Improved aeroponic culture of inocula of arbuscular mycorrhizal fungi

We compared conventional atomizing disc aeroponic technology with the latest ultrasonic nebulizer technology for production of Glomus intraradices inocula. The piezo ceramic element technology used in the ultrasonic nebulizer employs high-frequency sound to nebulize nutrient solution into microdroplets 1 μm in diameter. Growth of pre-colonized arbuscular mycorrhizal (AM) roots of Sudan grass was achieved in both chambers used but both root growth and mycorrhization were significantly faster and more extensive in the ultrasonic nebulizer system than in the atomizing disc system. Shearing of the AM fungi (AMF) infected roots in both the systems did not reduce inoculum viability, as evident from the MPN data. However, sheared roots from the ultrasonic nebulizer system had significantly more infective propagules than those produced in the atomizing disc system. Thus, the latest ultra-sonic nebulizer aeroponic technology appears to be superior and an alternative to conventional atomizing disc or spray nozzle systems for the production of high-quality AMF inocula. These can be used in small doses to produce a large response, which is a prerequisite for commercialization of AMF technology. Accepted: 11 January 2000     

The response of ectomycorrhizal fungal inoculum to long-term increases in nitrogen supply

The inoculum of ectomycorrhizal (EM) fungi was examined in a 16 y long nitrogen fertilization experiment maintained in a temperate oak savanna. To measure EM fungal inoculum, bur oak seedlings were grown in three types of bioassays: (i) intact soil cores that measure inoculum such as spores, mycelia and mycorrhizal roots; (ii) resistant propagule bioassays that measure inoculum types resistant to soil drying; and (iii) previously mycorrhizal root bioassays that measure the ability of EM fungi to colonize new roots from mycorrhizal roots. Colonization of bur oak seedlings was characterized by morphotyping and where necessary by restriction analysis and internal transcribed spacer (ITS) sequencing. Fourteen morphotypes were found in intact soil core bioassays with species of Cortinarius, Cenococcum and Russula abundant. Five morphotypes were found in resistant propagule bioassays with Cenococcum, a thelephoroid morphotype and a Wilcoxina-like ascomycete abundant and frequent. In intact soil core bioassays total percent root colonization and number of morphotypes were not affected by N supply in 2000 and 2001. However the composition of EM fungi colonizing oak seedling roots was different with increased N supply such that Russula spp. (primarily Russula aff. amoenolens) were most abundant at the highest level of N supply. Dominant Russula spp. did not colonize any roots in resistant propagule bioassays but did colonize oak seedling roots from previously mycorrhizal roots. Results suggest that in this savanna N supply can influence the kinds of inoculum propagules present and thereby might affect the dynamics of ectomycorrhizal communities by differentially influencing reproductive and colonization strategies.     

A shearing technique measuring resistance properties of plant stems

A shearing technique was developed to measure fracturing properties of plant stems. Shearing force measurements assess the amount of energy required to fragment plant tissue as an indication of resistance to particle breakdown during mastication. The objective of this study was to develop a rapid and inexpensive method of accurately measuring the shearing properties of forage stems. Shearing properties of alfalfa stems were measured by shearing 20 main stems from each of 24 cultivars. Stems were dried and divided into three 16 cm segments, top, middle and bottom. Each segment was sheared between two nodes at the approximate mid-point using an Ottawa Texture Measurement System with a Warner-Bratzler blade (dull grade) lowered at 1.35 mm s⁻¹. Instrument repeatability was determined using a homogenous material (plastic coffee stirrers) with mechanical and textural properties similar to those of a plant stem. The shearing technique provided a repeatable and rapid measurement of shearing force (coefficient of variation for plastic stirrers, 4.2%). For alfalfa stems harvested at similar physiological maturity, shearing forces for each shearing location on the stem differed (P < 0.001) among cultivars. At all shearing locations, shearing force varied considerably between cultivars; however, variations among shearing locations were similar. Shearing force was significantly correlated with diameter, weight, linear density and, to a lesser extent, with the cell wall chemical constituents of the stem.     

Evaluation of commercial arbuscular mycorrhizal inocula in a sand/peat medium

Eight commercial inocula of arbuscular mycorrhizal fungi (AMF) were tested for their ability to colonize plant roots in the sand/peat medium specified by the U.S. Golf Association for use in putting greens. Using the standard assay for potency of inocula (Zea mays grown for 6 weeks in containers), inocula were added at the rate recommended by the manufacturer as well as at five and ten times the recommended rate. To ensure that growth conditions were conducive to AM formation, a soil-based inoculum of native AMF also was assessed for inoculum potential. Only three of the commercial inocula formed mycorrhizas when used at the recommended rate, and the extent of colonization ranged from 0.4 to 8%. Increasing the amount of inoculum resulted in colonization levels of 8.6 to 72.5% at the highest rate (10×). Mean colonization using the native AMF was 60%. One inoculum that did not form mycorrhizas at the recommended rate or at 5× produced 8.6% colonization at 10×. An inoculum that did not produce mycorrhizas at any application rate did contain a fungus tentatively identified as a root pathogen (Olpidium brassicae) that colonized the corn roots. The failure of five of the eight commercial inocula to colonize roots when applied at the recommended rate suggests that preliminary trials should be made before commercial AMF inocula are used in important plantings.     

不同培养期深色有隔内生真菌链格孢菌对紫花苜蓿生长的影响

接种深色有隔内生真菌（dark septate endophytes,DSE）可以提高植物对养分的吸收利用,促进植物生长发育。为探讨接种不同培养时间的DSE链格孢菌Alternaria sp. CGMCC 17463对紫花苜蓿生长的影响,以紫花苜蓿为研究对象,接种培养4、6、8、10d的Alternaria sp. CGMCC 17463鲜样菌剂和风干样菌剂进行盆栽试验,以阐明不同培养时间的Alternaria sp. CGMCC 17463对紫花苜蓿出芽率、生长特性和营养含量的作用。结果表明,接种Alternaria sp. CGMCC 17463鲜样菌剂和风干样菌剂的紫花苜蓿根系内均有Alternaria sp. CGMCC 17463定殖,且均能增加紫花苜蓿株高、地上部干重、根部干重和根冠比。其中,接种培养10d的Alternaria sp. CGMCC 17463鲜样菌剂和培养8d的风干样菌剂对紫花苜蓿的促生效果最佳。同时,接种鲜样菌剂和风干样菌剂可以提高紫花苜蓿对养分吸收。随Alternaria sp. CGMCC 17463培养时间的增加,接种鲜样菌剂和风干样菌剂的紫花苜蓿的氮、磷、钾含量均呈先增加再降低的趋势。接种培养4-6d的Alternaria sp. CGMCC 17463菌剂,紫花苜蓿的氮、磷、钾含量相对较高。Pearson 相关性分析表明,Alternaria sp. CGMCC 17463培养时间与紫花苜蓿株高、根干重、根系Alternaria sp. CGMCC 17463总侵染率和菌丝侵染率呈显著正相关（P<0.05）,菌剂类型不是影响紫花苜蓿生长的主要因素,本研究为进一步开发利用Alternaria sp. CGMCC 17463菌剂提供了理论支撑。     

Influence of inoculum density and aeration volume on biomass and bioactive compound production in bulb-type bubble bioreactor cultures of Eleutherococcus koreanum Nakai

This study deals with the effects of initial inoculum density and aeration volume on biomass and bioactive compound production in adventitious roots of Eleutherococcus koreanum Nakai in bulb-type bubble bioreactors (3-L capacity). While the fresh and dry weights of the roots increased with increasing inoculum density, the highest percentage dry weight and accumulation of total target compounds (eleutheroside B and E, chlorogenic acid, total phenolics, and flavonoids) were noted at an inoculum density of 5.0 g L⁻¹. Poor aeration volume (0.05 vvm) stunted root growth, and high aeration volume (0.4 vvm) caused physiological disorders. Moreover, an inoculum density of 5.0 g L⁻¹ and an aeration volume of 0.1 vvm resulted in the highest concentration of total target compounds and least root death. Such optimization of culture conditions will be beneficial for the large-scale production of E. koreanum biomass and bioactive compounds.     

Production of glomus intraradices propagules, an arbuscular mycorrhizal fungus, in an airlift bioreactor

This work addresses the symbiotic culture of the arbuscular mycorrhizal (AM) fungus Glomus intraradices with Daucus carota hairy roots transformed by Agrobacterium rhizogenes, in two submerged culture systems: Petri dish and airlift bioreactor. AM fungi play an active role in plant nutrition and protection against plant pathogens. These fungi are obligate biotrophs as they depend on a host plant for their needs in carbohydrates. The effect of the mycorrhizal roots inoculum-to-medium volume ratio on the growth of both symbionts was studied. A critical inoculating condition was observed at approximately 0.6 g dry biomass (DW). L-1 medium, above which root growth was significantly reduced when using a low-salt minimal (M) liquid medium previously developed for hairy root-AM fungi co-culture. Below critical inoculum conditions the maximum specific root growth and specific G. intraradices spore production rates of 0.021 and 0.035 d-1, respectively, were observed for Petri dish cultures. Maximum spore production in the airlift bioreactor was ten times lower than that of Petri dish cultures and obtained with the lowest inoculum assessed (0.13 g DW. L-1 medium) with 1.82 x 10(5) +/- 4.05 x 10(4) (SEM) spores (g DW inoculum)-1 (L medium)-1 in 107 d. This work proposes a second-generation bioprocess for AM fungi propagule production in bioreactors. Copyright 1999 John Wiley & Sons, Inc.     

Optimization of Inocula Conditions for Enhanced Biosurfactant Production by Bacillus subtilis SPB1, in Submerged Culture, Using Box–Behnken Design

The inoculum age and density can influence considerably the production yield and cost of the fermentation process. Some literature studies report the use of two-stage inocula to enhance metabolite production. In the present study, optimization studies were done in order to define the best inocula conditions supporting a maximum biosurfactant production by Bacillus subtilis SPB1. Hence, by adjusting the levels of the two-stage inocula strategy, lipopeptide production was effectively enhanced to almost 3.4 g/l as estimated gravimetrically. The new defined parameters were as follows; a first inoculum age of 23 h followed by a second inoculum age and size of 4 h and 0.01, respectively. Thereby, we note an improved production as compared to the production yield described under non-optimized inocula conditions reported in our previous work.     

Quantitation of Adsorption of Rhizobia in Low Numbers to Small Legume Roots

Bacteria adsorbed in low numbers to alfalfa or clover root surfaces were counted after incubation of seedlings in mineral solution with very dilute inocula (less than 10⁵ bacteria per ml) of an antibiotic-resistant strain under defined conditions. After specified washing, bacteria which remained adsorbed to roots were selectively quantitated by culturing the roots embedded in yeast extract-mannitol-antibiotic agar and counting the microcolonies along the root surface; the range was from about 1 bacterium per root (estimated as the most probable number) to 50 bacteria per cm of root length (by direct counting). This simple procedure can be used with any pair of small-rooted plant and antibiotic-resistant bacterium, requires bacterial concentrations comparable to those frequently found in soils, and yields macroscopic localization and distribution data for adsorbed bacteria over the root surface. The number of adsorbed bacteria was proportional to the size of the inoculum. One of every four Rhizobium meliloti cells adsorbed in very low numbers to alfalfa roots resulted in the formation of a nodule. Overall adsorption of various symbiotic and nonsymbiotic bacterial strains to alfalfa and clover roots did not reflect the specificities of these legumes for their respective microsymbionts, R. meliloti and R. trifolii.     

Differences in the composition of arbuscular mycorrhizal fungal communities promoted by different propagule forms from a Mediterranean shrubland

As it is well known, arbuscular mycorrhizal (AM) colonization can be initiated from the following three types of fungal propagules: spores, extraradical mycelium (ERM), and mycorrhizal root fragments harboring intraradical fungal structures. It has been shown that biomass allocation of AM fungi (AMF) among these three propagule types varies between fungal taxa, as also differs the ability of the different AMF propagule fractions to initiate new colonizations. In this study, the composition of the AMF community in the roots of rosemary (Rosmarinus officinalis L., a characteristic Mediterranean shrub), inoculated with the three different propagule types, was analyzed. Accordingly, cuttings from this species were inoculated with either AMF spores, ERM, or colonized roots extracted from a natural soil. The AMF diversity within the rosemary roots was characterized using terminal restriction fragment length polymorphism (T-RFLP) of the small subunit (SSU) rDNA region. The AMF community established in the rosemary plants was significantly different according to the type of propagule used as inoculum. AMF taxa differed in their ability to initiate new colonizations from each propagule type. Results suggest different colonization strategies for the different AMF families involved, Glomeraceae and Claroideoglomeraceae colonizing mainly from colonized roots whereas Pacisporaceae and Diversisporaceae from spores and ERM. This supports that AMF taxa show contrasting life-history strategies in terms of their ability to initiate new colonizations from the different propagule types. Further research to fully understand the colonization and dispersal abilities of AMF is essential for their rational use in ecosystem restoration programs.     

A comparison of surface and rectal temperatures between sheared and non-sheared alpacas (Lama pacos)

The objective of this research was to determine if whole-body shearing would effect gross thermoregulation in alpacas. Eight mature, intact male alpacas were randomly assigned to one of two groups and maintained in outdoor pastures with adequate artificial shade from June through August (summer climate) in east central Alabama, USA. Group one animals (N=4) were sheared to remove all fiber to within 2 cm of their skin. Group 2 animals (N=4) were left non-sheared. Sheared alpacas tended to have lower rectal temperatures during high ambient temperatures than did non-sheared alpacas (P=0.06). Thermographic studies of the scrotum revealed cooler surface temperatures in sheared versus non-sheared alpacas (P=0.05). Temperatures in the right medial thigh of sheared animals were 0.9°C cooler than the thigh region of non-sheared animals in the morning (P<0.03). Right medial thigh temperatures were 1.6°C cooler in sheared alpacas in the afternoon (P<0.01). Significant positive correlations were found in non-sheared animals between ambient temperature and rectal temperature in the morning (r=0.612, P=0.014). In sheared animals during the morning significant positive correlations were established between the Heat Stress Index (HSI) and the right medial thigh surface temperatures (r=0.648, P=0.003), the HSI and rectal temperature (r=0.729, P=0.0003), the ambient temperature and right medial thigh surface temperature (r=0.485, P=0.04), and the ambient temperature and the rectal temperature (r=0.823, P<0.0001). In the afternoon a significant positive correlation was found in the sheared alpacas between the HSI and the right medial thigh surface temperature, rectal temperature and surface scrotal temperature (r=0.538, P=0.02, r=0.543, P=0.019 and r=0.522, P=0.045), respectively. These data indicate that whole-body shearing of alpacas could have a beneficial effect on thermoregulation when used as a preventative measure against heat stress. Shearing may assist heat dissipation resulting in a cooler surface body temperature and rectal temperature in alpacas when challenged by the heat and humidity experienced in the summer months in the southeastern United States.     

Arbuscular mycorrhizal infection changes the bacterial 16 S rDNA community composition in the rhizosphere of maize

Mycorrhizal and non-mycorrhizal (NM) maize plants were grown for 4 or 7 weeks in an autoclaved quartz sand-soil mix. Half of the NM plants were supplied with soluble P (NM-HP) while the other half (NM-LP), like the mycorrhizal plants, received poorly soluble Fe and Al phosphate. The mycorrhizal plants were inoculated with Glomus mosseae or G. intraradices. Soil bacteria and those associated with the mycorrhizal inoculum were reintroduced by adding a filtrate of a low P soil and of the inocula. At 4 and 7 weeks, plants were harvested and root samples were taken from the root tip (0-1 cm), the subapical zone (1-2 cm) and the mature root zone at the site of lateral root emergence. DNA was extracted from the roots with adhering soil. At both harvests, the NM-HP plants had higher shoot dry weight than the plants grown on poorly soluble P. Mycorrhizal infection of both fungi ranged between 78% and 93% and had no effect on shoot growth or shoot P content. Eubacterial community compositions were examined by polymerase chain reaction-denaturing gradient gel electrophoresis of 16 S rDNA, digitisation of the band patterns and multivariate analysis. The community composition changed with time and was root zone specific. The differences in bacterial community composition in the rhizosphere between the NM plants and the mycorrhizal plants were greater at 7 than at 4 weeks. The two fungi had similar bacterial communities after 4 weeks, but these differed after 7 weeks. The observed differences are probably due to changes in substrate composition and amount in the rhizosphere.     

Suppression of nodulation in soybeans by superoptimal inoculation with Bradyrhizobium japonicum

Symbiotic nodulation of the primary roots of soybeans ( Glycine max L. Merrill cv. Pride 216) is regulated by the plant, and is suppressed in response to a high inoculum dose of Bradyrhizobium japonicum USDA strain I–110 (ARS)⁺ applied at one time to the root. If an optimal dose is followed 10 h later by a superoptimal dose, nodules from the first inoculum near the base of the primary root are suppressed in a dose-dependent way similar to that observed after single inoculations. The nodules which appear are probably derived from infections initiated by the bacteria in both inocula.     

Differential effects of tropical arbuscular mycorrhizal fungal inocula on root colonization and tree seedling growth: implications for tropical forest diversity

The potential for mycorrhizae to influence the diversity and structuring of plant communities depends on whether their affinities and effects differ across a suite of potential host species. In order to assess this potential for a tropical forest community in Panama, we conducted three reciprocal inoculation experiments using seedlings from six native tree species. Seeds were germinated in sterile soil and then exposed to arbuscular mycorrhizal fungi in current association with naturally infected roots from adults of either the same or different species growing in intact forest. The tree species represent a range of life histories, including early successional pioneers, a persistent understory species, and emergent species, typical of mature forest. Collectively, these experiments show: (i) the seedlings of small-seeded pioneer species were more dependent on mycorrhizal inocula for initial survival and growth; (ii) although mycorrhizal fungi from all inocula were able to colonize the roots of all host species, the inoculum potential (the infectivity of an inoculum of a given concentration) and root colonization varied depending on the identity of the host seedling and the source of the inoculum; and (iii) different mycorrhizal fungal inocula also produced differences in growth depending on the host species. These differences indicate that host–mycorrhizal fungal interactions in tropical forests are characterized by greater complexity than has previously been demonstrated, and suggest that tropical mycorrhizal fungal communities have the potential to differentially influence seedling recruitment among host species and thereby affect community composition.     

Relationship between type and age of the inoculum cultures and betalains biosynthesis by Beta vulgaris hairy root culture

From a study of the relationship between the type and age of the inocula, and the growth and biosynthesis of betalains in a Beta vulgaris hairy root culture, the best results were achieved with a 14 d inoculum grown in submerged culture giving 42 mg betalains (16 mg betacyanins and 26 betaxanthins) and 1.5 g dry biomass in 40 ml medium.     

Mass production of Glomus mosseae spores

Five crops inoculated with Glomus mosseae were grown for 10 weeks and the development of mycorrhizal infection and sporulation were assessed. Infected roots from pot cultures of different ages were used to examine the host effect on the development of mycorrhizae. The effectiveness of each host was assessed by measuring spore numbers. For all hosts, the percentage of root length infected increased rapidly up to 10 weeks after sowing. Infectivity of root inocula increased with increasing percentage of root length infected with the inoculum for all crops, except where large numbers of mature spores (1755) had been produced on barley. The highest spore numbers were achieved in the rhizosphere of barley plants, followed by chickpea and beans. The lowest spore numbers were found in the rhizosphere of corn and okra plants. The type of the crop as well as the harvest date greatly influenced the size of the spore population and the extent of root colonization of G. mosseae.     

Role of VA-mycorrhiza in growth and mineral nutrition of apple (Malus pumila var.domestica) rootstock cuttings

One-year-old apple cuttings (Malus pumila var.domestica cv. M26) were grown for 6 months in pot culture with and without inoculum of the VA-mycorrhizal fungus (VAMF)Glomus macrocarpum in soil from a long-term fertilizer field experiment with different P availability (20, 210, and 280 mg CAL-extractable P kg⁻¹). The indigenous VAMF propagule density was reduced by 0.5 Mrad X-irradiation. At harvest, non-inoculated and inoculated plants had similar proportions of root length bearing vesicles. Net dry weight of tree cuttings was significantly increased by inoculation only at 20 mg P kg⁻¹ (+62%). Increasing P availability from 210 to 280 mg P kg⁻¹ led to a 4-week depression of shoot elongation rate only in the inoculated plants. Uptake of P was significantly enhanced by inoculation at 20 and 210 mg P kg⁻¹ (+64 and +12%, respectively). On average, inoculated plants had significantly higher concentrations of Zn in leaves and in roots (+16 and +14%, respectively) and of copper in stems and in roots (+13 and +126%, respectively). Proportion of vesicle bearing root length was significantly correlated with root caloric content. A lipid content of 0.9–4.5% in the root dry matter was attributed to the presence of vesicles corresponding to 1.6–8.2% of total root caloric content. As the control plants were also infected, the beneficial effect of VA-mycorrhiza on nutrient uptake and growth of apple cuttings was underestimated at all P levels. Furthermore, VAM-potential at the lowest P level was not fully exploited as onset of infection was most certainly delayed because of a decreased photosynthetic rate due to P deficiency. Energy drain by VAMF-infection was most probably underestimated considerably, due to, among others, loss of infected root cortex during root growth, sampling and staining. It is concluded that apple cuttings rely on VA-mycorrhizal P-uptake at least in low P soils. In high P soils, apple cuttings may profit predominantly from the uptake of Zn and Cu by the fungal symbionts.     

Patterns of diversity and adaptation in Glomeromycota from three prairie grasslands

Arbuscular mycorrhizal (AM) fungi are widespread root symbionts that often improve the fitness of their plant hosts. We tested whether local adaptation in mycorrhizal symbioses would shape the community structure of these root symbionts in a way that maximizes their symbiotic functioning. We grew a native prairie grass (Andropogon gerardii) with all possible combinations of soils and AM fungal inocula from three different prairies that varied in soil characteristics and disturbance history (two native prairie remnants and one recently restored). We identified the AM fungi colonizing A. gerardii roots using PCR amplification and cloning of the small subunit rRNA gene. We observed 13 operational taxonomic units (OTUs) belonging to six genera in three families. Taxonomic richness was higher in the restored than the native prairies with one member of the Gigaspora dominating the roots of plants grown with inocula from native prairies. Inoculum source and the soil environment influenced the composition of AM fungi that colonized plant roots. Correspondingly, host plants and AM fungi responded significantly to the soil–inoculum combinations such that home fungi often had the highest fitness and provided the greatest benefit to A. gerardii. Similar patterns were observed within the soil–inoculum combinations originating from two native prairies, where five sequence types of a single Gigaspora OTU were virtually the only root colonizers. Our results indicate that indigenous assemblages of AM fungi were adapted to the local soil environment and that this process occurred both at a community scale and at the scale of fungal sequence types within a dominant OTU.     

Bioreactor application on adventitious root culture of <Emphasis Type="Italic">Astragalus membranaceus</Emphasis>

Astragalus membranaceus is one of the most widely used traditional medicinal herbs in China, but the time required to generate a useful product in the field production is long. The growth of adventitious root cultures was compared between cultures grown in solid, liquid, or a 5-L balloon-type bubble bioreactor. The maximum growth ratio (final dry weight/initial dry weight) was determined for adventitious roots grown in the bioreactor. Studies carried out to optimize biomass production of adventitious roots compared adventitious root growth from various inoculum root lengths, inoculum densities, and aeration volume in the bioreactors. The maximum growth ratio occurred in treatments with a 1.5-cm inoculum root length, with 30 g (fresh weight) of inoculum per bioreactor or with an aeration volume of 0.1 vvm (air volume/culture medium volume per min). The polysaccharide, saponin, and flavonoid content of roots from bioreactor-grown cultures were compared to roots from field-grown plants grown for 1 and 3 yr. Total polysaccharide content of adventitious roots in the bioreactor (30.0 mg g⁻¹ dry weight (DW)) was higher than the roots of 1-yr-old (13.8 mg g⁻¹ DW) and 3-yr-old (21.1 mg g⁻¹ DW) plants in the field. Total saponin (3.4 mg g⁻¹ DW) and flavonoid (6.4 mg g⁻¹ DW) contents were nearly identical to 3-yr-old roots and higher than that of 1-yr-old roots under field cultivation.     

Breakage of transgenic tobacco roots for monoclonal antibody release in an ultra‐scale down shearing device

Transgenic tobacco roots offer a potential alternative to leaves for monoclonal antibody (MAb) production. A possible method for extraction of MAbs from roots is by homogenization, breaking the roots into fragments to release the antibody. This process was assessed by shearing 10 mm root sections (“roots”) in a 24 mL ultra‐scale down shearing device, including an impeller with serrated blade edges, intended to mimic the action of a large‐scale homogenizer. Size distributions of the remaining intact roots and root fragments were obtained as a function of shearing time. The data suggest that about 36% of the roots could not be broken under the prevailing conditions and, beyond these unbreakable roots, the fragmentation was approximately first order with respect to intact root number. It was postulated that root breakage in such a high shearing device was due to root‐impeller collisions and the particle size data suggest that roots colliding with the impeller were completely fragmented into debris particles of the order of 0.1 mm in length. IgG release normalized to release by grinding appeared to lag behind the number of roots that had fragmented, suggesting that a process of leakage followed fragmentation in the ultra‐scale down shearing device. Biotechnol. Bioeng. 2014;111: 196–201. © 2013 Wiley Periodicals, Inc.