Development of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) fibers for skin tissue engineering: effects of topography, mechanical, and chemical stimuli

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biodegradable polyester, was electrospun to form defect-free fibers with high surface-area-to-volume ratio for skin regeneration. Several parameters such as solvent ratio, polymer concentration, applied voltage, flow rate, and tip-to-target distance were optimized to achieve defect-free morphology. The average diameter of the PHBV fibers was 724 ± 91 nm. PHBV was also solvent-cast to form 2-D films, and its mechanical properties, porosity, and degradation rates were compared with PHBV fibers. Our results demonstrate that PHBV fibers exhibited higher porosity, increased ductility, and faster degradation rate when compared with PHBV 2-D films (p < 0.05). In vitro studies with PHBV fibers and 2-D films were carried out to evaluate the adhesion, viability, proliferation, and gene expression of human skin fibroblasts. Cells adhered and proliferated on both PHBV fibers and 2-D films. However, the proliferation of cells on the surface of PHBV fibers was comparable to tissue culture polystyrene (TCPS, control) (p > 0.05). The gene expression of collagen I and elastin was significantly up-regulated when compared with TCPS control, whereas collagen III was down-regulated on PHBV fibers and 2-D film after 14 days in culture. The less ductile PHBV 2-D films showed higher levels of elastin expression. Furthermore, the PHBV fibers in the presence and absence of an angiogenesis factor (R-Spondin 1) were evaluated for their wound healing capacity in a rat model. The wound contracture in R-Spondin-1-loaded PHBV fibers was found to be significantly higher when compared with PHBV fibers alone after 7 days (p < 0.05). Furthermore, the presence of fibers promoted an increase in collagen and aided re-epithelialization. Thus our results demonstrate that the topography and mechanical and chemical stimuli have a pronounced influence on the cell proliferation, gene expression, and wound healing.     

Comparative oxo-biodegradation study of poly-3-hydroxybutyrate-co-3-hydroxyvalerate/polypropylene blend in controlled environments

The potential use of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) copolymer as a biodegradable additive in polypropylene (PP) has been explored. The melt blending technique was used to produce the blend of PHBV/PP (PB10). The degradation studies of PB10 were done in the field as well as in controlled laboratory conditions. The structural changes were studied using scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and thermogravimetric analysis (TGA). SEM micrographs showed the formation of agglomerates, pits, grooves, and holes on the treated films as a result of microbial activity. FTIR spectra indicated clear evidences of oxo-biodegradation of polymer chains due to an increase in carbonyl peak index. Thermogravimetric analysis confirmed that the thermal stability of PB10 was increased after soil burial. This study contributed toward the prospective commercial applications of PHBV for use in the food packaging industry.     

Isolation and characterisation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) degrading actinomycetes and purification of PHBV depolymerase from newly isolatedStreptoverticillium kashmirense AF1

Streptoverticillium kashmirense AF1 with the ability to degrade a natural polymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was isolated from municipal sewage sludge by soil burial technique. The PHBV film was degraded by the action of extracellular enzymes secreted by the microorganisms. Degradation of PHBV was evident by the formation of clear zones of hydrolysis on the polymer containing mineral salt agar plates. The extent of PHBV degradation increased up to 30 days of incubation. Maximum production of PHBV depolymerase was observed both at pH 8 and pH 7, 45 °C, 1% substrate concentration and in the presence of lactose as an additional carbon source. Two types of extracellular PHBV depolymerases were purified fromS. kashmirense AF1 by gel permeation chromatography using Sephadex G-75. The molecular weights of the two proteins were found to be 35 and 45 kDa approximately, as determined by SDS-PAGE. The results of the Sturm test also showed more CO₂ production as a result of PHBV degradation, in the test as compared to control. The present findings indicated the degradation capabilities ofS. kashmirense AF1.     

Introducing amine functionalities on a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) surface: comparing the use of ammonia plasma treatment and ethylenediamine aminolysis

Amine functionalities were introduced onto the surface of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) films by applying radio frequency ammonia plasma treatment and wet ethylenediamine treatment. The modified surfaces were characterized by X-ray photoelectron spectroscopy (XPS) for chemical composition and Raman microspectroscopy for the spatial distribution of the chemical moieties. The relative amount of amine functionalities introduced onto the PHBV surface was determined by exposing the treated films to the vapor of trifluoromethylbenzaldehyde (TFBA) prior to XPS analysis. The highest amount of amino groups on the PHBV surface could be introduced by use of ammonia plasma at short treatment times of 5 and 10 s, but no effect of plasma power within the range of 2.5-20 W was observed. Ethylenediamine treatment yielded fewer surface amino groups, and in addition an increase in crystallinity as well as degradation of PHBV was evident from Fourier transform infrared spectroscopy. Raman maps showed that the coverage of amino groups on the PHBV surfaces was patchy with large areas having no amine functionalities.     

In vitro biocompatibility of electrospun poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) fiber mats

In the present contribution, the potential for use of the ultrafine electrospun fiber mats of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) as scaffolding materials for skin and nerve regeneration was evaluated in vitro using mouse fibroblasts (L929) and Schwann cells (RT4-D6P2T) as reference cell lines. Comparison was made with PHB and PHBV films that were prepared by solution-casting technique. Indirect cytotoxicity assessment of the as-spun PHB and PHBV fiber mats with mouse fibroblasts (L929) and Schwann cells (RT4-D6P2T) indicated that the materials were acceptable to both types of cells. The attachment of L929 on all of the fibrous scaffolds was significantly better than that on both the film scaffolds and tissue-culture polystyrene plate (TCPS), while RT4-D6P2T appeared to attach on the flat surfaces of TCPS and the film scaffolds much better than on the rough surfaces of the fibrous scaffolds. For L929, all of the fibrous scaffolds were superior in supporting the cell proliferation to the film counterparts, but inferior to TCPS at days 3 and 5, while, for RT4-D6P2T, the rough surfaces of the fibrous scaffolds appeared to be very poor in supporting the cell proliferation when comparing with the smooth surfaces of TCPS and the film scaffolds. Scanning electron microscopy was also used to observe the behavior of both types of cells that were cultured on both the fibrous and the film scaffolds and glass substrate for 24 h.     

A Tuneable Switch for Controlling Environmental Degradation of Bioplastics: Addition of Isothiazolinone to Polyhydroxyalkanoates

Controlling the environmental degradation of polyhydroxybutyrate (PHB) and polyhydroxyvalerate (P(HB-co-HV)) bioplastics would expand the range of their potential applications. Combining PHB and P(HB-co-HV) films with the anti-fouling agent 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOI, <10% w/w) restricted microbial colonisation in soil, but did not significantly affect melting temperature or the tensile strength of films. DCOI films showed reduced biofouling and postponed the onset of weight loss by up to 100 days, a 10-fold increase compared to unmodified films where the microbial coverage was significant. In addition, the rate of PHA-DCOI weight loss, post-onset, reduced by about 150%; in contrast a recorded weight loss of only 0.05% per day for P(HB-co-HV) with a 10% DCOI loading was observed. This is in stark contrast to the unmodified PHB film, where a recorded weight loss of only 0.75% per day was made. The ‘switch’ that initiates film weight loss, and its subsequent reduced rate, depended on the DCOI loading to control biofouling. The control of biofouling and environmental degradation for these DCOI modified bioplastics increases their potential use in biodegradable applications.     

乙草胺的微生物降解及其对土壤磷脂脂肪酸特性的影响

通过监测不同培养条件下（灭菌、加选择抑菌剂、未灭菌）土壤中乙草胺残留数量和培养期间微生物标识物磷脂脂肪酸（PLFAs）含量的变化,探讨了以田间推荐量施用乙草胺时,不同微生物群落对乙草胺降解的相对贡献以及乙草胺对黑土微生物群落结构的影响.结果表明：乙草胺易于被微生物降解,其中,细菌对乙草胺的降解作用显著大于真菌,而真菌对乙草胺有更强的耐受能力.施入乙草胺后,各磷脂脂肪酸数量发生了显著变化,C14∶0、C16∶0和C18∶0所指示的土壤微生物量均有所下降;细菌PLFAs数量在培养初期显著降低,而后与对照的差异显著减小,表明细菌活性随着乙草胺的降解有所恢复;在施用乙草胺后的整个培养过程中,土壤真菌PLFAs数量始终低于对照,暗示着乙草胺对真菌的抑制可能是长期而不可逆的.     

Comparison of cytocompatibility of zein film with other biomaterials and its degradability in vitro

Cytocompatibility of particle zein (Pzein) and film zein (Fzein) was evaluated and compared with polyhydroxybutyrate (PHB), its copolymer poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV), polylactic acid (PLA), and collagen, using HL-7702 cells, in terms of cell attachment rate within 3 h, and cell viabilities at 3 and 6 days determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. The zein degradation test was carried out using collagenase and trypsin, and the degradation product was added to the culture medium at different concentrations in order to examine the concentration-dependent cytotoxic effect. RESULT: The adhesion rate of the HL-7702 cells on both Pzein and Fzein was higher than that on collagen film. Cell viabilities were higher on both Pzein and Fzein than on films of PLA, PHB, PHBV, and collagen from fish skin. Zein can be degraded by both trypsin and collagenase, and the degradation product can enhance cell viability within a certain range of concentrations.     

Introducing RGD peptides on PHBV films through PEG-containing cross-linkers to improve the biocompatibility

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biodegradable polyester, has been a good candidate of biomaterial employed in tissue engineering. However, the PHBV film is hydrophobic and has no recognition sites for cell attachment. In this study, PHBV films are activated by ammonia plasma treatment to produce amino groups on the surface, followed by sequential reactions with a heterobifunctional cross-linker containing a segment of poly(ethylene glycol) (PEG) and further with RGD-containing peptides. XPS analyses of modified surfaces after each reaction step reveal that the RGD-containing peptides have been covalently grafted onto PHBV films. The result of cell viability assay indicates that the RGD-modified PHBV films exhibit a distinctly improved cellular compatibility. Moreover, according to the results of serum adsorption tests by optical waveguide lightmode spectroscopy (OWLS) and fibrinogen adsorption tests by enzyme-linked immunosorbent assay (ELISA) on unmodified and modified PHBV surfaces, the introduced PEG chains can significantly decrease the nonspecific adsorption of proteins from serum and fibrinogen from plasma, thus decreasing the risk of thrombus formation and improving the blood compatibility of implanted materials.     

Effect of organosoluble salts on the nanofibrous structure of electrospun poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

Electrospinning of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) in chloroform was investigated to develop non-woven biodegradable nanofibrous structures for tissue engineering. Ultrafine PHBV fibers were obtained by electrospinning of 20 wt.% PHBV solution in chloroform and the resulting fiber diameters were in the range of 1.0-4.0 microm. When small amounts of benzyl trialkylammonium chlorides were added to the PHBV solution, the average diameter was decreased to 1.0 microm and the fibers were amounted in a straight shape. Conductivity of the PHBV solution was a major parameter affecting the morphology and diameter of the electrospun PHBV fibers. PHBV non-woven structures electrospun with salt exhibited a higher degradation rate than those prepared without salt probably due to the increase of surface area of PHBV fibers.     

Nanoscale reduction in surface friction of polymer surfaces modified with Sc3 hydrophobin from Schizophyllum commune

Hydrophobins are amphipathic self-assembling proteins secreted by filamentous fungi that exhibit remarkable ability to modify synthetic surfaces. Thin coatings of Sc3 hydrophobin isolated from the wood-rotting fungus Schizophyllum commune were prepared via spin coating and adsorption techniques onto polymeric surfaces. Surface morphology and nanotribological characteristics of the films were evaluated using lateral force microscopy (LFM) and nanoindentation techniques. This paper reports the first observation of reduction in nanoscale relative surface friction of Sc3 hydrophobin protein modified polymeric surfaces. Relative friction coefficients were dramatically reduced and hydrophilicity increased for polymer surfaces modified with Sc3 hydrophobin thin films. Morphology of the protein films as well as degree of surface modification was observed to be a function of film formation technique and composition of the substrate.     

Microbial degradation of poly(d-3-hydroxybutyrate) by a new thermophilic Streptomyces isolate

A new thermophilic microorganism capable of degrading poly(D-3-hydroxybutyrate) (PHB) was isolated from soil. A phylogenetic analysis based on 16S rDNA sequences indicated that the new isolate belongs to genus Streptomyces. PHB film and powder were completely degraded after 6 and 3 d cultivation, respectively at 50 degrees C. Scanning micrographs showed adherence of the microbial cells to the entire film surface, indicating that biodegradation occurs by colonization of the PHB surface. The film was degraded both by microbial attack and by the action of an extracellular enzyme secreted by the microorganism. The strain can also degrade poly(ethylene succinate), poly(ester carbonate), polycaprolactone and poly(butylene succinate), but to a lesser extent.     

Sequence distribution in microbial poly(3-hydroxybutyrate-co-3-hydroxyvalerate) co-polyesters determined by NMR and MS

The microstructure of bacterial poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolyesters (PHBV) as well as a mixture of two PHBV copolyesters of different comonomer composition and sequence distribution was studied by 13C NMR based on dyad and triad analysis and multistage electrospray ionization mass spectrometry (ESI-MSn). Both techniques gave results that were in good agreement for all investigated samples. The effect of microstructure on PHBV thermal properties was investigated from the melting behavior of samples. A PHBV copolyester with randomly distributed hydroxyvalerate units (12.0 mol % HV) showed a single melting peak, whereas samples with nonrandom composition distribution showed multiple melting peaks in their thermograms. Such complex melting behavior suggested that the 12.9 and 27.1 mol % PHBV copolyesters were actually blends of several copolymers with widely different comonomer-unit composition.     

基于分子生态学网络探究亚高山草甸退化对土壤微生物群落的影响

土壤微生物群落在草地生态系统功能中发挥着至关重要的作用,但目前尚不清楚微生物群落的分子生态网络如何响应亚高山草甸退化。以五台山4个不同退化阶段(未退化、轻度退化、中度退化和重度退化)亚高山草甸为研究对象,利用高通量测序和随机矩阵网络构建理论构建土壤微生物群落分子生态网络。探讨草地退化对亚高山草甸土壤微生物群落结构及网络的影响,不同退化程度下微生物网络结构中的关键微生物变化规律以及该过程中微生物之间的互作关系。研究结果表明,不同退化程度亚高山草甸土壤微生物(细菌、真菌和细菌-真菌)网络拓扑属性存在差异。总体上,退化增加了土壤细菌内部、真菌内部以及细菌-真菌群落间的相互作用,导致其网络结构更为复杂。未退化草甸土壤微生物网络具有较长的平均路径距离和较高的模块性,使其比退化草甸更能抵抗外界环境的变化,在应对人为干扰或者气候变化时可能具有更高的稳定性。退化草甸中的网络关键物种(模块枢纽和连接器)与未退化草甸明显不同。土壤含水量和pH与亚高山草甸土壤细菌、真菌以及整个微生物网络连通度均显著相关(P<0.05),总氮和硝氨态氮含量与土壤真菌和微生物网络连通度呈显著相关(P<0.05)。亚...     

林地覆盖对雷竹林土壤微生物特征及其与土壤养分制约性关系的影响

为了探讨林地覆盖雷竹林退化机理,给退化雷竹林恢复提供理论参考,对不同覆盖年限(CK、1、3 a 和6 a) 雷竹林土壤微生物区系组成和生物量碳(C_mic)、氮(N_mic)、磷(P_mic)等特征因子进行了测定,并分析了其与土壤养分的制约性关系。结果表明:(1) 雷竹林土壤微生物以细菌为主,真菌次之,放线菌最少,分别占土壤微生物总量的90.11%-98.03%、1.04%-9.22%和0.67%-1.37%。随覆盖年限增加,细菌、放线菌比率呈下降趋势,真菌比率呈上升趋势;土壤微生物总数、细菌和放线菌数量及C_mic、N_mic、P_mic均呈先升高后降低的变化趋势,试验雷竹林间差异极显著,真菌数量总体呈极显著升高趋势。(2)雷竹林土壤微生物特征因子与土壤有机质(SOM)、全氮(TN)、全磷(TP)、碱解氮(Available nitrogen, AN)和pH均呈显著或极显著相关,其中,CK和覆盖1 a、3 a雷竹林土壤微生物特征因子与土壤养分主要呈正相关,与pH呈负相关,而覆盖6 a雷竹林则相反。(3)不同覆盖年限雷竹林土壤养分与土壤微生物的制约性关系存在一定的差异,CK雷竹林土壤SOM、TN、AN、速效钾(AK)和pH主要影响土壤C_mic、N_mic和细菌,覆盖1 a雷竹林土壤SOM、TN、TP和AK主要影响土壤P_mic、放线菌和细菌,覆盖3 a雷竹林土壤SOM、TN、速效磷(AP)和AN主要影响土壤N_mic、放线菌和真菌,覆盖6 a雷竹林土壤SOM、TN和pH主要影响土壤N_mic、真菌。研究表明:长期覆盖雷竹林土壤细菌、放线菌数量与比例明显降低,真菌数量与比例明显提高,土壤养分与土壤微生物的制约性作用关系会发生较为明显变化,产生土壤障害,这是覆盖雷竹林退化的主要原因之一。     

百菌清对落叶松人工防护林土壤微生物群落的影响

利用稀释平板法和Biolog-Eco检测技术,分析了喷施百菌清对落叶松人工防护林土壤微生物群落的影响。结果表明,土壤微生物中细菌数量占据绝对优势,其数量直接影响落叶松人工防护林土壤微生物总量的变化趋势。总体上,百菌清对细菌的数量具有抑制作用。喷洒后0d,仅抑制表层与上层(0-10cm)土壤细菌的增殖;喷洒后2个月,对表层与上层土壤细菌的抑制作用减弱,而喷洒后4个月时,其抑制作用最强。喷洒后0d与2个月时,百菌清对表层与上层土壤真菌数量的增殖均具有促进作用。喷洒后2个月、4个月时均对下层土壤放线菌数量具有抑制作用。将土壤3大类群微生物数量、土壤理化指标及群落多样性指数进行相关分析表明,土壤中有效磷和速效钾与放线菌数量、水解氮与细菌数量存在显著正相关性,而与群落多样性无显著相关性。喷洒百菌清2个月后,对表层土壤微生物群落碳源利用率的影响不显著,而对上层(0-10cm)土壤微生物存在显著影响,下层(10-20cm)土壤微生物群落的碳源利用率在百菌清喷洒2个月与4个月后具有显著差异;而不同土层微生物群落对碳源利用率的差异不显著。微生物群落利用31种碳源的主成分分析表明,喷洒百菌清后,微生物群落利用单一碳源的能力显著提高,且喷洒2个月后微生物群落对某些碳源如肝糖与2-羟基苯甲酸等的利用率明显增加。方差分析表明,喷洒百菌清后,不同作用时间及不同处理,不同土层间土壤微生物对碳源的利用率具有显著差异。喷洒百菌清前(5月)土壤微生物对多聚物与氨基酸、碳水类、芳香类及胺类的利用率差异较大(P<0.05),且对多聚物的利用率最高;处理组的表层土壤微生物对碳源的利用率高于其它处理组;而喷洒2个月后,对胺类、芳香类的利用率与其它4类碳源具有显著差异。     

Soil–litter mixing and microbial activity mediate decomposition and soil aggregate formation in a sandy shrub-invaded Chihuahuan Desert grassland

Drylands account globally for 30% of terrestrial net primary production and 20% of soil organic carbon. Present ecosystem models under predict litter decay in drylands, limiting assessments of biogeochemical cycling at multiple scales. Overlooked decomposition drivers, such as soil–litter mixing (SLM), may account for part of this model-measurement disconnect. We documented SLM and decomposition in relation to the formation of soil-microbial films and microbial extracellular enzyme activity (EEA) in the North American Chihuahuan Desert by placing mesh bags containing shrub (Prosopis glandulosa) foliar litter on the soil surface within contrasting vegetation microsites. Mass loss (in terms of k, the decay constant) was best described by the degree of SLM and soil-microbial film cover. EEA was greatest during periods of rapid litter decomposition and associated SLM. Soil-microbial film cover on litter surfaces increased over time and was greater in bare ground microsites (50% litter surface area covered) compared to shrub and grass microsites (37 and 33% covered, respectively). Soil aggregates that formed in association with decomposing leaf material had organic C and N concentrations 1.5–2× that of local surface soils. Micrographs of soil aggregates revealed a strong biotic component in their structure, suggesting that microbial decomposition facilitates aggregate formation and their C and N content. Decomposition drivers in arid lands fall into two major categories, abiotic and biotic, and it is challenging to ascertain their relative importance. The temporal synchrony between surface litter mass loss, EEA, biotic film development, and aggregate formation observed in this study supports the hypothesis that SLM enhances decomposition on detached litter by promoting conditions favorable for microbial processes. Inclusion of interactions between SLM and biological drivers will improve the ability of ecosystem models to predict decomposition rates and dynamics in drylands.     

Biodegradability studies of polyhydroxyalkanoate (PHA) film produced by a marine bacteria using Jatropha biodiesel byproduct as a substrate

Polyhydroxyalkanoates are water-insoluble, hydrophobic polymers and can be degraded by microorganisms that produce extracellular PHA depolymerase. The present work was aimed to evaluate the degradability of Polyhydroxyalkanoate film produced by Halomonas hydrothermalis using Jatropha biodiesel byproduct as a substrate. PHB films were subjected to degradation in soil and compared with the synthetic polymer (acrylate) and blend prepared using the synthetic polymer (acrylate) and PHB. After 50 days, 60% of weight loss in PHB film and after 180 days 10% of blended film was degraded while no degradation was found in the synthetic film. Scanning electron microscopy and confocal microscopy revealed that after 50 days the PHB film and the blended film became more porous after degradation while synthetic film was not porous. The degradative process was biologically mediated which was evident by the control in which the PHB films were kept in sterile soil and the films showed inherent integrity over time. The TGA and DSC analysis shows that the melting temperatures were changed after degradation indicating physical changes in the polymer during degradation.     

Spatio-temporal variation of rhizosphere soil microbial abundance and enzyme activities under different vegetation types in the coastal zone,Shandong, China

In coastal sandy soils, the establishment of a plant cover is fundamental to avoid degradation and desertification processes. A better understanding of the ability of plants to promote soil microbial process in these conditions is necessary for successful soil reclamation. The current study was to investigate the ability of four different plant species to regenerate the microbiological processes in the rhizosphere soil and to discuss which species were the most effective for the reclamation of the coastal zone. The rhizosphere soils were studied by measuring microbial abundance (bacteria, fungi, actinomycetes, and ammonifiers), enzyme activities (invertase, catalase, urease, and phosphatase) and their relationship. Microbial abundance greatly varied among rhizospheres of different plant species (p < 0.05). Phragmites australis supported the highest amount of bacterial, actinomycetes, and ammonifiers abundance, and Echinochloa crusgalli supported the highest fungi abundance. In addition, the significant differences in rhizosphere enzyme activities of different plant species were also observed. There was a significant linear correlation between rhizosphere soil microbial abundances and enzyme activities between bacteria and urease and between fungi and catalase, but no such significant relationship was found between all rhizosphere soil microbial abundance and phosphatases. It was concluded that different plant species in coastal areas have different rhizosphere soils due to the impact of the different root exudates and plant residues of the microbial properties. In addition, natural grasslands (P. australis and E. crusgalli) are the most effective for revegetating coastal sandy soils.     

Fermentation of plant cell walls by ruminal bacteria, protozoa and fungi and their interaction with fibre particle size

The objective of the experiment was to evaluate the contribution of various ruminal microbial groups to the fermentation of cell walls of corn stover with different particle sizes based on ruminal gas production in vitro. Physical, chemical, and antibiotical methods were used to differentiate groups of bacteria, protozoa and fungi in rumen fluid, offering following rumen microbial groups: whole rumen fluid (WRF), bacterial (B), protozoal (P), fungal (F), bacterial plus protozoal (B + P), bacterial plus fungal (B + F), protozoal plus fungal (P + F), and negative control (CON). Cell walls from corn stover were ground and ball milled to produce two different particle sizes. The results showed that digestion of the cell walls was undertaken by the interaction among ruminal bacteria, protozoa and fungi, and such co-actions seemed to fail alternation by one of three microbial groups or any combinations. However, B + P group showed a significant contribution to the degradation of milled cell walls, and B + F group revealed a great synergy effect on the ground cell walls degradation. Particle size of cell walls also had a considerable influence on their fermentation extent instead of the fermentative patterns by various rumen microbial groups.