Sequencing and comparative analysis of three Chlorella genomes provide insights into strain-specific adaptation to wastewater.

Microalgal Chlorella has been demonstrated to process wastewater efficiently from piggery industry, yet optimization through genetic engineering of such a bio-treatment is currently challenging, largely due to the limited data and knowledge in genomics. In this study, we first investigated the differential growth rates among three wastewater-processing Chlorella strains: Chlorella sorokiniana BD09, Chlorella sorokiniana BD08 and Chlorella sp. Dachan, and the previously published Chlorella sorokiniana UTEX 1602, showing us that BD09 maintains the best tolerance in synthetic wastewater. We then performed genome sequencing and analysis, resulting in a high-quality assembly for each genome with scaffold N50 > 2 Mb and genomic completeness ≥91%, as well as genome annotation with 9,668, 10,240, 9,821 high-confidence gene models predicted for BD09, BD08, and Dachan, respectively. Comparative genomics study unravels that metabolic pathways, which are involved in nitrogen and phosphorus assimilation, were enriched in the faster-growing strains. We found that gene structural variation and genomic rearrangement might contribute to differential capabilities in wastewater tolerance among the strains, as indicated by gene copy number variation, domain reshuffling of orthologs involved, as well as a ~1 Mb-length chromosomal inversion we observed in BD08 and Dachan. In addition, we speculated that an associated bacterium, Microbacterium chocolatum, which was identified within Dachan, play a possible role in synergizing nutrient removal. Our three newly sequenced Chlorella genomes provide a fundamental foundation to understand the molecular basis of abiotic stress tolerance in wastewater treatment, which is essential for future genetic engineering and strain improvement.

The biological activities of prothioconazole enantiomers and their toxicity assessment on aquatic organisms.

Chiral fungicide prothioconazole has a wide range of antifungal spectrum; however, little research has been conducted to evaluate prothioconazole on an enantiomeric level. Five target pathogens and three common aquatic organisms were tested for the enantioselective bioactivity and toxicity of prothioconazole in this work. The antifungal activity of the enantiomers against wheat phytoalexin, rice blast fungus, exserohilum turcicum, Alternaria triticina, and Fusarium avenaceum was determined, and it was found that (-)-prothioconazole were 85 to 2768 times more active than (+)-prothioconazole toward these target organisms. In order to reflect the risk to aquatic ecosystem, the acute toxicity of the enantiomers to Daphnia magna, Chlorella pyrenoidosa, and Lemna minor L. was assessed. It was observed that the toxicity of (-)-prothioconazole to D. magna was 2.2 times higher than (+)-prothioconazole, but it was lower to C. pyrenoidosa and L. minor L. The toxicities of (+)-enantiomer and (-)-enantiomer to D. magna and C. pyrenoidosa were synergy, indicating that the racemate had higher threat to the organisms. It could be concluded that the effects of prothioconazole on target organisms and the acute toxicity to nontarget species were enantioselective with (-)-enantiomer possessing higher efficiency and lower toxicity. Such enantiomeric differences should be taken into consideration when assessing the performance of prothioconazole.

Modulation of lipid content and lipid profile by supplementation of iron, zinc, and molybdenum in indigenous microalgae.

The effects of iron (Fe), zinc (Zn), and molybdenum (Mo) on the biomass yield, lipid content, lipid yield, and fatty acid composition of Chlorella sp. NC-MKM, Graesiella emersonii NC-M1, Scenedesmus acutus NC-M2, and Chlorophyta sp. NC-M5 were studied. Among them, G. emersonii NC-M1 recorded the highest percentage increase in lipid content (140.3%) and neutral lipid (50.9%) under Zn-supplemented condition compared to the control. Also, it showed a 105% and 41.88% increase in lipid yield and neutral lipid under Fe-supplemented condition compared to the control. However, Chlorella sp. NC-MKM recorded an elevation in lipid yield (70.3% rise) and neutral lipid (24.32% rise) compared to the control in Mo-supplemented condition. The enhanced production of reactive oxygen species (ROS) and antioxidant enzyme (SOD and POD) under Fe-, Zn-, and Mo-supplemented condition supports the lipid accumulation. FAME analysis showed that the overall percentage of SFA and MUFA increased after the addition of Fe, Zn, and Mo in a culture medium compared to the control which is vital for a good-quality biodiesel. Further, biodiesel properties derived from FAMEs such as CN, SV, IV, CFPP, OS, υ, ρ, and HHV were found in accordance with biodiesel standard.

Excessive phosphorus caused inhibition and cell damage during heterotrophic growth of Chlorella regularis.

A high phosphorus concentration is widely accepted as favorable for enhancing both microalgae growth and lipid accumulation; however, excessively high P could be counter-productive. In this study, we investigated the effects of increasing P levels (5.4, 25, 45, 150, and 250 mg-P L-1) on the heterotrophic cultivation of Chlorella regularis. Microalgae growth was inhibited and cells were severely damaged in response to highly excessive P levels (≥150 mg-P L-1). In particular, 250 mg-P L-1 resulted in a ∼40% decrease in cell density and a ∼70% loss of cell viability. Microalgae damage induced by excessive phosphorus included enlarged cell size, deformation of cell walls, and disorganization of organelles. These negative effects were associated with the over-accumulation of polyphosphates within cells, which may further cause binding of P to intracellular components. Although P is an essential nutrient, excessive P lowers cell growth and viability.

Binary effect of titanium dioxide nanoparticles (nTio2) and phosphorus on microalgae (Chlorella 'Ellipsoides Gerneck, 1907).

The wide application of titanium dioxide nanoparticles and phosphorus in the manufacturing of many industrial products mainly used in agricultural sector has resulted in the release of considerable amounts of these compounds into freshwater aquatic ecosystem. These compounds may cause some unexpected effects to aquatic organisms. This study assessed the binary effects of Titanium nanoparticles (nTiO2) and Phosphorus on Chlorella ellipsoides. Toxicological assay test of the compounds nTiO2 (1.25 µM) alone and the combination of Titanium dioxide (1.25 µM) and Phosphorus (16, 32, 80, 160, 240 µM) was assessed, after 96 h exposures, for optical density (OD680), specific growth rate, chlorophyll levels and lipid peroxidation via Malondialdehyde (MDA) activity. Superoxide dismutase (SOD), peroxidase (POD) and glutathione-s-transferase (GST) activities were also measured. Two-way ANOVA showed a significant interaction (P < 0.05) between binary mixture. Co-exposure showed a decreased phosphorus bioconcentration in the microalgae with significant increase (P < 0.05) in chlorophyll a/b and total chlorophyll contents. A significant decrease (P < 0.05) in specific growth rate and optical density were recorded whereas, antioxidant enzymes (MDA, SOD, POD, GST) activities were significantly (P < 0.05) increased. These results showed that the addition of nTiO2 to Phosphorus affected the physiology of microalgae and should be of great concern for freshwater biodiversity.

Influence of Iron-Doped Apatite Nanoparticles on Viral Infection Examined in Bacterial Versus Algal Systems.

The Centers for Disease Control and Prevention have estimated that each year, two million people in the United States become infected with antibiotic-resistant bacteria, of which, approximately 23000 die as a direct result of these infections. Phage therapy, or the treatment of bacterial infection by specific, antagonistic viruses, provides one alternative to traditional antibiotics. Bacteriophages, or phages, are bacteria-specific viruses that possess biological traits that allow for not only the removal of bacterial infection, but also the evasion of bacterial resistance, which renders antibiotics ineffective. Previous research has shown the addition of iron-doped apatite nanoparticles (IDANPs) to bacteria prior to phage exposure results in increased bacterial plaques in vitro. Coupled with the biocompatible nature of apatite, these results provide promise for future use of IDANPs as adjuvants to phage therapy along with anti-bacterial applications yet to be explored. Although IDANP enhancement of phage infection has been replicated many times in gram-positive and gram-negative prokaryotic hosts as well as with the utilization of both RNA and DNA viruses, the specific mechanisms involved remain elusive. To further understand increased phage infections in a prokaryotic system, and to evaluate the safety of IDANPs as a treatment used in a eukaryotic system, we have replicated plaque assay experiments in an algal system using Chlorella variabilis NC64A and its virus, Paramecium bursaria chlorella virus 1 (PBCV-1). Statistical modeling was used to evaluate alteration in numbers of plaques observed after viral introduction in IDANP-exposed versus non-IDANP-exposed bacterial and algal cell cultures. While IDANPs synthesized between 25°C-45°C and doped with 30% iron have been shown to influence dramatic increases in phage-induced bacterial death, experiments replicated in an algal system indicated viral infections do not increase when C. variabilis cells are pre-exposed to IDANPs. It is essential to potential use of IDANPs as an antibacterial adjuvant that IDANPs do not increase viral infection of eukaryotic host cells during treatment.

Hyperosmosis and its combination with nutrient-limitation are novel environmental stressors for induction of triacylglycerol accumulation in cells of Chlorella kessleri.

Triacylglycerols of oleaginous algae are promising for production of food oils and biodiesel fuel. Air-drying of cells induces triacylglycerol accumulation in a freshwater green alga, Chlorella kessleri, therefore, it seems that dehydration, i.e., intracellular hyperosmosis, and/or nutrient-limitation are key stressors. We explored this possibility in liquid-culturing C. kessleri cells. Strong hyperosmosis with 0.9 M sorbitol or 0.45 M NaCl for two days caused cells to increase the triacylglycerol content in total lipids from 1.5 to 48.5 and 75.3 mol%, respectively, on a fatty acid basis, whereas nutrient-limitation caused its accumulation to 41.4 mol%. Even weak hyperosmosis with 0.3 M sorbitol or 0.15 M NaCl, when nutrient-limitation was simultaneously imposed, induced triacylglycerol accumulation to 61.9 and 65.7 mol%, respectively. Furthermore, culturing in three-fold diluted seawater, the chemical composition of which resembled that of the medium for the combinatory stress, enabled the cells to accumulate triacylglycerol up to 24.7 weight% of dry cells in only three days. Consequently, it was found that hyperosmosis is a novel stressor for triacylglycerol accumulation, and that weak hyperosmosis, together with nutrient-limitation, exerts a strong stimulating effect on triacylglycerol accumulation. A similar combinatory stress would contribute to the triacylglycerol accumulation in air-dried C. kessleri cells.

Synergistic dynamics of nitrogen and phosphorous influences lipid productivity in Chlorella minutissima for biodiesel production.

The study synergistically optimized nitrogen and phosphorous concentrations for attainment of maximum lipid productivity in Chlorella minutissima. Nitrogen and phosphorous limited cells (N(L)P(L)) showed maximum lipid productivity (49.1±0.41mg/L/d), 1.47 folds higher than control. Nitrogen depletion resulted in reduced cell size with large sized lipid droplets encompassing most of the intracellular space while discrete lipid bodies were observed under nitrogen sufficiency. Synergistic N/P starvations showed more prominent effect on photosynthetic pigments as to individual deprivations. Phosphorous deficiency along with N starvation exhibited 17.12% decline in carbohydrate while no change in nitrogen sufficient cells were recorded. The optimum N(L)P(L) concentration showed balance between biomass and lipid by maintaining intermediate cell size, pigments, carbohydrate and proteins. FAME profile showed C14-C18 carbon chains in N(L)P(L) cells with biodiesel properties comparable to plant oil methyl esters. Hence, synergistic N/P limitation was effective for enhancing lipid productivity with reduced consumption of nutrients.

Effect of Tris-(hydroxymethyl)-amino methane on microalgae biomass growth in a photobioreactor.

One of the buffers namely Tris (Tris-(hydroxymethyl)-amino methane) was used to increase the growth of microalgae by stabilizing the pH value in microalgae cultures. The objective of this research is to determine the growth rate and biomass productivity of Chlorella sp. with and without Tris addition. Both conditions function at various N:P ratios cultured in photobioreactors (carbon dioxide of 5%(v/v), light intensity of 3.3 Klux). Daily variations in nutrient removal (nitrogen and phosphorus), cell concentration, DO, temperature and pH were measured for data analysis. The results show that the largest yield of biomass was achieved at the N:P ratio of 15:1 with and without Tris. After cultivation lasting 92 h, the algae concentration at this ratio was 1250 mg L(-1) and 3568 mg L(-1) with and without Tris, respectively. This indicates that adding Tris to the photobioreactor greatly reduces algae biomass due to bacterial competition.

Enhancing growth rate and lipid yield of Chlorella with nuclear irradiation under high salt and CO2 stress.

In order to produce biodiesel from microalgae cultured with abundant seawater, Chlorella sp. was mutated with (137)Se-γ ray irradiation and domesticated with f/2 seawater culture medium (salinity=3 wt.%) under 15 vol.% CO2 stress. Biomass yield of the mutant increased by 25% compared with wild species and lipid content increased to 54.9%. When nitrogen and phosphorus concentrations in the initial substrate increased, the increased propagation speed of the mutant resulted in decreased cell diameter by 26.6% and decreased cell wall thickness by 69.7%. The dramatically increased biomass yield of the mutant with sufficient initial substrate and relative nitrogen starvation in the later growth period with continuous 15 vol.% CO2 led to an increased lipid yield of 1.0 g/L. The long-chain unsaturated fatty acids increased, whereas short-chain saturated fatty acids decreased.

Photorespiration participates in the assimilation of acetate in Chlorella sorokiniana under high light.

The development of microalgae on an industrial scale largely depends on the economic feasibility of mass production. High light induces productive suspensions during cultivation in a tubular photobioreactor. Herein, we report that high light, which inhibited the growth of Chlorella sorokiniana under autotrophic conditions, enhanced the growth of this alga in the presence of acetate. We compared pigments, proteomics and the metabolic flux ratio in C. sorokiniana cultivated under high light (HL) and under low light (LL) in the presence of acetate. Our results showed that high light induced the synthesis of xanthophyll and suppressed the synthesis of chlorophylls. Acetate in the medium was exhausted much more rapidly in HL than in LL. The data obtained from LC-MS/MS indicated that high light enhanced photorespiration, the Calvin cycle and the glyoxylate cycle of mixotrophic C. sorokiniana. The results of metabolic flux ratio analysis showed that the majority of the assimilated carbon derived from supplemented acetate, and photorespiratory glyoxylate could enter the glyoxylate cycle. Based on these data, we conclude that photorespiration provides glyoxylate to speed up the glyoxylate cycle, and releases acetate-derived CO2 for the Calvin cycle. Thus, photorespiration connects the glyoxylate cycle and the Calvin cycle, and participates in the assimilation of supplemented acetate in C. sorokiniana under high light.

Synchronized growth and neutral lipid accumulation in Chlorella sorokiniana FC6 IITG under continuous mode of operation.

Synchronized growth and neutral lipid accumulation with high lipid productivity under mixotrophic growth of the strain Chlorella sorokiniana FC6 IITG was achieved via manipulation of substrates feeding mode and supplementation of lipid elicitors in the growth medium. Screening and optimization of lipid elicitors resulted in lipid productivity of 110.59mgL(-1)day(-1) under the combined effect of lipid inducers sodium acetate and sodium chloride. Fed-batch cultivation of the strain in bioreactor with intermittent feeding of limiting nutrients and lipid inducer resulted in maximum biomass and lipid productivity of 2.08 and 0.97gL(-1)day(-1) respectively. Further, continuous production of biomass with concomitant lipid accumulation was demonstrated via continuous feeding of BG11 media supplemented with lipid inducers sodium acetate and sodium chloride. The improved biomass and lipid productivity in chemostat was found to be 2.81 and 1.27gL(-1)day(-1) respectively operated at a dilution rate of 0.54day(-1).

Carbon-to-nitrogen ratio affects the biomass composition and the fatty acid profile of heterotrophically grown Chlorella sp. TISTR 8990 for biodiesel production.

Chlorella sp. TISTR 8990 was cultivated heterotrophically in media with various initial carbon-to-nitrogen ratios (C/N ratio) and at different agitation speeds. The production of the biomass, its total fatty acid content and the composition of the fatty acids were affected by the C/N ratio, but not by agitation speed in the range examined. The biomass production was maximized at a C/N mass ratio of 29:1. At this C/N ratio, the biomass productivity was 0.68gL(-1)d(-1), or nearly 1.6-fold the best attainable productivity in photoautotrophic growth. The biomass yield coefficient on glucose was 0.62gg(-1) during exponential growth. The total fatty acids (TFAs) in the freeze-dried biomass were maximum (459mgg(-1)) at a C/N ratio of 95:1. Lower values of the C/N ratio reduced the fatty acid content of the biomass. The maximum productivity of TFAs (186mgL(-1)d(-1)) occurred at C/N ratios of 63:1 and higher. At these conditions, the fatty acids were mostly of the polyunsaturated type. Allowing the alga to remain in the stationary phase for a prolonged period after N-depletion, reduced the level of monounsaturated fatty acids and the level of polyunsaturated fatty acids increased. Biotin supplementation of the culture medium reduced the biomass productivity relative to biotin-free control, but had no effect on the total fatty acid content of the biomass.

[Toxicity of Coptis chinensis Rhizome Extracts to Green Algae].

Coptis chinensis contains antiseptic alkaloids and thus its rhizomes and preparations are widely used for the treatment of.fish diseases. In order to realize the risk of water ecosystems produced by this medical herb and preparations used in aquaculture, the present experiment was carried out to study the toxicity of Coptis chinensis rhizome extract (CRE) to Scenedesmus oblique and Chlorella pyrenoidosa grown in culture solution with 0.00 (CK), 0.088 (Tl), 0.44 (T2) and 1.76 mg · L(-1) (T3) of CRE, respectively. The results show that low concentration of CRE (T1) inhibited the growth rate of the alga and high CRE (T2 and T3) ceased growth and reproductions. CRE also decreased the chlorophyll and proteins in alga cells, indicating the inhibition of photosynthesis and protein biosynthesis, which could be direct reasons for the low growth rate and death of green alga. The efflux of protons and substances from alga cells led to pH reduction and conductivity increment in culture solution with CRE. Furthermore, the activity of superoxide dismutase in alga increased at the beginning of CRE in T1 and T2 treatments but decreased as time prolonged which was in contrast to high CRE treatment. And the long exposure to low CRE treatment behaved otherwise. This suggests that the low concentration of CRE could induce the resistant reactions in alga at initial time but high CRE concentration or long exposure even at low CRE concentration could inhibit the enzyme synthesis. Similarly, malondialdehyde in alga increased as CRE concentrations increased in culture solutions, implying the damage and high permeability of cell membrane. In general, Chlorella pyrenoidosa was more sensitive to CRE. The abuse of rhizomes and preparations in aquaculture and intensive cultivation of Coptis chinensis plants in a large scale might produce ecological risks to primary productivity of water ecosystems.

Screening and characterization of oleaginous Chlorella strains and exploration of photoautotrophic Chlorella protothecoides for oil production.

The growth and oil production of nine Chlorella strains were comparatively assessed and Chlorellaprotothecoides CS-41 demonstrated the greatest lipid production potential. The effects of different nitrogen forms and concentrations, phosphorus concentrations and light intensities on growth and oil production were studied in laboratory columns. C. protothecoides CS-41 accumulated lipids up to 55% of dry weight, with triacylglycerol and oleic acid being 71% of total lipids and 59% of total fatty acids, respectively. High biomass and lipid productivities were achieved in outdoor panel PBRs, up to 1.25 and 0.59 g L(-1) day(-1), or 44. 1 and 16.1 g m(-2) day(-1), respectively. A two-stage cultivation strategy was proposed to enhance the algal biomass and lipid production. This is the first comprehensive investigation of both indoor and outdoor photoautotrophic C. protothecoides cultures for oil production, and C. protothecoides CS-41 represents a promising biofuel feedstock worthy of further exploration.

Potential lipid accumulation and growth characteristic of the green alga Chlorella with combination cultivation mode of nitrogen (N) and phosphorus (P).

This study aimed to evaluate the potential lipid accumulation of an oleaginous Chlorella protothecoides by combination cultivation mode of nitrogen (N) and phosphorus (P). Under co-deficiency of N and P, the largest lipid content (55.8%) was accomplished in C. protothecoides, which was higher than either sole P-deficiency (32.77%) or N-deficiency (52.5%), or co-repletion of N and P (control) (22.17%). However, the highest lipid productivity (224.14mg/L/day) with combination mode of N-deficiency and P-repletion represented 1.19-3.70-fold more than that of control, P-deficiency/limitation, and co-deficiency of N and P, respectively. This indicating N-deficiency plus P-repletion was a promising lipid trigger to motivate lipid accumulation in C. protothecoides cells. Further, difference gel electrophoresis (DIGE)-based proteomics was employed to reveal the molecular pathways associated with lipid biosynthesis. These results provide the foundation to develop engineering strategies targeting lipid productivity for industrial production of microalgae-based biodiesel.

Adaptability of growth and nutrient uptake potential of Chlorella sorokiniana with variable nutrient loading.

Chlorella sorokiniana can sustain growth in conditions hostile to other species, and possesses good nutrient removal and lipid accumulation potentials. However, the effects of variable nutrient levels (N and P) in wastewaters on growth, productivity, and nutrient uptake by C. sorokiniana have not been studied in detail. This study demonstrates the ability of this alga to sustain uniform growth and productivity, while regulating the relative nutrient uptake in accordance to their availability in the bulk medium. These results highlight the potential of C. sorokiniana as a suitable candidate for fulfilling the coupled objectives of nutrient removal and biomass production for bio-fuel with wastewaters having great variability in nutrient levels.

Herbicidal effects of harmaline from Peganum harmala on photosynthesis of Chlorella pyrenoidosa: probed by chlorophyll fluorescence and thermoluminescence.

The herbicidal effects of harmaline extracted from Peganum harmala seed on cell growth and photosynthesis of green algae Chlorella pyrenoidosa were investigated using chlorophyll a fluorescence and thermoluminescence techniques. Exposure to harmaline inhibited cell growth, pigments contents and oxygen evolution of C. pyrenoidosa. Oxygen evolution was more sensitive to harmaline toxicity than cell growth or the whole photosystem II (PSII) activity, maybe it was the first target site of harmaline. The JIP-test parameters showed that harmaline inhibited the donor side of PSII. Harmaline decreased photochemical efficiency and electron transport flow of PSII but increased the energy dissipation. The charge recombination was also affected by harmaline. Amplitude of the fast phase decreased and the slow phase increased at the highest level of harmaline. Electron transfer from QA(-) to QB was inhibited and backward electron transport flow from QA(-) to oxygen evolution complex was enhanced at 10 µg mL(-1) harmaline. Exposure to 10 µg mL(-1) harmaline caused appearance of C band in thermoluminescence. Exposure to 5 µg mL(-1) harmaline inhibited the formation of proton gradient. The highest concentration of harmaline treatment inhibited S3QB(-) charge recombination but promoted formation of QA(-)YD(+) charge pairs. P. harmala harmaline may be a promising herbicide because of its inhibition of cell growth, pigments synthesis, oxygen evolution and PSII activities.

The effect of iron on growth, lipid accumulation, and gene expression profile of the freshwater microalga Chlorella sorokiniana.

The effects of iron on the growth, lipid accumulation, and gene expression profiles of the limnetic Chlorella sorokiniana CCTCC M209220 under photoautotrophy were investigated. The addition of iron up to 10(-5) mol l(-l) increased final cell densities by nearly 2-fold at 2.3 × 10(7) cells/ml, growth rate by 2-fold, and the length of the exponential phase by 5 days as compared to unsupplemented controls while 10(-3) mol l(-1) iron was toxic. The lipid content increased from 12 % for unsupplemented cultures to 33 % at 10(-4) mol l(-1) iron while the highest overall lipid yield reached 179 mg l(-1). A genefishing and qPCR comparison between the C. sorokiniana at low and high iron levels indicated increases in the expression of several genes, including carbonic anhydrase involved in microalgal cell growth, as well as acc1 and choline transporter related to lipid synthesis. This study provides insights into changes in gene expression and metabolism that accompany iron supplementation to Chlorella as well as potential metabolic engineering targets for improving growth and lipid synthesis in microalgae.

The effect of nutrition pattern alteration on Chlorella pyrenoidosa growth, lipid biosynthesis-related gene transcription.

Heterotrophy to photoautotrophy transition leads to the accumulation of lipids in Chlorella, which has potential to produce both healthy food and biofuels. Therefore, it is of key interest to study the metabolism shift and gene expression changes that influenced by the transition. Both total and neutral lipids contents were increased rapidly within 48 h after the switch to light environment, from 24.5% and 18.0% to 35.3% and 27.4%, respectively, along with the sharp decline of starch from 42.3% to 10.4% during 24h photoinduction phase. By analyzing the correlation between lipid content and gene expression, results revealed several genes viz. me g3137, me g6562, pepc g6833, dgat g3280 and dgat g7566, which encode corresponding enzymes in the de novo lipid biosynthesis pathway, are highly related to lipid accumulation and might be exploited as target genes for genetic modification. These results represented the feasibility of lipid production through trophic converting cultivation.