Hop Cultivars Research Articles

Cross-Infectivity of Powdery Mildew Isolates Originating from Hemp (Cannabis sativa) and Japanese Hop (Humulus japonicus) in New York

In the recent decade, agricultural production of both hemp (Cannabis sativa) and hop (Humulus lupulus) has expanded throughout the Pacific Northwest, Midwest, and Eastern United States to support the growing industries for which these plants are key components. The significant and rapidly expanding overlap of production regions of these two Cannabaceae plant family members creates a potential dispersal route for organisms that are pathogenic to both hosts. Powdery mildew is a disease of high economic impact in both hemp and hop production systems, yet it was largely unknown whether the powdery mildew fungi commonly associated with hemp could also be pathogenic on hop, and vice versa. We isolated Golovinomyces spadiceus growing upon hemp in New York production greenhouses and Podosphaera macularis from feral hop (H. japonicus) plantings also in New York. Herein, we report the pathogenicity of P. macularis associated with hop to C. sativa cultivars ‘Anka’ and ‘Wild Horse’ and pathogenicity of G. spadiceus toward hop. The potential for P. macularis to establish, produce viable, infectious conidia, and undergo sexual recombination on hemp could complicate efforts to exclude the MAT1-2 mating type of P. macularis from western North America and could facilitate the spread of races pathogenic toward ‘Cascade’ hop, and hop cultivars with R6-based resistance to P. macularis, including ‘Nugget’. Further assessment of the pathogenicity of diverse P. macularis isolates, in both geographic origin and the range of hop species, is necessary to better understand the dispersal risk of P. macularis on hemp.

Disentangling photoperiod from hop vernalization and dormancy for global production and speed breeding

Humulus lupulus L. (hop) flowers are a key ingredient in beer, imparting the beverage’s aroma and bitterness profile. Photoperiod is known to interact with temperature to control flowering in hops. Studies have stipulated that resting dormant buds on hops require a minimum chilling duration for their meristems to break dormancy and grow fruitfully. This assertion, in part, led to a long-held notion that hops require vernalization and/or dormancy for the meristem to change from a vegetative to floral state. The research in this study aims to separate photoperiod from vernalization and dormancy through a series of experiments that artificially control photoperiod to prevent the onset of dormancy and chilling exposure. Six experiments were performed to assess flower yield and quality for seven diverse hop cultivars (with and without exposure to chilling and dormancy) to quantify the impact on flowering performance. Vernalization and dormancy, two plant traits previously considered necessary to the proliferation of hop flowers, do not influence hop flower yield and quality. The findings have broad implications; global hop production can be distributed more widely and it paves the way for speed breeding and controlled-environment production to achieve 4 hop generation cycles per year, as opposed to 1 under field-grown conditions.

Bioactive compounds and antioxidant activities of Brazilian hop (Humulus lupulus L.) extracts

SummaryBioactive compounds from Brazilian hop (Humulus lupulus L.) cultivars were extracted by ultrasound and their phenolic profile compared with commercial hop from the USA. The most effective extraction conditions (solution of ethanol 49%, at 52 °C and a solid/liquid ratio of 1 g per 34 mL) for the total phenolic compounds (TPC) were determined using a Central Composite Rotatable Design. The Brazilian hop showed higher content of TPC (33.93 ± 0.67 mg GAE g−1), total flavonoids (54.47 ± 0.10 mg QE g−1) and higher antioxidant activity (ABTS: EC50 21.29 ± 1.36 μL mL−1; DPPH: EC50 3.91 ± 0.17 μL mL−1) when compared with the USA hop. The main phenolic compounds present in the extracts were the flavonoids isoquercitrin and quercetin. The antioxidant properties of the Brazilian hop extract had not been reported yet in the literature for this raw material, thus showing potential to be incorporated in polymeric films used as active packaging.

Temporal and spatial assessment of defence responses in resistant and susceptible hop cultivars during infection with Verticillium nonalfalfae

Hop (Humulus lupulus L.) is an important industrial plant providing ingredients for brewing and pharmaceutical industry worldwide. Its intensive production is challenged by numerous diseases. One of the most lethal and difficult to control is verticillium wilt, a vascular disease caused by the fungal pathogen Verticillium nonalfalfae. The disease can be successfully controlled by the host resistance. Despite various studies that already researched resistance mechanisms of hops, only limited number of resistance genes and markers that could be utilized for efficient resistance breeding has been identified. In this study we aimed to follow fungus colonization pattern and the differential expression of selected genes during pre-symptomatic period of susceptible (Celeia) and resistant (Wye Target) hop cultivars. Results of gene expressions and fungal colonisation of compatible and incompatible interactions with V. nonalfalfae suggest that the hop plant is challenged already at the very early fungal colonisation stages. In total, nine out of 17 gene targets investigated in our study resulted in differential expression between inoculated and control plants of susceptible and resistant cultivars. The difference was the most evident in stems at an early stage of colonisation (6 dpi), showing relatively stronger changes in targeted gene expression to infection in the resistant cultivar than in the susceptible one. Analysed gene targets are involved in the overall defence response processes of nucleic acid binding, signalling, protein ubiquitination, cell oxidative burst, hydroxylation, peroxidation, alternative splicing, and metabolite biosynthesis. The up-regulation of some genes (e.g. glycine-rich RNA-binding family protein, protein phosphatase, cysteine-rich receptor-like protein kinase, zinc finger CCCH domain-containing protein 40, cinnamic acid 4-hydroxylase, class III peroxidase, putative MAPK2, peroxiredoxin-2F) upon infection in incompatible interactions might reflect defence activation, restriction of disease spreading throughout the plant and successful response of resistant genotype.

Utilization of molecular methods for hop (Humulus lupulusL.) genotype evaluation

In recent years, the utilization of molecular genetic methods is the best tool for the evaluation of individual genotypes. Modern molecular methods allow us to check the changes caused by the combination of parents, evaluate the relationships of individual genotypes or variability (diversity) within the population. Recently, we have developed an efficient marker system to control the authenticity of Czech hop (Humulus lupulus L.) cultivars, which we introduced into the system for the identification and control of hop cultivar purity. In our presented study, we evaluated molecular genetic variability within 109 world hop cultivars by hierarchical clustering techniques and principal coordinates analysis (PCoA). For molecular analyses, we used 269 amplified marker of SSR, STS and EST-SSR loci in total. Hop cultivars were divided into two major groups with North American or European origin and breeding influence by cluster analysis, as common knowledge from previous published results. PCoA more distinguished two breeding cultivar groups of ‘Saaz’ and ‘Fuggle’ hops in Europe. The usage of some cultivars during breeding history (‘Brewers Gold’, ‘Northern brewer’, ‘Cascade’, ‘Columbus’) was also noticeable.

Hop aroma characterization of dry hopped beers using Headspace-Trap GC-MS

In the brewing process hop is a major ingredient which provides a distinctive aroma and characteristic bitter taste. The breeding of new hop cultivars with a multitude of aromas and the increasing popularity of dry hopped beers called for detailed insights into beer aroma influenced by hop. Headspace sampling is a suitable technique for extraction of aroma compounds from complex matrices. The benefit of the use of a trap device in combination with headspace is the improvement of detection limits. In this study, a headspace (HS)-trap gas chromatography (GC)-mass spectrometry (MS) method was developed to study dry hopping aroma in beer. Analysis of monoterpenes, sesquiterpenes, terpene alcohols, esters, and ketones in various beer samples was performed. The influence of different hop cultivars (‘Mandarina Bavaria’, ‘Lemondrop’, ‘Bravo’, ‘Hallertau Cascade’, ‘Polaris’, ‘Smaragd’, ‘Saphir’, ‘Calypso’, and ‘Hallertau Blanc’) on the aroma profile of beer as well as the impact of hop addition time for the hop cultivar ‘Lemondrop’ was investigated. The HS-trap GC-MS technique covers a broad range of hop-derived aroma compounds and enables to monitor the volatiles during the whole brewing process.

Differences in photosynthetic and transpiration activity of bine and branch leaves of hop plant (Humulus lupulus L.)

This paper evaluates photosynthetic and transpiration rate of bine and branch leaves of hop (cultivar 'Agnus'). Monitoring of photosynthetic rate helps us determine phases of vegetation and environmental conditions under which plants are most active, most productive, or what influences their activity. By measuring, using LC pro+ (ADC Bio Scientific Ltd.), we detected that branch leaves show a higher rate of photosynthesis than bine leaves. Branch leaves' photosynthetic rate in the period 2011-2013 ranged between 120-184% compared with bine leaves. However, we did not find any dependence of yield on the rate of photosynthesis during the experiment. In some years when lower photosynthetic rate was measured, higher yields were achieved than in years with higher photosynthetic activity. Yield formation is therefore a multifactorial complex, involving main influence of year and weather conditions. Photosynthetic rate of bine leaves ranged from 3.29 to 8.33 μmol CO2 m(-2) s(-1). Photosynthetic rate of branch leaves ranged from 6.05 to 9.89 μmol CO(2) m(-2) s(-1). Transpiration rate of branch leaves ranged between 104-119% compared with bine leaves.

Comparison of biomass in Czech hop cultivars ‘Premiant’ and ‘Sládek’ grown in low and tall trellises

Spring field operations in a tall wirework frame require excessive labor, which may be often difficult to have in an optimal time and a good quality. Especially stringing and training are expensive and lengthy field operations, which can cause problems with optimum timing of treatments against downy mildew, hop flea beetle and weevils. Effectiveness of Czech hybrid tall cultivars ‘Premiant’ and ‘Sladek’ grown in a low trellis was observed. The results in the third year of cultivation (2011) showed different distribution of hop biomass. Hop cones, leaves, laterals and bines were picked by hand and weighed. Distribution of the individual parts in ‘Premiant’ (P) and ‘Sladek’ (S) was compared: 1. Tall trellis: cones 36% in both cultivars, leaves 28% (P), resp. 31% (S), bine 19% (P), resp. 18% (S), laterals 17% (P), resp. 15% (S); 2. Low trellis: cones 13% (P), resp. 24% (S), leaves 37% (P), resp. 34% (S), bine 38% (P), resp. 25% (S), laterals 12% (P), resp. 17% (S). The height of a trellis severely influenced the yield potential of hop plants. Vegetative tops did not train themselves and started to bend after reaching the height of 3 m. If we compare the productivity of cones in the both tested cultivars we can conclude that ‘Premiant’ is less suitable for low trellis cultivation.

Polyploid breeding strategies for developing hop cultivars in New Zealand

The main objective of the New Zealand hop breeding programme is to develop triploid cultivars with unique brewing properties. Created from crosses between diploid and tetraploid parents, these genotypes have the advantage of being seedless as well as high yielding. Population improvement procedures are used independently for both diploid and tetraploid populations. Restricted maximum likelihood statistical procedures are used for the selection of both male and female genotypes for tetraploid and diploid parental material. In this regard, particular emphasis is placed on identifying sexually derived tetraploids to widen the gene pool. Crosses using tetraploids, as both female and male parents, hybridised with diploid genotypes, are described. Gender segregation ratios from recent triploid seedling populations vary considerably. The statistical analysis of triploid seedling female genotypes from these populations for several key traits is presented. A summary of main chemistry features of New Zealand bred polyploid cultivars is presented. Future breeding strategies for triploid hops are discussed.

Performance of sexually derived tetraploids for parental development of triploid hop cultivars in New Zealand

Performance of sexually derived tetraploids for parental development of triploid hop cultivars in New Zealand

Genome-wide association study on ten traits of economic importance in hop (Humulus lupulus L.)

Hop is used primarily for bittering and flavoring of beer although new uses of hop have been implemented in poultry production, sugar processing and pharmaceuticals. Traditional hop breeding is a lengthy, expensive process. Modern selection methods such as marker-assisted selection are needed to greatly improve genetic gains per selection cycle as well as reduce the number of experimental lines evaluated in the field. Recent implementation of next generation sequencing enables the scanning of whole genomes for markers linked to traits of interest using an analysis called genome-wide association studies (GWAS). This study was initiated to identify markers associated with expression of 10 different traits in hop. Illumina Inc. technology sequencing of 116 genetically distinct hop cultivars from multiple regions of the world was performed. SNP marker identification was achieved using the TASSEL v3.0 UNEAK pipeline. Filtering of SNP markers resulted in a working group of 32,206 SNP markers screened across all 116 cultivars. Phenotypic data for all cultivars were collected over a 35-year period and data from four years were used. Mixed linear models analyses in TASSEL v5.0.0 were used for five of the traits while the remaining five traits were evaluated using general linear models. A total of 86 SNP markers were identified and reported as significantly associated with one of the 10 traits. Heritability for traits matched with those previously published. This is the first reported genome wide association study in hop and as such represents an important step forward in aiding marker assisted selection. Nevertheless, it remains that further validation of these SNP markers is needed prior to implementation in a breeding program.

New and resurgent insect pests on low trellis hops

Abstract Selective insecticides have largely displaced broad‐spectrum products on cultivated hops, allowing a resurgence of historic pests and new ones to arise. The present study reports the incidence of 30 insect species from four insect Orders and 14 Families feeding on aphid susceptible and/or aphid‐resistant low trellis hop cultivars in south‐east England and at three West Midland sites. Three are Kent Red Data Book species [Adoxophyes orana (Lepidoptera: Tortricidae), Hypena rostralis (Lepidoptera: Noctuidae) and Psylliodes attenuatus (Coleoptera: Chrysomelidae)] and three are DEFRA biodiversity action plan priority insects [Acronicta rumicis, Melanchra persicariae (Lepidoptera: Noctuidae) and Spilosoma lubricipedia (Lepidoptera: Arctiidae)]. Six common lepidopteran species severely damaged hop cones (Adoxophyes orana, Autographa gamma, Epiphyas postvittana, Eupitheca assimilata, Mamestra brassicae and S. lubricipedia), whereas Biston betularia, H. rostralis, Melanchra persicariae and Phlogophora meticulosa did not. European corn borer (Ostrinia nubilalis) feeding on hops in Kent are the so‐called E‐pherotype. Lygocoris pabulinus (Hemiptera: Miridae) caged on developing flowering lateral shoots reduced numbers of developing hop cones by 38% and cone weight by 42%, whereas Liocoris tripustulatus caused no losses. Companion plants in the drive alleys between rows of hops reduced the incidences of L. tripustulatus and S. lubricipedia.

White Hop Shoot Production in Slovenia: Total Phenolic, Microelement and Pesticide Residue Content in Five Commercial Cultivars.

SUMMARYHarvesting of white hop shoots might be justified if they can be shown to be beneficial to human health. The aim of the present study is to determine the effects of hop cultivars and year of production on total phenolics, antioxidant potential, microelements and pesticide residues. Biomass per plant was highly variable across the cultivars (3.1-7.1 g dry mass per plant) and depended on hop cultivar and year (2009-2011). Total phenolics as chlorogenic acid equivalents (CAE) on dry mass basis varied from 0.60 to 1.80 mg/g, and showed significant effects across hop cultivar and year. The radical scavenging activities of the samples collected in years 2010-2012 ranged from 11 to 19 µg CAE. Ferric reducing activity was <0.01, with significantly different effects across hop cultivars (pC≤0.05) and year (py≤0.05) observed only in 2012. Traces of microelements and potentially active compounds from the use of pesticides in white hop shoots of Humulus lupulus ‘Dana’ were analysed. The content of zinc in the hop shoots on dry mass basis was very low (4 mg/kg), and it was below the limit of detection in the soil. The content of copper in the hop shoots was also very low (2.3 mg/kg), while in the soil it was below the critical emission (100 vs 300 mg/kg, respectively). All 182 active ingredients from the residues of the previously used pesticides were below the limits of detection. It can be concluded that these white hop shoots are better antioxidants than hop cones and hop leaves, and that they do not contain any pesticide residues.

β-Eudesmol, an Oxygenized Sesquiterpene, Reduces the Increase in Saliva 3-Methoxy-4-Hydroxyphenylglycol After the “Trier Social Stress Test” in Healthy Humans: A Randomized, Double-Blind, Placebo-Controlled Cross-Over Study

Hops, the immature inflorescences of the female hop plant (Humulus lupulus L.) are one of the main components of beer and provides flavor and bitterness. β-Eudesmol, an oxygenated sesquiterpene, is reported to accumulate in a particular hop cultivar. Recently, we revealed that β-Eudesmol ingestion affected autonomic nerve activity in an animal model. The effect on humans has not been elucidated, therefore, we investigated the effects of β-Eudesmol on reducing objective and subjective markers related to sympathetic nerve activity after the application of mental stress in healthy participants. Fifty participants (male and female aged 20 to 50 years) were randomly assigned to two groups. Five minutes before taking the Trier Social Stress Test (TSST) as a mental stressor, participants in each group ingested a beverage containing β-Eudesmol, the active beverage, or a placebo beverage that did not contain β-Eudesmol. Saliva 3-methoxy-4-hydroxyphenylglycol (MHPG), a major product of noradrenaline breakdown and a representative marker of sympathetic nerve activity, was significantly lower just after the TSST in the active group compared with the placebo group. Saliva cortisol, a marker of the endocrine stress response system, was not significantly different between the two groups. No adverse events related to test beverage ingestion were observed. This is the first experimental evidence of β-Eudesmol effect for mental stress in human.

Effect of drying temperature on the content and composition of hop oils

In terms of content, the paper is aimed at analysing and comparing the quality of fresh green hops and hops dried at two drying temperatures – 55°C (in the traditional manner) and 40°C (using the so-called gentle drying), regarding the maximum preservation of hop essential oils. Comparative experiments were carried out in an experimental chamber dryer with two Czech hop cultivars Saaz and Harmonie. The moisture content of hops at the beginning of drying was 75% and at the end of drying it was 9–10%. By lowering the drying temperature from 55°C to 40°C, the drying time in cv. Saaz prolonged from 8 to 10 h and for the cv. Harmonie from 9 h to 12 h. Compared to fresh hops, the amount of hop oils decreased by 10% when dried at 40°C and by 36% (cv. Saaz) and 43% (cv. Harmonie) when dried at 55°C. These losses can be considered significant, especially for hops intended for late and dry beer hopping. However, by drying the hops at different temperatures, the ratios between various components of the essential oils and thus also their sensory character remained approximately unchanged. Due to the reduced amount of essential oils, the drying effect reduces the intensity of hop aroma depending on the drying temperature.

A Cultivar-Based Screening of Hops for Dextrin Degrading Enzymatic Potential

Enzymes in hops have recently been demonstrated to hydrolyze beer dextrins, produce fermentable sugars in finished beer, and pose significant safety and quality challenges for brewers. In an effort to mitigate beer refermentation following dry-hopping in the presence of yeast and the effect of “hop creep,” brewers look to adjust recipes, selection of ingredients, and dry-hopping conditions. With phytochemical/secondary metabolite based flavor differences known to exist across hop cultivars, it was posited that there may also exist unique enzymatic activities based upon cultivar and that brewers may use these differences to guide their hopping practices. The enzymatic power of 30 hop cultivars was screened using specific enzyme assay kits (α-amylase, β-amylase, amyloglucosidase) on hops as well as quantifying via liquid chromatography the nonfermentable dextrin degradation and sugar production in beer dry-hopped with different hops. Differences across all cultivars were found and cluster analysis revealed groupings that were not based on pedigree, genetic makeup, or specific enzyme activities. The influence of growing and harvest practices, processing conditions, and presumably age post processing on hop enzymatic power warrants more investigation. These findings indicate that the choice of hop cultivar may influence the degree of refermentation in finished beer as a result of dry-hopping.

Assessment of the phytochemical profiles of novel hop (Humulus lupulus L.) cultivars: A potential route to beer crafting

This study investigated the volatile phytochemical diversity of 30 samples obtained from experimental hybrid and commercial H. lupulus L. plants. Essential oils distilled from these samples were analysed by high resolution gas chromatography coupled with accurate mass time-of-flight mass spectrometry (GC–accTOFMS). A total of 58 secondary metabolites, mainly comprising 18 esters, 6 monoterpene hydrocarbons, 2 oxygenated monoterpenes, 20 sesquiterpene hydrocarbons, 7 oxygenated sesquiterpenes, and 4 ketones, were positively or tentatively identified. A total of 24 metabolites were detected in all samples, but commercial cultivars (selected for brewing performance) had fewer compounds identified compared to experimental genotypes. Chemometrics analyses enabled distinct differentiation of experimental hybrids from commercial cultivars, discussed in terms of the different classes of compounds present in different genotypes. Differences among the mono- and sesquiterpenoids, appear to be related to either: i) the genetic origin of the plants; or ii) the processes of bioaccumulation of the identified secondary metabolites.

Registration of High Alpha‐Acid Male Hop Germplasm USDA 21267M

USDA 21267M (Reg. No. GP‐37, PI 559093) male hop (Humulus lupulus L. var. lupulus) germplasm was released to the public 6 Dec. 2011. This germplasm has been used by the USDA–ARS for hop cultivar development for several decades. Nonagency interest in this germplasm was expressed, and release of this material is considered beneficial for the US public. USDA 21267M arose from a cross, made in 1973 involving the hop cultivar Comet and the USDA–ARS hop male germplasm 21110M. The defining characteristics of USDA 21267M are its high alpha acid levels observed in lupulin glands as well as good breeding value for yield, alpha and beta acid levels, and high xanthohumol levels. It is expected that hop breeders will use this germplasm as a parent for high‐yielding, high‐alpha hop breeding material. The release of USDA 21267M will give both public and private breeders new germplasm for varietal development.

Registration of Downy Mildew–Resistant Male Hop Germplasm USDA 21087M

USDA 21087M (Reg. No. GP‐36, PI 558607) male hop (Humulus lupulus L. var. lupulus) germplasm was released to the public on 6 Dec. 2011. This germplasm has been used by the USDA–ARS for hop cultivar development for several decades. Nonagency interest in this germplasm was expressed, and release of this material is considered beneficial for the US public. USDA 21087M was obtained from the former Yugoslavia by the USDA–ARS in 1974 as Selection 3/3. The defining characteristics of USDA 21087M are its high resistance to downy mildew in the US Pacific Northwest, potential resistance to non‐v4/v6 strains of powdery mildew, and potential good storage ability in hop offspring. It is expected that hop breeders will use this germplasm as a parent for development of downy mildew–resistant hop cultivars. The release of USDA 21087M will give both public and private breeders new germplasm for cultivar and germplasm development.

Aroma profile and bitter acid characterization of hop cones (Humulus lupulus L.) of five healthy and infected Polish cultivars

The characteristic hoppy aroma is directly correlated with the chemical composition of its volatile profile and essential oil. The aim of this work was to analyse the essential oils and volatile fractions of five popular Polish hop cultivars (Sybilla, Lubelski, Pulawski, Magnat and Marynka), healthy and infected by viruses and viroids by SPME and GC–MS. Hop fraction of hard resins and alpha- and beta acids were determined according to the official analytical method (modified Wöllmer method EBC 7.7 and HPLC method EBC 7.5, respectively). Hop Mosaic Virus (HpMV), Apple Mosaic Virus (ApMV), Hop Latent Viroid (HLVd) are commonly found in plants of all hop cultivars commercially produced in Poland. Monoterpenes hydrocarbons showed higher percentages in the aroma profile of infected cones (82.5%–94.3%) than in the healthy ones (81.1%–91.4%) and increased from the healthy to the infected plants, except for ‘Sybilla’ that had an opposite trend (from 90.1% in healthy to 77.9% in infected hop). Hydrogenated sesquiterpenes were the second main class of aroma constituents with major amount in healthy cones with the exception for ‘Sybilla’. Myrcene was always the most abundant chemical constituent in all the infected cultivars, except for ‘Sybilla’ (from 86.3% in infected to 74.6% in healthy plants), followed by α-humulene in four of the five cultivars except for ‘Marynka’, where (E)-β-farnesene showed higher amount.The EO yields related to the five healthy cultivars showed variations between 0.2% in ‘Lubelski’ up to 1.1% in ‘Marynka’, very similar to those observed in the five infected cultivars. Monoterpenes predominated over sesquiterpenes in healthy and infected ‘Sybilla’ and ‘Marynka’ EOs, while in healthy and infected ‘Lubelski’ and ‘Magnat’ prevailed an opposite trend. ‘Magnat’ is the only one cultivar that showed different amount of the two main classes of constituents between the healthy and infected plants (sesquiterpenes 64.6% in healthy cones and 50.5% of monoterpenes in the infected ones). Myrcene was the main constituent in all the infected EO samples (29.9%–67%), while α-humulene predominated only in three healthy cultivars (‘Lubelski’, ‘Pulawski’, and ‘Magnat’ with 34.2, 36.8, 31.0%, respectively).The content of bitter substances (α and β-acids as well as hard and soft resins) varied in healthy and infected hop plants and this behaviour was strongly dependent on the selected cultivars but differences between healthy and infected plants were not statistically significant. In conclusion virus/viroid infection seems not to interfere in a substantial way with the normal hop secondary metabolite biosynthesis, but only in the biomass yields.