger CH-A-2010 inulinases, and that this procedure impacts positively on the production of ethanol.     

Efficient chemical and enzymatic saccharification of the lignocellulosic residue from <Emphasis Type="Italic">Agave tequilana</Emphasis> bagasse to produce ethanol by <Emphasis Type="Italic">Pichia caribbica</Emphasis>

Bagasse of Agave tequilana (BAT) is the residual lignocellulosic waste that remains from tequila production. In this study we characterized the chemical composition of BAT, which was further saccharified and fermented to produce ethanol. BAT was constituted by cellulose (42%), hemicellulose (20%), lignin (15%), and other (23%). Saccharification of BAT was carried out at 147°C with 2% sulfuric acid for 15 min, yielding 25.8 g/l of fermentable sugars, corresponding to 36.1% of saccharificable material (cellulose and hemicellulose contents, w/w). The remaining lignocellulosic material was further hydrolyzed by commercial enzymes, ~8.2% of BAT load was incubated for 72 h at 40°C rendering 41 g/l of fermentable sugars corresponding to 73.6% of the saccharificable material (w/w). Mathematic surface response analysis of the acid and enzymatic BAT hydrolysis was used for process optimization. The results showed a satisfactory correlation (R ² = 0.90) between the obtained and predicted responses. The native yeast Pichia caribbica UM-5 was used to ferment sugar liquors from both acid and enzymatic hydrolysis to ethanol yielding 50 and 87%, respectively. The final optimized process generated 8.99 g ethanol/50 g of BAT, corresponding to an overall 56.75% of theoretical ethanol (w/w). Thus, BAT may be employed as a lignocellulosic raw material for bioethanol production and can contribute to BAT residue elimination from environment.     

Low-Input Fermentations of <Emphasis Type="Italic">Agave tequilana</Emphasis> Leaf Juice Generate High Returns on Ethanol Yields

During tequila production, up to 75 % w/w of the Agave plant is discarded when leaves are removed from the stem. The discarded leaves represent an extensive amount of unexploited biomass that was used here for bioethanol production in no-input fermentations, where no acid or enzymatic hydrolysis, supplementation of nutrients or standardization of carbohydrate content occur. Ethanol yield from Agave leaf juice is unaffected by sterilization but reduced if fermentation is reliant solely on endogenous microorganisms. Non-Saccharomyces yeasts, including Kluyveromyces marxianus and Candida akabanensis, proved to be more robust than standard Saccharomyces spp. and yielded up to 88 % of the theoretical maximum ethanol from leaf juice. Combining leaf and stem juice, as from a whole plant, was predicted to maximize yield at up to 19,439 L/ha of ethanol from mature plants.     

Potential of Plants from the Genus Agave as Bioenergy Crops

Agave is a succulent genus within the monocot family Agavaceae. The plants have a large rosette of thick fleshy leaves, each ending generally in a sharp point, and are native to arid and semi-arid regions from the southern USA to northern South America. The most important commercial species is Agave tequilana grown for production of tequila. Several cultivated species of Agave such as Agave sislana and Agave salmiana can perform well in areas where rainfall is insufficient for the cultivation of many C₃ and C₄ crops. The key feature of the crassulacean acid metabolism photosynthetic pathway used by agaves is the stomata opening and CO₂ uptake during the night, thus allowing less water to be lost by transpiration. Alcoholic beverages, sweeteners, fibers, and some specialty chemicals are currently the main products coming from agave plants. The recovered information related to productivity, biofuel processability, by-products, etc. suggests that some Agave species have a real potential to compete economically with other bioenergy crops. But more than compete, it could complement the list of bioenergy crops due to its capacity to grow with very little rainfall and/or inputs and still reach good amount of biomass, so unused semi-arid land could be productive. Although Agave has great potential to be developed as a bioenergy crop, more laboratory and field research are needed.     

Marginal land bioethanol yield potential of four crassulacean acid metabolism candidates (Agave fourcroydes,Agave salmiana,Agave tequilana and Opuntia ficus‐indica) in Australia

The Nobel environmental productivity index (EPI) was used as a framework for the development of a predictive geospatial model to estimate the bioethanol yield potential of four crassulacean acid metabolism (CAM) candidates in Australia (Agave fourcroydes, Agave salmiana, Agave tequilana, and Opuntia ficus‐indica). GIS software was used to integrate climate datasets with titratable acidity responses to changes in photosynthetically active radiation (PAR), temperature, and water availability. Additional refinements to Nobel's approach were made to accommodate spatial and temporal fluctuations in soil water potential (ψs) as a function of soil particle size distribution and precipitation, and CO₂ uptake response to a range of day and night temperatures. A scalar factor for CO₂ persistence during periods of drought was also introduced to model the capacity of succulent species of Agave to buffer against fluctuations in ψs. Macro‐scale criteria were applied to estimate environmentally responsible (ER) bioethanol yield potential on lands that are not suitable for food production. Consideration was given to indigenous vascular plant species richness and endemism scores at ER sites of interest. The highest mean ER bioethanol yield was achieved by A. fourcroydes (μ: 3.89, max. 7.17 kL ha^‐1 yr^‐1) while the highest maximum yield was achieved by A. tequilana (μ: 3.78, max. 7.63 kL ha^‐1 yr^‐1). This research indicated the CAM pathway may produce significant yields (≥≥ 5 kL ha^‐1 yr^‐1) at ER sites totalling 57,700 km² (0.7% land area of Australia).     

Environmental influences on CO2 uptake by agaves,cam plants with high productivities

Agaves have long been utilized for their leaf fiber and for beverage production. As first reported in 1968 for Agave americana, they are Crassulacean Acid Metabolism (CAM) plants, for which stomatal opening and CO₂ uptake occur primarily at night when the lower temperatures greatly reduce water loss. More recently, the influences of rainfall, temperature, and photosynthetically active radiation on CO₂ uptake by agaves have been determined and incorporated into an Environmental Productivity Index (EPI). Nutrient effects on CO₂ uptake and growth can be quantified by a Nutrient Index, which multiplies EPI to account for soil element effects. New growth data forAgave victoriae-reginae are consistent with the Nutrient Index, except that high soil potassium levels inhibited dry weight increases and sodium was somewhat more inhibitory than expected. Productivities of agaves are high, the 25 tons dry weight hectare^-1 yr^-1 achievable byAgave mapisaga, A. salmiana, and A. tequilana exceeding the productivity of most annual agricultural crops. Often interest is focused on a specific harvestable plant part, such as the stems ofA. tequilana, which are harvested for tequila production. These stems have threefold higher levels of nonstructural carbohydrates such as sugars and polysaccharides than do the leaves; levels of such carbohydrates tend to be higher at times of the year with higher EPI, new data that can affect traditional harvesting practices. In conclusion, because of CAM, agaves can have high productivities in regions of moderate annual rainfall, and because of EPI, such productivity can be predicted, which augurs well for the increased future cultivation of agaves.     

Fuzzy GIS‐based multi‐criteria evaluation for US Agave production as a bioenergy feedstock

In the United States, renewable energy mandates calling for increased production of cellulosic biofuels will require a diversity of bioenergy feedstocks to meet growing demands. Within the suite of potential energy crops, plants within the genus Agave promise to be a productive feedstock in hot and arid regions. The potential distributions of Agave tequilana and Agave deserti in the United States were evaluated based on plant growth parameters identified in an extensive literature review. A geospatial suitability model rooted in fuzzy logic was developed that utilized a suite of biophysical criteria to optimize ideal geographic locations for this new crop, and several suitability scenarios were tested for each species. The results of this spatially explicit suitability model suggest that there is potential for Agave to be grown as an energy feedstock in the southwestern region of the United States – particularly in Arizona, California, and Texas – and a significant portion of these areas are proximate to existing transportation infrastructure. Both Agave species showed the highest state‐level renewable energy benefit in Arizona, where agave plants have the potential to contribute 4.8–9.6% of the states' ethanol consumption, and 2.5–4.9% of its electricity consumption, for A. deserti and A. tequilana, respectively. This analysis supports the feasibility of Agave as a complementary bioenergy feedstock that can be grown in areas too harsh for conventional energy feedstocks.     

Effect of <Emphasis Type="Italic">Agave tequilana</Emphasis> juice on cell wall polysaccharides of three <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains from different origins

In this study, a characterization of cell wall polysaccharide composition of three yeasts involved in the production of agave distilled beverages was performed. The three yeast strains were isolated from different media (tequila, mezcal and bakery) and were evaluated for the β(1,3)-glucanase lytic activity and the β-glucan/mannan ratio during the fermentation of Agave tequilana juice and in YPD media (control). Fermentations were performed in shake flasks with 30 g l⁻¹ sugar concentration of A. tequilana juice and with the control YPD using 30 g l⁻¹ of glucose. The three yeasts strains showed different levels of β-glucan and mannan when they were grown in A. tequilana juice in comparison to the YPD media. The maximum rate of cell wall lyses was 50% lower in fermentations with A. tequilana juice for yeasts isolated from tequila and mezcal than compared to the bakery yeast.     

Expression of the 1-SST and 1-FFT genes and consequent fructan accumulation in Agave tequilana and A. inaequidens is differentially induced by diverse (a)biotic-stress related elicitors

The expression of genes coding for sucrose:sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99) and fructan:fructan 1-fructosyltransferase (1-FFT; EC 2.4.1.100), both fructan biosynthesizing enzymes, characterization by TLC and HPAEC-PAD, as well as the quantification of the fructo-oligosaccharides (FOS) accumulating in response to the exogenous application of sucrose, kinetin (cytokinin) or other plant hormones associated with (a)biotic stress responses were determined in two Agave species grown in vitro, domesticated Agave tequilana var. azul and wild A. inaequidens. It was found that elicitors such as salicylic acid (SA), and jasmonic acid methyl ester (MeJA) had the strongest effect on fructo-oligosaccharide (FOS) accumulation. The exogenous application of 1 mM SA induced a 36-fold accumulation of FOS of various degrees of polymerization (DP) in stems of A. tequilana. Other treatments, such as 50 mM abscisic acid (ABA), 8% Sucrose (Suc), and 1.0 mg L⁻¹ kinetin (KIN) also led to a significant accumulation of low and high DP FOS in this species. Conversely, treatment with 200 μM MeJA, which was toxic to A. tequilana, induced an 85-fold accumulation of FOS in the stems of A. inaequidens. Significant FOS accumulation in this species also occurred in response to treatments with 1 mM SA, 8% Suc, and 10% polyethylene glycol (PEG). Maximum yields of 13.6 and 8.9 mg FOS per g FW were obtained in stems of A. tequilana and A. inaequidens, respectively. FOS accumulation in the above treatments was tightly associated with increased expression levels of either the 1-FFT or the 1-SST gene in tissues of both Agave species.     

Ethanol production by Escherichia coli from detoxified lignocellulosic teak wood hydrolysates with high concentration of phenolic compounds

Teak wood residues were subjected to thermochemical pretreatment, enzymatic saccharification, and detoxification to obtain syrups with a high concentration of fermentable sugars for ethanol production with the ethanologenic Escherichia coli strain MS04. Teak is a hardwood, and thus a robust deconstructive pretreatment was applied followed by enzymatic saccharification. The resulting syrup contained 60 g l^–1 glucose, 18 g l^–1 xylose, 6 g l^–1 acetate, less than 0.1 g l^–1 of total furans, and 12 g l^–1 of soluble phenolic compounds (SPCs). This concentration of SPC is toxic to E. coli, and thus two detoxification strategies were assayed: (1) treatment with Coriolopsis gallica laccase followed by addition of activated carbon and (2) overliming with Ca(OH)₂. These reduced the phenolic compounds by 40% and 76%, respectively. The detoxified syrups were centrifuged and fermented with E. coli MS04. Cultivation with the overlimed hydrolysate showed a 60% higher volumetric productivity (0.45 g_ETOH l^–1 hr^–1). The bioethanol/sugar yield was over 90% in both strategies.     

Watermelon juice: a promising feedstock supplement, diluent, and nitrogen supplement for ethanol biofuel production

Background  

Two economic factors make watermelon worthy of consideration as a feedstock for ethanol biofuel production. First, about 20% of each annual watermelon crop is left in the field because of surface blemishes or because they are misshapen; currently these are lost to growers as a source of revenue. Second, the neutraceutical value of lycopene and L-citrulline obtained from watermelon is at a threshold whereby watermelon could serve as starting material to extract and manufacture these products. Processing of watermelons to produce lycopene and L-citrulline, yields a waste stream of watermelon juice at the rate of over 500 L/t of watermelons. Since watermelon juice contains 7 to 10% (w/v) directly fermentable sugars and 15 to 35 μmol/ml of free amino acids, its potential as feedstock, diluent, and nitrogen supplement was investigated in fermentations to produce bioethanol.     

Comparison between Yeasts from Grape and Agave Musts for Traits of Technological interest

Summary In Mexico there are different alcoholic beverages produced from agave juices from different agave plants, which are cooked, fermented and distilled. For tequila production only Agave tequilana is allowed. In this study we compared yeast strains of different species from different origin (agave and grape juice) for parameters of technological interest, such as SO₂ and copper resistance, ethanol tolerance and enzymatic activities. All agave strains were found to be more resistant to SO₂ and agave non-Saccharomyces yeasts were more tolerant to ethanol, whereas grape strains exhibited positive results for β-glucosidase and β-xylosidase activities. As regards fermentations of Agave tequilana juice with ethanol added at different concentrations, only agave Saccharomyces strains were more tolerant to ethanol than grape strains.     

Fermentation of Agave tequilana juice by Kloeckera africana: influence of amino-acid supplementations

This study aimed to improve the fermentation efficiency of Kloeckera africana K1, in tequila fermentations. We investigated organic and inorganic nitrogen source requirements in continuous K. africana fermentations fed with Agave tequilana juice. The addition of a mixture of 20 amino-acids greatly improved the fermentation efficiency of this yeast, increasing the consumption of reducing sugars and production of ethanol, compared with fermentations supplemented with ammonium sulfate. The preference of K. africana for each of the 20 amino-acids was further determined in batch fermentations and we found that asparagine supplementation increased K. africana biomass production, reducing sugar consumption and ethanol production (by 30, 36.7 and 45%, respectively) over fermentations supplemented with ammonium sulfate. Therefore, asparagine appears to overcome K. africana nutritional limitation in Agave juice. Surprisingly, K. africana produced a high concentration of ethanol. This contrasts to poor ethanol productivities reported for other non-Saccharomyces yeasts indicating a relatively high ethanol tolerance for the K. africana K1 strain. Kloeckera spp. strains are known to synthesize a wide variety of volatile compounds and we have shown that amino-acid supplements influenced the synthesis by K. africana of important metabolites involved in the bouquet of tequila. The findings of this study have revealed important nutritional limitations of non-Saccharomyces yeasts fermenting Agave tequilana juice, and have highlighted the potential of K. africana in tequila production processes.     

Production of Fermentable Sugars and Hydrogen-Rich Gas from <Emphasis Type="Italic">Agave tequilana</Emphasis> Biomass

The Mexican tequila industry annually processes approximately 1 × 10⁶ Agave tequilana plants, generating approximately 1.78 × 10⁸ kg of bagasse per year. This biomass is considered an attractive alternative to fossil fuels as an energy source and to produce biofuels and/or chemical products because it is produced and used without adversely affecting the environment. The first aim of the present work was to determine the effect of temperature, the concentration of H₂SO_4, and reaction time on the hydrolysis of agave bagasse to maximize the fermentable sugars using a steam explosion. This step process generated 71.11 g/L of reducible sugars in the supernatant (59.29 % glucose, 29.05 % xylose, and 11.66 % fructose) and unconverted organic matter of enzymatic hydrolysis bagasse (35.4 % α-cellulose, 7.33 % hemicellulose, 49.91 % lignin, and 7.31 % ashes). A mathematical surface response analysis of the hydrolysis was used for process optimization. The second aim involves the study of the thermodynamics of the reforming of unconverted organic matter from enzymatic hydrolysis of Agave tequilana bagasse (ATB) evaluated by the Gibbs free energy minimization method for hydrogen production. The effect of the parameters on the system performance measures, such as reaction temperature (T), Water/Biomass ratio (WBR), and pressure (P), were also investigated. The maximum H₂ production obtained was 23.2 mol of H₂/271.5 g ATB with a WBR ≥ 11 and a temperature of 740 °C. These findings indicate that the temperature and WBR are essential factors in the production of H₂, which was reflected in the efficiency of the process.     

Productivity and water use efficiency of Agave americana in the first field trial as bioenergy feedstock on arid lands

Agave species are high‐yielding crassulacean acid metabolism (CAM) plants, some of which are grown commercially and recognized as potential bioenergy species for dry regions of the world. This study is the first field trial of Agave species for bioenergy in the United States, and was established to compare the production of Agave americana with the production of Agave tequilana and Agave fourcroydes, which are produced commercially in Mexico for tequila and fiber. The field trial included four experimental irrigation levels to test the response of biomass production to water inputs. After 3 years, annual production of healthy A. americana plants reached 9.3 Mg dry mass ha^?1 yr^?1 (including pup mass) with 530 mm of annual water inputs, including both rainfall and irrigation. Yields in the most arid conditions tested (300 mm yr^?1 water input) were 2.0–4.0 Mg dry mass ha^?1 yr^?1. Agave tequilana and Agave fourcroydes were severely damaged by cold in the first winter, and produced maximum yields of only 0.04 Mg ha^?1 yr^?1 and 0.26 Mg ha^?1 yr^?1, respectively. The agave snout weevil (Scyphophorus acupunctatus) emerged as an important challenge for A. americana cropping, killing a greater number of plants in the higher irrigation treatments. Physiological differences in A. americana plants across irrigation treatments were most evident in the warmest season, with gas exchange beginning up to 3 h earlier and water use efficiency declining in treatments with the greatest water input (780 mm yr^?1 water input). Yields were lower than previous projections for Agave species, but results from this study suggest that A. americana has potential as a bioenergy crop and would have substantially reduced irrigation requirements relative to conventional crops in the southwestern USA. Challenges for pest management and harvesting must still be addressed before an efficient production system that uses Agave can be realized.     

Comparative Secretome Analysis of Trichoderma reesei and Aspergillus niger during Growth on Sugarcane Biomass

Background

Our dependence on fossil fuel sources and concern about the environment has generated a worldwide interest in establishing new sources of fuel and energy. Thus, the use of ethanol as a fuel is advantageous because it is an inexhaustible energy source and has minimal environmental impact. Currently, Brazil is the world''s second largest producer of ethanol, which is produced from sugarcane juice fermentation. However, several studies suggest that Brazil could double its production per hectare by using sugarcane bagasse and straw, known as second-generation (2G) bioethanol. Nevertheless, the use of this biomass presents a challenge because the plant cell wall structure, which is composed of complex sugars (cellulose and hemicelluloses), must be broken down into fermentable sugar, such as glucose and xylose. To achieve this goal, several types of hydrolytic enzymes are necessary, and these enzymes represent the majority of the cost associated with 2G bioethanol processing. Reducing the cost of the saccharification process can be achieved via a comprehensive understanding of the hydrolytic mechanisms and enzyme secretion of polysaccharide-hydrolyzing microorganisms. In many natural habitats, several microorganisms degrade lignocellulosic biomass through a set of enzymes that act synergistically. In this study, two fungal species, Aspergillus niger and Trichoderma reesei, were grown on sugarcane biomass with two levels of cell wall complexity, culm in natura and pretreated bagasse. The production of enzymes related to biomass degradation was monitored using secretome analyses after 6, 12 and 24 hours. Concurrently, we analyzed the sugars in the supernatant.

Results

Analyzing the concentration of monosaccharides in the supernatant, we observed that both species are able to disassemble the polysaccharides of sugarcane cell walls since 6 hours post-inoculation. The sugars from the polysaccharides such as arabinoxylan and β-glucan (that compose the most external part of the cell wall in sugarcane) are likely the first to be released and assimilated by both species of fungi. At all time points tested, A. niger produced more enzymes (quantitatively and qualitatively) than T. reesei. However, the most important enzymes related to biomass degradation, including cellobiohydrolases, endoglucanases, β-glucosidases, β-xylosidases, endoxylanases, xyloglucanases, and α-arabinofuranosidases, were identified in both secretomes. We also noticed that the both fungi produce more enzymes when grown in culm as a single carbon source.

Conclusion

Our work provides a detailed qualitative and semi-quantitative secretome analysis of A. niger and T. reesei grown on sugarcane biomass. Our data indicate that a combination of enzymes from both fungi is an interesting option to increase saccharification efficiency. In other words, these two fungal species might be combined for their usage in industrial processes.     

Seed germination of Agave species as influenced by substrate water potential

Background

Plants of Agave spp. perform Crassulacean acid metabolism (CAM) and are highly drought-tolerant, but little is known concerning seed germination under low water availability. The aim of this study was to assess the effect of substrate water potential (Ψ_W) on seed germination and contrast hydrotime parameters of seven valuable and commercially-important Agave species from different geographical distributions and climatic regions of Mexico. Our hypothesis was that seed germination of Agave species is not affected by low water availability independently of seed biomass and the climate of their distribution area.

Results

Seed germination (at 25°C and in the dark) between 85 and 100% for all species occurred within 80–180 h at -0.03 MPa and 250–430 h at -1.0 MPa. Seed germination at -1.5 MPa declined to less than 50% (p < 0.05) for A. asperrima and A. cupreata but did not change significantly for A. americana var. marginata, A. lechuguilla and A. striata, although they showed the lowest mean base water potential (-2.01 to -2.64 MPa). Seed germination of 40% Agave species, from arid and semi-arid climates in this study, was not affected by the lower Ψ_W.

Conclusion

Germination of seeds of Agave species is moderately affected by low water availability, is partially dependent of their ecological distribution, and is independent of seed mass.     

Performance evaluation of Pichia kluyveri, Kluyveromyces marxianus and Saccharomyces cerevisiae in industrial tequila fermentation

Traditionally, industrial tequila production has used spontaneous fermentation or Saccharomyces cerevisiae yeast strains. Despite the potential of non-Saccharomyces strains for alcoholic fermentation, few studies have been performed at industrial level with these yeasts. Therefore, in this work, Agave tequilana juice was fermented at an industrial level using two non-Saccharomyces yeasts (Pichia kluyveri and Kluyveromyces marxianus) with fermentation efficiency higher than 85 %. Pichia kluyveri (GRO3) was more efficient for alcohol and ethyl lactate production than S. cerevisiae (AR5), while Kluyveromyces marxianus (GRO6) produced more isobutanol and ethyl-acetate than S. cerevisiae (AR5). The level of volatile compounds at the end of fermentation was compared with the tequila standard regulation. All volatile compounds were within the allowed range except for methanol, which was higher for S. cerevisiae (AR5) and K. marxianus (GRO6). The variations in methanol may have been caused by the Agave tequilana used for the tests, since this compound is not synthesized by these yeasts.     

Relative genetic diversity of the rare and endangered Agave shawii ssp. shawii and associated soil microbes within a southern California ecological preserve

Shaw''s Agave (Agave shawii ssp. shawii) is an endangered maritime succulent growing along the coast of California and northern Baja California. The population inhabiting Point Loma Peninsula has a complicated history of transplantation without documentation. The low effective population size in California prompted agave transplanting from the U.S. Naval Base site (NB) to Cabrillo National Monument (CNM). Since 2008, there are no agave sprouts identified on the CNM site, and concerns have been raised about the genetic diversity of this population. We sequenced two barcoding loci, rbcL and matK, of 27 individual plants from 5 geographically distinct populations, including 12 individuals from California (NB and CNM). Phylogenetic analysis revealed the three US and two Mexican agave populations are closely related and have similar genetic variation at the two barcoding regions, suggesting the Point Loma agave population is not clonal. Agave‐associated soil microbes used significantly more carbon sources in CNM soil samples than in NB soil likely due to higher pH and moisture content; meanwhile, soil type and soil chemistry analysis including phosphorus, nitrate nitrogen, organic matter, and metals revealed significant correlations between microbial diversity and base saturation (p < 0.05, r ² = 0.3676), lime buffer capacity (p < 0.01, r ² = 0.7055), equilibrium lime buffer capacity (p < 0.01, r ² = 0.7142), and zinc (p < 0.01, r ² = 0.7136). Soil microbiome analysis within the CNM population revealed overall expected richness (H′ = 5.647–6.982) for Agave species, while the diversity range (1 − D = 0.003392–0.014108) suggests relatively low diversity marked by high individual variation. The most prominent remaining US population of this rare species is not clonal and does not seem to be threatened by a lack of genetic and microbial diversity. These results prompt further efforts to investigate factors affecting Agave''s reproduction and fitness.     

Recalcitrance Assessment of the Agro-industrial Residues from Five <Emphasis Type="Italic">Agave</Emphasis> Species: Ionic Liquid Pretreatment,Saccharification and Structural Characterization

Agave has recently shown its potential as a bioenergy feedstock with promising features such as higher biomass productivity than leading bioenergy feedstock while at the same time being drought-resistant with low water requirements and high sugar to ethanol conversion using ionic liquid (IL) pretreatment. IL pretreatment was studied to develop the first direct side-by-side comparative recalcitrance assessment of the agro-industrial residues from five Agave species [Agave americana (AME), A. angustifolia (ANG), A. fourcroydes (FOU), A. salmiana (SAL), and A. tequilana (TEQ)] using compositional analysis, X-ray diffraction, and the lignin syringyl/guaiacyl subunit ratio (S/G) by pyrolysis molecular beam mass spectrometry (PyMBMS). Prominent calcium oxalate peaks were found only in unpretreated AME, SAL, and TEQ. The S/G ratios of all five unpretreated Agave species were between 1.27 and 1.57 while the IL-pretreated samples were from 1.39 to 1.72. The highest overall sugar production was obtained with IL-pretreated FOU with 492 mg glucose/g biomass and 157 mg xylose/g biomass at 120 °C and 3 h using 1-ethyl-3-methylimidazolium acetate ([C₂C₁Im][OAc]). An estimated theoretical ethanol yield from the studied agro-industrial residues from the five Agave species was in the range of 1060 to 5800 L ethanol/ha/year. These comparison results demonstrate the potential of the Agave spp. as a suitable biofuel feedstock which can be employed within a biorefinery scheme.