Green Microalga Botryococcus Braunii Research Articles

Enhancing biomass, hydrocarbon and biodiesel properties of green microalga Botryococcus braunii KMITL through gamma and UV radiation exposure.

This study investigated the effects of gamma (137Cs, 0-250Gy) and UV (UV-C, 0-12h) radiation on growth and biodiesel properties of Botryococcus braunii KMITL. For gamma radiation, maximum biomass (1.37 ± 0.02g L-1) was achieved with 50Gy, while a dose of 200Gy resulted in the highest hydrocarbon content (51.84 ± 0.20%) and yield (0.66 ± 0.01g L-1). For UV radiation, a 9h exposure produced the highest biomass (2.45 ± 0.05g L-1), hydrocarbon content (55.01 ± 1.22%), and yield (1.35 ± 0.04g L-1). Algae exposed to gamma radiation within the range of 0-150Gy exhibited C16:0 as the dominant fatty acid methyl ester (FAME), similar to those exposed to UV radiation, while algae exposed to 200-250Gy displayed C18:1n9t as the dominant FAME. High levels of gamma and UV radiation were observed to lengthen fatty acid chains and increase unsaturated fatty acids. The cetane values of biodiesel from algae exposed to gamma and UV radiation ranged from 64.55 ± 0.14-66.47 ± 0.20 and 59.43 ± 0.04-65.27 ± 0.22, respectively, all meeting standard criteria. Both gamma and UV radiation also improved the saponification value and cold flow properties of the biodiesel. These findings suggest that controlled levels of gamma and UV radiation effectively enhance hydrocarbon yields with significant implications for biofuel production.

Reclassification of Botryococcus braunii chemical races into separate species based on a comparative genomics analysis.

The colonial green microalga Botryococcus braunii is well known for producing liquid hydrocarbons that can be utilized as biofuel feedstocks. B. braunii is taxonomically classified as a single species made up of three chemical races, A, B, and L, that are mainly distinguished by the hydrocarbons produced. We previously reported a B race draft nuclear genome, and here we report the draft nuclear genomes for the A and L races. A comparative genomic study of the three B. braunii races and 14 other algal species within Chlorophyta revealed significant differences in the genomes of each race of B. braunii. Phylogenomically, there was a clear divergence of the three races with the A race diverging earlier than both the B and L races, and the B and L races diverging from a later common ancestor not shared by the A race. DNA repeat content analysis suggested the B race had more repeat content than the A or L races. Orthogroup analysis revealed the B. braunii races displayed more gene orthogroup diversity than three closely related Chlamydomonas species, with nearly 24-36% of all genes in each B. braunii race being specific to each race. This analysis suggests the three races are distinct species based on sufficient differences in their respective genomes. We propose reclassification of the three chemical races to the following species names: Botryococcus alkenealis (A race), Botryococcus braunii (B race), and Botryococcus lycopadienor (L race).

Effect of nitrogen source and nickel concentration on green microalga Botryococcus braunii growth and its remediation potential

Effect of nitrogen source and nickel concentration on green microalga Botryococcus braunii growth and its remediation potential

High-efficiency production of biomass and biofuel under two-stage cultivation of a stable microalga Botryococcus braunii mutant generated by ethyl methanesulfonate-induced mutation

Green microalga Botryococcus braunii is a eukaryotic photosynthetic microorganism considered as a rich source of biofuel compounds, such as hydrocarbons and lipids, despite its rather slow growth. To improve the biomass, hydrocarbon, and lipid productivity, an ethyl methanesulfonate-induced mutagenesis was carried out on the indigenous microalga, Botryococcus braunii AARL G036. Among those mutants, EMS-mutant E1.0H15 gave the highest biomass and hydrocarbon productivity, which was higher than 1.17, 1.45, and 1.16-fold, respectively, when compared with the wild type (WT). Subsequently, two-stage cultivation with crucial stress factors was successfully exploited to boost the biofuel productivity of EMS-mutant E1.0H15 by increasing the lipid content by 1.22-fold (up to 48.6%) and by 1.20-fold for the hydrocarbon content (up to 71.6%) compared to one-stage cultivation. More importantly, after 24-month maintenance, the high biofuel productivity in EMS-mutant E1.0H15 remains unchanged. In addition, the fatty acid composition (>96% C16–C18) and fuel properties of the mutated microalgal lipid-derived biodiesel indicated good oxidation stability (17.5 h) and high heating values (38.6 MJ kg−1), which were in accordance with those of the international standards, i.e., EN-14214 and ASTM D675140. This strategy could contribute greatly to the further development of the microalgal strain, allowing improvement and cultivation for the desired biofuel properties.

In situ extraction (milking) of the two promising Botryococcus braunii strains Showa and Bot22 under optimized extraction time

In situ extraction or “milking” of microalgae is a promising approach to reduce downstream costs in order to produce low-value substances such as lipids from microalgae in an economical way. Due to its ability to secrete high amounts of long-chain hydrocarbons to an extracellular matrix, the green microalga Botryococcus braunii is suitable for the process of in situ extraction as the cost intensive steps of harvesting, dewatering, and cell disruption could be omitted. Based on a previous study investigating various B. braunii strains in terms of growth, lipid accumulation, and solvent compatibility, the B. braunii strains Showa and Bot22 (both B race) were identified as potential candidates for the process of in situ extraction. In order to prove the suitability of these two strains for the process of in situ extraction, this study first determined the optimal extraction time using short-term in situ extraction over 7 days at different starting biomass concentrations of 1.5 and 2.5 g L−1. Furthermore, both strains were treated applying the optimal extraction time in long-term in situ extractions for 30 days to confirm the results from the short-term extractions. The results indicate a strain-dependent optimal extraction time of 300 min day−1 for strain Showa and 200 min day−1 for strain Bot22. During long-term in situ extraction for 30 days, hydrocarbon productivity was 16.99 mg L−1 day−1 (10.53 mg gDW−1 day−1) for strain Showa and 14.53 mg L−1 day−1 (10.48 mg gDW−1 day−1) for strain Bot22. Furthermore, a direct correlation between hydrocarbon productivity achieved by in situ extraction and the hydrocarbon concentration in the biomass of the respective strain could be established. It could be shown that the consideration of the effective extraction time and the phase boundary area is required to calculate an extraction system independent value for the comparison of different extraction setups.

Structure of the polysaccharide sheath from the B race of the green microalga Botryococcus braunii

The green microalga Botryococcus braunii forms a colony of cells held together by an intricate extracellular matrix. This extracellular matrix also acts as a storage location for liquid hydrocarbons initially produced inside the cells. These hydrocarbons can be used as feedstocks to produce transportation fuels. In the B race of B. braunii, the extracellular matrix is composed of several elements: a cross-linked hydrocarbon network filled with the liquid hydrocarbons, a retaining wall to hold the hydrocarbons in the extracellular matrix, an arabinogalactan polysaccharide fibrillar sheath that extends outward from the retaining wall, and a single protein known as polysaccharide associated protein is found at the base of the polysaccharide fibers where they connect to the retaining wall. During growth, colonies of the B race of B. braunii shed fragments of the retaining wall with polysaccharide fibrils attached into the media. Here we describe the isolation and structural analysis of these polysaccharides using a series of chemical degradations in combination with glycosyl residue composition and linkage analysis, as well as 2-dimensional nuclear magnetic resonance spectroscopy. The results demonstrate that the polysaccharide is highly branched, consisting of a galactan backbone with short branches comprised of d-galactopyranose, l-arabinofuranose, 4-O-methyl-d-glucopyranuronic acid, and 6-deoxy-d-altropyranose. To our knowledge, this is the first description of 6-deoxyaltrose in algae or plants.

Identification of suitable Botryococcus braunii strains for non-destructive in situ hydrocarbon extraction

The process of milking microalgae is a promising approach to reduce the downstream costs for the production of valuable substances from microalgae by avoiding the steps of harvest, dewatering, and cell disruption of the common process chain (cultivation, harvesting, dewatering, cell disruption, extraction, purification). The green microalga Botryococcus braunii is particularly suited for this process due to its ability to produce large amounts of long-chain hydrocarbons accumulating in an extracellular matrix. The extracellular location of hydrocarbons is an enormous advantage in comparison with other microalgae that accumulate lipids in intracellular lipid bodies. At present, only a few B. braunii strains (UTEX 572, CCAP 807/2, SAG 807/1, FACHB 357, Bot22, and SCCAP 1761) have been examined for the process of long-term repetitive milking. In order to identify promising candidates for the milking process, twelve different B. braunii strains (SAG 30.81, SAG 807/1, UTEX 572, UTEX 2441, CCAP 807/2, ACOI 58, ACOI 1257, SCCAP K-1489, var. Showa, Bot22, SCCAP K-1761, and CCALA 779) were investigated in terms of growth, lipid accumulation, nutrient uptake, solvent compatibility, and extracellular hydrocarbon extractability. Based on these results, a ranking was defined in view of eligibility for non-destructive hydrocarbon extraction. Results indicate a particular potential for hydrocarbon milking for two of those twelve B. braunii strains. The strain Showa (71 out of 75 ranking points), which has not yet been examined for long-term repetitive milking, and the strain Bot22 (64 out of 75 ranking points) seem to be the most suitable strains for the milking process. They both possess good extractant compatibility including hydrocarbon extractability as well as high biomass and lipid productivity.

Improving the Energy Balance of Hydrocarbon Production Using an Inclined Solid–Liquid Separator with a Wedge-Wire Screen and Easy Hydrocarbon Recovery from Botryococcus braunii

The green colonial microalga Botryococcus braunii produces large amounts of hydrocarbons and has attracted attention as a potential source of biofuel. When this freshwater microalga is cultured in a brackish medium, the hydrocarbon recovery rate increases; furthermore, the colony size becomes large. In this study, the effects of such changes on the energy balance of harvesting and hydrocarbon recovery were studied via filtrate experiments on an inclined separator and extraction from a concentrated slurry. The inclined separator was effective for harvesting large-colony-forming algae. The water content on the wire screen of slit sizes larger than 150 µm was <80% and a separation rate of >85% could be achieved. The input energy of the harvesting using the brackish medium with this separator was ≈44% of that using the freshwater medium with vacuum filtration, while the input energy of the hydrocarbon recovery using the brackish medium was ≈88% of that using the freshwater medium with pre-heating before n-hexane extraction. Furthermore, the energy profit ratio of the process in the brackish medium was 2.92, which was ≈1.2 times higher than that in the freshwater medium. This study demonstrated that filtration techniques and hydrocarbon recovery from B. braunii with a low energy input through culture in a brackish medium are viable.

Isotopically enriched n-alkan-2-ones with even chain predominance in a torbanite from the Sydney Basin, Australia

n-Alkan-2-ones (methyl ketones) ranging from C14 to C31, were recovered from the polar fraction of an extract of a torbanite from the Sydney Basin, Australia. There was a very slight even-over-odd predominance between C20 and C24n-alkan-2-ones, together with remarkably high contents of the C24, C26 and C28 homologues. A range of C16 to C28 macrocyclic alkanes, as well as their corresponding methylated counterparts (C19–C24), was also detected in the saturate fraction obtained after molecular sieve treatments. Due to the relatively high contents of the n-alkan-2-ones and macrocyclic alkanes, we were able to carry out GC–irMS analysis to determine the δ13C values of specific compounds. The even-numbered n-alkan-2-ones from C24 to C28 were significantly 13C-enriched, compared to the other n-alkan-2-ones, which had similar isotopic values to the macrocyclic alkanes. The majority of the n-alkan-2-ones detected here probably had the same algal source as the macrocyclic alkanes, which is likely to be lipids and algaenan of the green microalga Botryococcus braunii. However, the distribution and 13C enrichment of C24, C26 and C28n-alkan-2-ones indicates that an additional source must be present.

Outdoor open pond batch production of green microalga Botryococcus braunii for high hydrocarbon production: enhanced production with salinity

The aim of this work was to enhance the biodiesel quality and hydrocarbon content of green microalga B. braunii strain KMITL 2 cultivated outdoor under several salinity conditions in a batch production. The enhancement would be such that the microalgal biodiesel qualities met or exceeded the current standard so that it would be a good raw material for biodiesel production. The microalga production was in 300 L open oval ponds, among various salinity levels tested (0–20 ppt), 5 ppt was the best for hydrocarbon production, yielding 54.2 ± 0.9% hydrocarbon content and 5.1 ± 0.4 g L−1 biomass. As the microalga production was scaled up by cultivation in 3,675 L open raceway pond under the 5 ppt condition, the microalga yielded a bit higher hydrocarbon content (58.8 ± 2.9%) but much lower biomass (2.5 ± 0.5 g L−1). The production in both oval and raceway ponds yielded a nearly identical biodiesel property (61.06–67.42 cetane number) which exceeded the value specified in international standards. Therefore, it was concluded that B. braunii strain KMITL 2 can be batch cultivated in an open pond at optimum salinity to yield sufficient hydrocarbon and biomass for biodiesel production.

Detection of the oil-producing microalga Botryococcus braunii in natural freshwater environments by targeting the hydrocarbon biosynthesis gene SSL-3

The green microalga Botryococcus braunii produces hydrocarbon oils at 25–75% of its dry weight and is a promising source of biofuel feedstock. Few studies have examined this species’ ecology in natural habitats, and few wild genetic resources have been collected due to difficulties caused by its low abundance in nature. This study aimed to develop a real-time PCR assay for specific detection and quantification of this alga in natural environments and to quantify spatiotemporal variations of wild B. braunii populations in a tropical pond. We designed PCR primers toward the hydrocarbon biosynthesis gene SSL-3 and examined amplification specificity and PCR efficiency with 70 wild strains newly isolated from various environments. The results demonstrated that this PCR assay specifically amplified B. braunii DNA, especially that of B-race strains, and can be widely used to detect wild B. braunii strains in temperate and tropical habitats. Field-testing in a tropical pond suggested a diurnal change in the abundance of B. braunii in surface water and found B. braunii not only in surface water, but also at 1–1.5 m deep and in bottom sediments. This method can contribute to efficient genetic resource exploitations and may also help elucidate the unknown ecology of B. braunii.

Docking analysis of models for 4-hydroxy-3-methylbut-2-enyl diphosphate reductase and a ferredoxin from Botryococcus braunii, race B.

The green microalga Botryococcus braunii Showa, which produces large amounts of triterpene hydrocarbons, exclusively uses the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosyntheses, and the terminal enzyme in this pathway, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR), is regarded as a light-dependent key regulatory enzyme. In order to investigate the possible association of HDR and ferredoxin in this organism, we constructed tertiary structure models of B. braunii HDR (BbHDR) and one of ferredoxin families in the alga, a photosynthetic electron transport F (BbPETF)-like protein, by using counterparts from E. coli and Chlamydomonas reinhardtii as templates, respectively, and performed docking analysis of these two proteins. After docked models are superimposed onto their counterpart proteins in a non-photosynthetic organism, Plasmodium falciparum, the BbPETF-like protein comes in contact with the backside of BbHDR, which was defined in a previous report (Rekittke et al. 2013), and the distance of the two Fe-S centers is 14.7 Å. This distance is in almost the same level as that for P. falicarum, 12.6 Å. To our knowledge, this is the first model suggesting the possible association of HDR with a ferredoxin in O2-evolving photosynthetic organisms.

Isolation and characterization of 4-hydroxy-3-methylbut-2-enyl diphosphate reductase gene from Botryococcus braunii, race B.

The B race of a green microalga Botryococcus braunii Kützing produces triterpene hydrocarbons that is a promising source for biofuel. In this algal race, precursors of triterpene hydrocarbons are provided from the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. The terminal enzyme of this pathway, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is regarded as one of the key enzymes that affect yields of products in terpene biosynthesis. In order to better understand the MEP pathway of the alga, cDNA and genomic clones of HDR were obtained from B. braunii Showa strain. B. braunii HDR (BbHDR) is encoded on a single copy gene including a 1509-bp open reading frame that was intervened by 6 introns. The exon-intron structure of BbHDR genes did not show clear relation to phylogeny, while its amino acid sequence reflected phyla and classes well. BbHDR sequence was distinctive from that of the HDR protein from Escherichia coli in the residues involved in hydrogen-bond network that surrounds substrate. Introduction of BbHDR cDNA into an E. coli HDR deficient mutant resulted in recovery of its auxotrophy. BbHDR expression level was upregulated from the onset of liquid culture to the 24th day after inoculation with a 2.5-fold increase and retained its level in the subsequent period.

Immobilization of the green microalga Botryococcus braunii in polyester wadding: Effect on biomass, fatty acids, and exopolysaccharide production

Abstract Botryococcus braunii is a renowned source of biomass, lipids and hydrocarbons for biofuel production. However, this microalga also produces exopolysaccharides (EPS) which might be used industrially. The artificial immobilization of B. braunii has proven to influence its growth and metabolite yield. In this work, B. braunii was immobilized using 3 g/L of polyester wadding, a recyclable material no reported before as fixing matrix for this microalga. This inexpensive polymer was non-toxic to the cells and allowed their fixing during 2 months. After 24 days, the final biomass yield (g/L) was statistically higher (P

Polysaccharide associated protein (PSAP) from the green microalga Botryococcus braunii is a unique extracellular matrix hydroxyproline-rich glycoprotein

The green colonial microalga Botryococcus braunii produces large amounts of liquid hydrocarbons that can be converted into transportation fuels. Colony cells are held together by a complex extracellular matrix (ECM) made up of a cross-linked long-chain hydrocarbon network around which liquid hydrocarbons are stored, a retaining wall for holding hydrocarbons within the cross-linked hydrocarbon network, and a polysaccharide fibrillar sheath radiating from the retaining wall and surrounding the entire colony. Analysis of “shells” shed from cell apical regions during cell division and containing the retaining wall and polysaccharide fibers shows association of a single protein where the fibers meet the retaining wall, suggesting involvement of this protein in polysaccharide fiber formation. Here we use peptide mass fingerprinting and bioinformatics to identify this protein called polysaccharide associated protein (PSAP). PSAP does not show similarity to any protein in databases, but contains several Proline-rich domains. Staining studies confirm PSAP as a glycoprotein, and mass spectrometry analysis identified ten N-linked glycosylation sites comprising seven different glycans containing mainly mannose and N-acetylglucosamine. Three of these glycans also contain fucose, with one of these glycans being unusual since it also contains arabinose. Additionally, four hydroxyproline residues have short O-linked glycans of mainly arabinose and galactose, with one also containing a 6-deoxyhexose. PSAP secretion and localization to shell material is confirmed using western blot analysis and microscopy. These studies indicate PSAP contains unique glycans and suggest its involvement in ECM polysaccharide fiber biosynthesis.

Molecular cloning and functional characterization of NADPH-dependent cytochrome P450 reductase from the green microalga Botryococcus braunii, B race

The green microalga Botryococcus braunii of the B race accumulates various lipophilic compounds containing a 10,11-oxidosqualene epoxide moiety in addition to large amounts of triterpene hydrocarbons. While 2,3-squalene epoxidases have already been isolated and characterized from the alga, the enzyme that catalyzes the 10,11-epoxidation of squalene has remained elusive. In order to obtain a molecular tool to explore a 10,11-squalene epoxidase, cDNA cloning of an NADPH-dependent cytochrome P450 reductase (CPR) that is required by both squalene epoxidases and cytochrome P450 enzymes was carried out. The isolated cDNA contained an open reading frame (1998 bp) that encoded for a protein with 665 amino acid residues with a predicted molecular weight of 71.46kDa and a theoretical pI of 5.49. Analysis of the deduced amino acid sequence revealed the presence of conserved motifs, including FMN, FAD, and NADPH binding domains, which are typical of other CPRs and necessary for enzyme activity. By truncation of the N-terminal transmembrane anchor and addition of a 6× His-tag, BbCPR was heterologously produced in Escherichia coli and purified by Ni-NTA affinity chromatography. The purified recombinant enzyme showed optimal reducing activity of cytochrome c at around a neutral pH at a temperature range of 30-37°C. For steady state kinetic parameters, the recombinant enzyme had a km for cytochrome c and NADPH of 11.7±1.6 and 9.4±1.4μM, and a kcat for cytochrome c and NADPH of 2.78±0.09 and 3.66±0.11μmol/min/mg protein, respectively. This is the first study to perform the functional characterization of a CPR from eukaryotic microalgae.

Utilization of ammonium by the hydrocarbon-producing microalga, Botryococcus braunii Showa

As a prospective feedstock for sustainable biofuels, the green microalga Botryococcus braunii race B has attracted significant attention since this colony-forming alga has the ability to accumulate considerable amounts of triterpene hydrocarbons, botryococcenes, in its extracellular matrix. However, its primary metabolism, nutrient uptake and utilization relevant to growth, are still poorly understood in this alga. From this perspective, we investigated the utilization of ammonium by B. braunii as a more reduced form of nitrogen source compared to nitrate, which is generally used for cultures of the alga. Isotope-ratio mass spectrometry analyses indicated that the uptake rate of ammonium was higher than that of nitrate at a certain concentration. When ammonium was added to culture medium, however, it inhibited algal growth even in the presence of nitrate. Buffered culture media ameliorated the toxicity of ammonium, and the alga accumulated the same levels of hydrocarbons and secondary carotenoids, irrespective of nitrogen source. To characterize utilization of ammonium by B. braunii at the molecular level, putative ammonium transporter (AMT) genes were screened from a cDNA library and four isogenes (BbAMT1;1, 1;2, 1;3 and 1;4) were cloned. Two of them, BbAMT1;1 and 1;2, were upregulated under nitrogen deficient conditions. BbAMT1;1 complemented the growth of an ammonium uptake-defective yeast strain, showing it to be a functional gene coding for AMT. These results could pave the way for culturing B. braunii more efficiently using ammonium as a sole nitrogen source by improving ammonium uptake through our understanding of AMT.

ROS Detection in Botryococcus braunii Colonies with CellROX Green Reagent.

We analyzed the reactive oxygen species (ROS) accumulation in the colony-forming green microalga Botryococcus braunii in response to several stress inducers such as NaCl, NaHCO3, salicylic acid (SA), methyl jasmonate, and acetic acid. A staining assay using the fluorescent dye CellROX Green was used. CellROX Green is a fluorogenic probe used for measuring oxidative stress in live cells. The dye is weakly fluorescent inside cells in a reduced state but exhibits bright green photostable fluorescence upon oxidation by ROS and subsequent binding to DNA. The large amount of liquid hydrocarbons produced and excreted by B. braunii, creates a highly hydrophobic extracellular environment that makes difficult to study short times defense responses on this microalga. The procedure developed here allowed us to detect ROS in this microalga even within a short period of time (in minutes) after treatment of cells with different stress inducers.

Stress responses of the oil-producing green microalga Botryococcus braunii Race B.

Plants react to biotic and abiotic stresses with a variety of responses including the production of reactive oxygen species (ROS), which may result in programmed cell death (PCD). The mechanisms underlying ROS production and PCD have not been well studied in microalgae. Here, we analyzed ROS accumulation, biomass accumulation, and hydrocarbon production in the colony-forming green microalga Botryococcus braunii in response to several stress inducers such as NaCl, NaHCO3, salicylic acid (SA), methyl jasmonate, and acetic acid. We also identified and cloned a single cDNA for the B. braunii ortholog of the Arabidopsis gene defender against cell death 1 (DAD1), a gene that is directly involved in PCD regulation. The function of B. braunii DAD1 was assessed by a complementation assay of the yeast knockout line of the DAD1 ortholog, oligosaccharyl transferase 2. Additionally, we found that DAD1 transcription was induced in response to SA at short times. These results suggest that B. braunii responds to stresses by mechanisms similar to those in land plants and other organisms.

Effects of 2-azahypoxanthine on extracellular terpene accumulations by the green microalga Botryococcus braunii, race B

The B race of the green microalga Botryococcus braunii accumulates large amounts of triterpene hydrocarbons that are attractive as a source for biofuels. The alga, however, exhibits rather slow growth and this characteristic has hindered its practical application for biofuel production. In order to test a phytohormone-like compound that may regulate the growth or hydrocarbon production of this alga, the effects of 2-azahypoxanthine (AHX) on algal biomass and terpene contents in the extracellular matrix (ECM) in the Showa strain of B. braunii were investigated. The biomass of the Showa strain increased with AHX supplementation under static culture conditions although such effects were not observed in cultures with aeration or agitation. AHX supplementation significantly induced the accumulation of secondary carotenoids, including species-specific tetramethylsqualene (TMS)-conjugated carotenoids, botryoxanthin A and braunixanthin 1. Conversely, the addition of AHX showed a tendency to slightly lower the triterpene hydrocarbon (botryococcenes) content in the ECM. These metabolic changes in tri- and tetraterpene accumulation could be caused by a reduction in nitrogen uptake from the culture medium due to AHX addition. Thus, there is a possibility that AHX could be used as a reagent to modulate terpene biosynthesis in the B race of B. braunii.