Monitoring granule formation in anaerobic upflow bioreactors using oligonucleotide hybridization probes

The process of granule formation in upflow anaerobic sludge blanket (UASB) reactors was studied using oligonucleotide hybridization probes. Two laboratory-scale UASB reactors were inoculated with sieved primary anaerobic digester sludge from a municipal wastewater treatment plant and operated similarly except that reactor G was fed glucose, while reactor GP was fed glucose and propionate. Size measurements of cell aggregates and quantification of different populations of methanogens with membrane hybridization targeting the small-subunit ribosomal RNA demonstrated that the increase in aggregate size was associated with an increase in the abundance of Methanosaeta concilii in both reactors. In addition, fluorescence in situ hybridization showed that the major cell components of small aggregates collected during the early stages of reactor startup were M. concilii cells. These results indicate that M. concilii filaments act as nuclei for granular development. The increase in aggregate size was greater in reactor GP than in reactor G during the early stages of startup, suggesting that the presence of propionate-oxidizing syntrophic consortia assisted the formation of granules. The mature granules formed in both reactors exhibited a layered structure with M. concilii dominant in the core, syntrophic consortia adjacent to the core, and filamentous bacteria in the surface layer. The excess of filamentous bacteria caused delay of granulation, which was corrected by increasing shear through an increase of the recycling rate.     

Biological sulfate reduction using synthesis gas as energy and carbon source

Biological sulfate reduction was studied in laboratory-scale gas-lift reactors. Synthesis gas (gas mixtures of H(2)/CO/CO(2)) was used as energy and carbon source. The required biomass retention was obtained by aggregation and immobilization on pumice particles. Special attention was paid to the effect of CO addition on the sulfate conversion rate, aggregation, and aggregate composition.Addition of 5% CO negatively affected the overall sulfate conversion rate; i.e., it dropped from 12-14 to 6-8 g SO(2-) (4)/L day. However, a further increase of CO to 10 and 20% did not further deteriorate the process. With external biomass recycling the sulfate conversion rate could be improved to 10 g SO(2-) (4)/L day. Therefore biomass retention clearly could be regarded as the rate-limiting step. Furthermore, CO affected the aggregate shape and diameter. Scanning electron microscopy (SEM) photographs showed that rough aggregates pregrown on H(2)/CO(2) changed into smooth aggregates upon addition of CO. Addition of CO also changed the aggregate Sauter mean diameter (d(32)) from 1.7 mm at 5% CO to 2.1 mm at 20% CO. After addition of CO, a layered biomass structure developed. Acetobacterium sp. were mainly located at the outside of the aggregates, whereas Desulfovibrio sp. were located inside the aggregates. (c) 1996 John Wiley & Sons, Inc.     

Structure and Dynamics of Anaerobic Bacterial Aggregates in a Gas-Lift Reactor

Anaerobic mixed-culture aggregates, which converted glucose to acetic, propionic, butyric, and valeric acids, were formed under controlled conditions of substrate feed (carbon limitation) and hydraulic regimen. The continuous-flow system used (anaerobic gas-lift reactor) was designed to retain bacterial aggregates in a well-mixed reactor. Carrier availability (i.e., liquid-suspended sand grains) proved necessary for bacterial aggregate formation from individual cells during reactor start-up. Electron microscopic examination revealed that incipient colonization of sand grains by bacteria from the bulk liquid occurred in surface irregularities, conceivably reflecting local quiescence. Subsequent confluent biofilm formation on sand grains proved to be unstable, however. Substrate depletion in the bulk liquid is assumed to weaken deeper parts of the biofilm due to cellular lysis, after which production of gas bubbles and liquid shearing forces cause sloughing. The resulting fragments, although sand free, were nevertheless large enough to be retained in the reactor and gradually grew larger through bacterial growth and by clumping together with other fragments. In the final steady state, high cell densities were maintained in the form of aggregates, while sand had virtually disappeared due to sampling losses and wash-out. Numerical cell densities within aggregates ranged from 10¹²/ml at the periphery to very low values in the center. The cells were enmeshed in a polymer matrix containing polysaccharides; nevertheless, carbon sufficiency was not a prerequisite to sustain high hold-up ratios.     

南亚热带人工林种植对赤红壤团聚体分布及稳定性的影响

本文选取南亚热带地区桉树、杉木、马尾松、米老排和红锥5种典型人工林为研究对象,采用Elliott湿筛法和Le Bissonnais(LB)法研究了人工林对土壤团聚体稳定性的影响。结果表明: 5种人工林土壤经湿筛法处理后,水稳性团聚体(WR_>0.25)均在62.2%以上,团聚体平均重量直径(MWD)和几何平均直径(GMD)分别介于1.58～3.71和0.57～2.02 mm,表现为杉木林最大,桉树林最小。各林分土壤团聚体结构破坏率(PAD)介于4.6%～31.5%之间;采用转移矩阵法评价5种人工林土壤团聚体,得出土壤团聚体稳定性指数(ASI)为杉木林>红锥林>米老排林>马尾松林>桉树林。在LB法3种处理下,快速湿润处理(FW)对土壤团聚体的破坏程度最大,说明消散作用是研究区团粒崩解的主要机制;预湿润振荡处理(WS)的破坏程度最小;慢速湿润处理(SW)介于两者之间,MWD和GMD值变化一致,均表现为WS>SW>FW,且随土层的加深,呈现逐渐降低的趋势。5种人工林土壤在LB法FW处理下团聚体GMD值与湿筛法ASI、MWD、GMD均达到显著正相关,表明湿筛法与LB法的FW处理具有很好的相关性,可用于表征南亚热带赤红壤地区土壤团聚体的稳定性。综合MWD、GMD、PAD和ASI等团聚体稳定性指标,杉木人工林比其他4种人工林更有助于土壤团聚水平的提高,其土壤结构相对较为稳定。     

Formation of bacterial aggregates in a continuous-flow reactor: Influence of carrier availability

Summary A dilution-rate shift-up was employed to induce bacterial hold-up in a continuous-flow gas-lift reactor. A minimum carrier concentration (sand, 2–5 g/l) was found a prerequisite for formation of bacterial aggregates, which fermented glucose either to propionate/acetate or to butyrate/acetate. Higher levels of sand did not affect the onset of propionate/acetate-forming aggregates, but decreased the rate at which they subsequently grew. Reversely, butyrate/acetate-producing aggregates grew at a constant rate but the onset of their formation was progressively retarded by increasing sand concentrations. In both cases, completion of start-up was most rapid at low sand concentrations.     

冻融交替对黑土团聚体稳定性的影响

应用Le Bissonnais法分析冻融循环(0、1、3、5和9)对3～5 mm黑龙江省黑土团聚体稳定性的影响,采用蜡封法分析黑土孔隙度的变化.结果表明: 不同粒径团聚体含量随冻融循环次数增加均呈波动状态,团聚体含量变化系数随冻融循环次数的增加逐渐趋于稳定;快速湿润、慢速湿润、预湿润震荡3种处理土壤中>0.25 mm团聚体含量有明显差异;孔隙度随冻融循环次数的增加而增大,变化范围在32.4%～41.4%.随冻融次数的增加,不同破碎方式下团聚体含量变化程度较低,团聚体平均重量直径与孔隙度呈负相关,表明冻融条件下孔隙度是影响团聚体稳定性的重要因素.     

Biomass retention in a glucose acidifying anaerobic gas-lift reactor: isolation of the organism responsible for granule formation

Summary A study was undertaken of the microbial composition of aggregates from an acidifying anaerobic gas-lift reactor. For this purpose a simple 100 ml anaerobic gas lift reactor was developed. It was found that the predominant organism in the aggregates was Selenomonas ruminantium. Both, microscopical observations and a newly developed enumeration technique led to the conclusion that the mixed granules consisted mainly of this organism. Grown in pure culture, S. ruminantium was capable of forming aggregates. These aggregates resembled the mixed aggregates both macro- as well as microscopically. Furthermore the fermentation pattern of this pure aggregated culture was similar to that of a mixed aggregated culture.     

长白山阔叶红松林不同演替阶段土壤团聚体粒径组成及有机碳含量变化

阔叶红松林是我国东北重要的原生群落,其土壤团聚体在森林生态系统碳固定中具有重要作用.本研究采用空间代替时间的方法,选取白桦幼龄林、白桦中龄林、白桦成熟林、阔叶红松成熟林和阔叶红松过熟林5个不同演替序列,通过湿筛法研究长白山天然针阔混交林群落恢复演替中土壤团聚体粒径组成及有机碳含量的变化.结果表明: 土壤团聚体粒径组成受演替过程影响较大,不同演替阶段下土壤团聚体各粒级所占比例差异显著.团聚体平均质量直径随演替的进行表现为先升高再降低的单峰形式,且最高点出现在白桦成熟林阶段.土壤中不同粒级的团聚体内有机碳含量随着演替的进行呈先增加后略有下降的趋势,且团聚体内有机碳含量最大值出现在阔叶红松成熟林阶段.在同一演替阶段下,0～5和5～10 cm土层(除演替末期的阔叶红松过熟林外)中的各粒径团聚体内有机碳含量都随着粒径的减小而增加,而10～20 cm土层中的各粒径团聚体内有机碳含量都随着粒径的减小而减小.从演替初期的白桦幼龄林到演替末期的阔叶红松过熟林,每个样地内的同一粒径团聚体内有机碳含量均具有明显的垂直分布特性,均随着土层深度的增加而显著降低.     

长期秸秆还田对水稻土团聚体及氮磷钾分配的影响

为了探究秸秆还田配施化肥条件下水稻土团聚体组成及其稳定性,以及土壤团聚体氮、磷、钾养分特征,本研究进行了34年定位试验,设置无肥(CK)、化肥(NPK)、秸秆还田+化肥(NPKS)3个处理。采用湿筛法测定0～20、20～40 cm土层水稳性团聚体组成,量化分析氮、磷、钾养分分配特征、贡献率和活化度。结果表明: 水稻土水稳性团聚体以>2 mm和0.25～1 mm粒级为主,<0.053 mm粒级含量最低。与CK相比,在0～20 cm和20～40 cm土层,NPKS处理增加了>2 mm和1～2 mm团聚体含量,降低了0.053～0.25 mm和<0.053 mm团聚体含量;NPK处理在0～20 cm土层也表现出与NPKS类似的规律。NPKS较NPK处理0～20 cm和20～40 cm土层团聚体平均重量直径(MWD)提高3.9%～15.5%,几何平均直径(GMD)提高6.3%～41.7%,不稳定团粒指数(E_LT)降低5.7%～28.7%。NPKS处理显著提高了团聚体全氮、有效磷和速效钾含量,尤其是直径>0.25 mm部分,但对碱解氮和全钾的提升效果与NPK处理差异不显著。水稻土团聚体养分贡献率受到其团聚体组成的影响,NPKS处理明显增大了>1 mm团聚体氮、磷、钾养分贡献率。秸秆还田配施化肥可提高0～20 cm和20～40 cm土层团聚体稳定性,并增加团聚体氮、磷、钾养分含量,尤其是大团聚体(>1 mm)。该研究结果为调节土壤碳氮比来保障稻田土壤质量和资源可持续发展提供了科学依据。     

CANON and Anammox in a gas-lift reactor

Anoxic ammonium oxidation (Anammox) and Completely Autotrophic Nitrogen removal Over Nitrite (CANON) are new and promising microbial processes to remove ammonia from wastewaters characterized by a low content of organic materials. These two processes were investigated on their feasibility and performance in a gas-lift reactor. The Anammox as well as the CANON process could be maintained easily in a gas-lift reactor, and very high N-conversion rates were achieved. An N-removal rate of 8.9 kg N (m(3) reactor)(-1) day(-1) was achieved for the Anammox process in a gas-lift reactor. N-removal rates of up to 1.5 kg N (m(3) reactor)(-1) day(-1) were achieved when the CANON process was operated. This removal rate was 20 times higher compared to the removal rates achieved in the laboratory previously. Fluorescence in situ hybridization showed that the biomass consisted of bacteria reacting to NEU, a 16S rRNA targeted probe specific for halotolerant and halophilic Nitrosomonads, and of bacteria reacting to Amx820, specific for planctomycetes capable of Anammox.     

黄土高原天然次生林植被演替过程中土壤团聚体有机碳动态变化

土壤团聚体物理保护是促进有机碳积累主要机制之一。以黄土高原子午岭林区天然次生林植被演替群落为对象,研究从农田、草地（白羊草,Bothriochloa ischaemum）、灌木林（沙棘,Hippophae rhamnoides）、先锋林（山杨,Populus davidiana）到顶级林（辽东栎,Quercus liaotungensis）5个植被演替阶段0-20 cm土壤团聚体稳定性和团聚体有机碳的动态变化,并分析团聚体有机碳的影响因素。结果表明：土壤团聚体稳定性随着植被演替显著提高（P<0.05）,顶级林的团聚体稳定性最高;土壤有机碳含量和各粒径土壤团聚体（> 2 mm、2-0.25 mm、0.25-0.053 mm、<0.053 mm）有机碳含量均随着植被演替而增加。除草地0.25-0.053 mm团聚体有机碳含量最高外,其他演替阶段均为0.25-2 mm粒径最高。根系生物量、凋落物生物量、微生物生物量碳、团聚体稳定性均与团聚体有机碳含量呈显著正相关关系（P<0.05）。总体而言,长期植被演替有助于团聚体稳定性和团聚体有机碳累积。     

The influence of link protein stabilization on the viscometric properties of proteoglycan aggregate solutions

The dynamic, steady-shear and transient shear flow properties of precisely prepared link-stable (s0 136, 66% aggregate) and link-free (s0 93, 59% aggregate) proteoglycan aggregate solutions at concentrations ranging from 10 to 50 mg/ml were determined using a cone-on-plate viscometer in a mechanical spectrometer. All proteoglycan solutions tested possessed: (1) linear viscoelastic properties - as measured by the dynamic complex modulus under small amplitude steady oscillatory conditions (1 less than or equal to omega less than or equal to 100 rad/s) - and (2) nonlinear shear-rate dependent apparent viscosities and primary normal stress difference under steady shearing conditions (0.25 less than or equal to gamma less than or equal to 250 s-1). Our transient flow data show that all proteoglycan aggregate solutions exhibited transient stress overshoot effects in shear stress and normal stress. From these steady and transient flow data, we conclude that link protein stabilized aggregates have significant effects on their dynamic and steady-shear properties as well as transient flow properties. The transient stress overshoot data provide a measure of the energy per unit volume of fluid required to overcome the proteoglycan networks in solution from a resting state. Thus we found that link-stable aggregates form much stronger networks than link-free aggregates. This is corroborated by the fact that link-stable aggregates form more elastic (lower than delta) and stiffer (higher [G*]) networks than link-free aggregates. The complete spectrum of viscometric flow data is entirely compatible with the proposed role of link protein in adding structural stability to the proteoglycan-hyaluronate bond. In cartilage, the enhanced strength of the networks formed by link-stable aggregates may play an important role in determining the material properties of the tissue and thereby contribute to the functional capacity of cartilage in diarthrodial joints.     

pH and Glucose Profiles in Aggregates of Bacillus laevolacticus

Size distributions and glucose and pH profiles of aggregates of the d-(-)-lactic acid-producing organism Bacillus laevolacticus were measured. The organisms were grown in continuous culture with a medium glucose concentration of either 280 or 110 mM. A maximal aggregate diameter of 2.2 mm, with a Sauter mean of 1.46 mm, was determined for the former culture condition, whereas aggregates from a culture with 110 mM glucose input had a maximal diameter of 1.9 mm (Sauter mean of 1.07 mm). A pH gradient of approximately 2 U was observed for large aggregates (above 1.5 mm). In smaller aggregates (0.75 mm), the pH value in the interior part was approximately 0.4 U lower than that in the culture fluid. It could be concluded that, in cultures with the high glucose input, lactic acid accumulated within the aggregates to such an extent that metabolism in the central region of the larger aggregates could not proceed further. In these cultures, approximately 90% of the total biomass was active. In aggregates from cultures with a low glucose input, glucose only partly penetrated the larger-sized aggregates, and the activity of this culture was reduced to approximately 70% of the biomass. These aggregates were found to decrease in size after prolonged periods of cultivation. It is suggested that this is caused by glucose depletion in the interior of the aggregates. It is concluded that the availability of glucose is an important factor in determining the size of aggregates of B. laevolacticus.     

Distribution of sulfate-reducing and methanogenic bacteria in anaerobic aggregates determined by microsensor and molecular analyses.

Using molecular techniques and microsensors for H(2)S and CH(4), we studied the population structure of and the activity distribution in anaerobic aggregates. The aggregates originated from three different types of reactors: a methanogenic reactor, a methanogenic-sulfidogenic reactor, and a sulfidogenic reactor. Microsensor measurements in methanogenic-sulfidogenic aggregates revealed that the activity of sulfate-reducing bacteria (2 to 3 mmol of S(2-) m(-3) s(-1) or 2 x 10(-9) mmol s(-1) per aggregate) was located in a surface layer of 50 to 100 microm thick. The sulfidogenic aggregates contained a wider sulfate-reducing zone (the first 200 to 300 microm from the aggregate surface) with a higher activity (1 to 6 mmol of S(2-) m(-3) s(-1) or 7 x 10(-9) mol s(-1) per aggregate). The methanogenic aggregates did not show significant sulfate-reducing activity. Methanogenic activity in the methanogenic-sulfidogenic aggregates (1 to 2 mmol of CH(4) m(-3) s(-1) or 10(-9) mmol s(-1) per aggregate) and the methanogenic aggregates (2 to 4 mmol of CH(4) m(-3) s(-1) or 5 x 10(-9) mmol s(-1) per aggregate) was located more inward, starting at ca. 100 microm from the aggregate surface. The methanogenic activity was not affected by 10 mM sulfate during a 1-day incubation. The sulfidogenic and methanogenic activities were independent of the type of electron donor (acetate, propionate, ethanol, or H(2)), but the substrates were metabolized in different zones. The localization of the populations corresponded to the microsensor data. A distinct layered structure was found in the methanogenic-sulfidogenic aggregates, with sulfate-reducing bacteria in the outer 50 to 100 microm, methanogens in the inner part, and Eubacteria spp. (partly syntrophic bacteria) filling the gap between sulfate-reducing and methanogenic bacteria. In methanogenic aggregates, few sulfate-reducing bacteria were detected, while methanogens were found in the core. In the sulfidogenic aggregates, sulfate-reducing bacteria were present in the outer 300 microm, and methanogens were distributed over the inner part in clusters with syntrophic bacteria.     

The Effect of Shear Rate on the Molecular Mass Distribution of Heat-Induced Aggregates of Mixtures Containing Whey Proteins and κ-Carrageenan

The present study attempts to characterize the effect of shear rate on the composition, size, and molecular weight of the protein aggregates present in the upper layer after phase separation of 5% whey protein isolate (WPI) mixed with 0.5% κ-carrageenan (κ-car) at pH 7.0. The mixtures were heated and sheared under different shearing rates. Size exclusion chromatography (SEC), dynamic light scattering, and static light scattering were employed to describe the effect of shear rate on the size and molecular mass of WPI aggregates. At the molecular level, the size of the aggregates increased with an increase in shear rate. Shear rate also caused a decrease in turbidity of the upper layer after centrifugation. SEC combined with multi-angle laser light scattering showed that the WPI aggregates molecular mass was between 10⁶and 10⁷ g/mol when the shear rate increased from 3.6 to 86.4 s⁻¹. Two empirical models described well the effect of shear rate on the size of WPI aggregates, and both models gave comparable results. By varying process parameters such as flow behavior and temperature, it is possible to control WPI aggregation and, thus, obtain aggregates with a range of different characteristics (size).     

马尾松人工林土壤各粒径团聚体湿筛后的有机碳分配

选取25a、45a和65a马尾松人工林为研究对象,采用湿筛法对各粒径土壤团聚体分别湿筛。探究了马尾松人工林各粒径团聚体湿筛后的团聚体有机碳分配,以探讨各粒径团聚体湿筛后分配到同一粒级团聚体有机碳含量及其对团聚体水稳性的贡献差异。结果表明：种植年限增加显著降低土壤团聚体水稳性（P<0.05）;各粒径团聚体湿筛后分配的有机碳随粒级减小含量呈先降后增趋势,以保持原粒级团聚体有机碳（12.96-32.01 g/kg）含量最高,其次是<0.25 mm粒级（8.08-23.53 g/kg）。各粒径团聚体湿筛分配到同一粒级的有机碳以保持原粒级的含量最高（P<0.05）;土壤团聚体水稳性与各粒径团聚体湿筛后保持原粒径的有机碳呈显著或极显著正相关（P<0.05或0.01）,分配到越小的粒级正相关性越不显著。此外,团聚体水稳性与各粒径团聚体湿筛分配到同一粒级的有机碳呈正相关,以保持原粒级相关性最高（P<0.01或0.05）;回归方程及相关性系数表明,有机碳与保持原粒径团聚体呈显著呈或极显著正相关（P<0.05或0.01）,与消散到其他粒级的团聚体呈负相关或极显著负相关（P>0.05或<0.01）。本研究得出有机碳含量增加促进更大粒径团聚体形成。反之,促使大粒径团聚体向较小粒径团聚体转化。同一粒级团聚体间,保持原粒级团聚体比易转化形成更大粒级团聚体有更高的有机碳含量和更强的水稳性,这对团聚体的固碳提供了新的认识。     

应用Le Bissonnais 法研究黄土丘陵区植被类型对土壤团聚体稳定性的影响

植被类型直接影响土壤特性,对土壤团聚体的形成和稳定性有重要影响,水稳性团聚体是反映黄土高原土壤抗蚀性的最佳指标。本文选择黄土丘陵区延河流域作为研究区域,应用Le Bissonnais(LB)法和Yoder法测定了森林、森林草原两种植被类型下土壤水稳性团聚体稳定性,对比分析了LB法3种处理的结果,并计算土壤团聚体平均重量直径（MWD）和可蚀性因子K值。结果表明：在LB法3种湿润处理下,预湿后扰动处理（WS）对土壤团聚体结构的破坏程度最大,处理后土壤水稳性团聚体以<0.2 mm为主;快速湿润处理（FW）对团聚体的破坏程度次之;慢速湿润处理(SW)对团聚体的破坏程度最小,处理后土壤水稳性团聚体主要以>2 mm团聚体为主;说明黄土丘陵区延河流域土壤团聚体破坏的主要机制是气爆作用（消散作用）和机械扰动。LB法的3种处理结果中预湿后扰动的测定结果与传统的湿筛法（Yoder法）更接近。LB法包含Yoder法的基本原理,能够全面、准确的测定土壤团聚体结构,适宜作为黄土丘陵区土壤团聚体测定方法。森林植被类型的土壤团聚体平均重量直径（MWD）大于森林草原植被类型,而且SW>FW>WS,但可蚀性因子K值却是森林植被类型小于森林草原植被类型。土壤水稳性团聚体由小颗粒向大颗粒转变,土壤结构趋于稳定。不同植被类型下土壤有机质含量不同,土壤团聚体形成过程及土壤团聚度也有差异,因而造成土壤可蚀性和土壤抗蚀性能不同。     

峡谷型喀斯特不同生态系统土壤团聚体稳定性及有机碳特征

通过野外调查与室内分析相结合的方法,对峡谷型喀斯特水田(ST)、旱地(HD)、草地(CD)、灌丛(GC)、人工林(RGL)、次生林(CSL)6种生态系统的土壤团聚体及其有机碳的分布特点进行了研究.结果表明: 干筛处理下(除HD外)均以>8 mm的土壤团聚体含量最高,总体上不同粒径土壤团聚体含量呈现随粒径减小先降低后增加再降低的趋势;湿筛处理下(除HD外)均以>5 mm的土壤团聚体含量最高,总体上不同粒径土壤团聚体含量随粒径减少而降低.干筛处理土壤团聚体的平均质量直径(MMD)为ST>CD>RGL>CSL>GC>HD;几何平均直径(GMD)为ST>CD>RGL>CSL>HD>GC;湿筛处理的MMD为RGL>CSL>GC>CD>ST>HD,GMD为CSL>RGL>GC>CD>ST>HD,用湿筛的MMD特别是GMD评价喀斯特石灰土土壤团聚体质量性状比干筛指标更准确.团聚体机械稳定性的分形维数D表现为CD>HD>ST>RGL>CSL>GC,水稳定性表现为GC>CSL>RGL>HD>CD>ST.土壤SOC含量越高,D、MMD和GMD值越大,土壤结构越合理.不同生态系统下各粒径团聚体SOC含量均以0.053～0.25 mm粒径最高,部分>5 mm粒径含量最低,但以>5 mm团聚体对土壤SOC的贡献率最高,且贡献率随着粒径的减小逐渐降低.     

Characterization of spatial growth and distribution of chondrocyte cells embedded in collagen gels through a stereoscopic cell imaging system

The stereoscopic image analysis of fluorescence-labeled chondrocyte cells for cytoplasm and nucleus was performed for the quantitative determination of spatial cell distribution as well as cell aggregate size in the collagen-embedded culture. The three-dimensional histomorphometric data indicated that the cells in the gels formed aggregates by cell division, and the size of aggregates increased with elapsed culture time. In the culture seeded at 2.0 x 10(6) cells/cm(3), the cells showed a semilunar shape that is a typical chondrocytic morphology, and formed the dense cell aggregates producing collagen type II. From the quantitative analysis of aggregate size, in addition, it was found that the cell division caused the aggregate growth with an increase of cell number in respective aggregates at 7 days, and some of aggregates made coalescence at 14 days. In the gel surface region, further coalescence of aggregates accompanied with cell division produced larger cell clusters, creating cell layers on the gel surface at the end of culture (21 days). In the culture seeded at 2.0 x 10(5) cells/cm(3), the different manner of aggregation was observed. At 14 days, the loose clusters of spindle-shaped cells emerged in the deeper region of gels, suggesting that the cell migration and gathering occurred in the gels. This loose-clustered aggregates did not produce collagen type II. Our results suggest that the seeding density is a factor to cause different mechanisms of cell distribution accompanied with the formation of aggregates as well as collagen type II.     

Aggregation kinetics of human mesenchymal stem cells under wave motion

Human mesenchymal stem cells (hMSCs) are primary candidates in cell therapy and regenerative medicine but preserving their therapeutic potency following culture expansion is a significant challenge. hMSCs can spontaneously assemble into three‐dimensional (3D) aggregates that enhance their regenerative properties. The present study investigated the impact of hydrodynamics conditions on hMSC aggregation kinetics under controlled rocking motion. While various laboratory methods have been developed for hMSC aggregate production, the rocking platform provides gentle mixing and can be scaled up using large bags as in wave motion bioreactors. The results show that the hMSC aggregation is mediated by cell adhesion molecules and that aggregate size distribution is influenced by seeding density, culture time, and hydrodynamic conditions. The analysis of fluid shear stress by COMSOL indicated that aggregate size distribution is inversely correlated with shear stress and that the rocking angle had a more pronounced effect on aggregate size distribution than the rocking speed due to its impact on shear stress. hMSC aggregates obtained from the bioreactor exhibit increased stemness, migratory properties, and expression of angiogenic factors. The results demonstrate the potential of the rocking platform to produce hMSC aggregates with controlled size distribution for therapeutic application.