ABSTRACT
AIMS: To evaluate carbon source complexity as a process lever to impact the microstructure, chemical composition and water retention capacity of biofilms produced by Neurospora discreta. METHODS AND RESULTS: Biofilms were produced by nonpathogenic fungus N. discreta, using sucrose, cellulose or lignin as carbon source. The increase in complexity of carbon source from sucrose to lignin resulted in decreased water retention values (WRV) and wet weights of harvested biofilms. Confocal laser scanning microscopy was used to calculate porosity from bright-field images, and relative stained areas of cells and carbohydrates from fluorescence imaging of samples stained with Trypan blue and Alexa Fluor 488. Porosity and relative quantity of cells increased with increase in carbon source complexity while the amount of carbohydrates decreased. The chemical analysis of the extracted extracellular matrix (ECM) showed that biofilms grown on more complex carbon sources had lower carbohydrate and protein content, which also explains the lower WRV trend, as carbohydrates are hydrophilic. CONCLUSIONS: The nature of carbon source impacts the metabolic pathway of cells, thereby influencing the relative proportions of ECM and cells. This in turn impacts the microstructure, composition and water content of biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: This work shows that carbon source can be used as process lever to control the properties of biofilms and presents a novel view of biofilms as potentially useful biomaterials.
Subject(s)
Biofilms , Carbon/metabolism , Extracellular Matrix/chemistry , Neurospora/physiology , Carbohydrates/chemistry , Carbon/analysis , Hydrophobic and Hydrophilic Interactions , Microscopy, Confocal , Neurospora/chemistry , Neurospora/metabolism , Neurospora/ultrastructure , Polysaccharides/analysis , Polysaccharides/metabolism , Porosity , Water/analysisABSTRACT
Primary porcine hepatocytes cultured in suspension self-assemble into multicellular aggregates or spheroids that display enhanced liver-specific functional capability and remain viable for an extended period of time in vitro. The molecular events underlying the process of spheroid formation were explored by differential gene expression analysis. Critical time points in spheroid formation were first identified by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of stress-related gene expression levels at different stages of spheroid formation. Suppression subtractive hybridization was used to identify transcripts up- or down-regulated at different stages of spheroid formation. Subsequently, three sets of reciprocal subtractions, comparing freshly isolated hepatocytes, spheroid-forming hepatocytes, and mature spheroids were carried out, and differentially expressed transcripts were isolated, cloned, sequenced, and annotated. A total of 65 genes and 14 novel transcripts were identified as differentially expressed, and very high sequence conservation between pig and human transcripts was observed. The resultant expressed sequence tags (ESTs) revealed a rapid decrease in the transcript levels of a subset of liver-specific genes, cytochrome P450s, and enzymes involved in heme biosynthesis, as well as up-regulation of genes involved in calcium-dependent vesicle trafficking and a number of acute-phase proteins in mature spheroids. Previous morphological and functional data on hepatocyte spheroid formation support cellular polarization of the hepatocyte into apical and basolateral domains in spheroids. This is important for the re-emergence of differentiated functions in vitro and is reflected by differences in gene expression patterns.
Subject(s)
Gene Expression , Hepatocytes/cytology , Hepatocytes/metabolism , Spheroids, Cellular/cytology , Spheroids, Cellular/metabolism , Animals , Base Sequence , Cell Polarity , Cells, Cultured , DNA, Complementary/genetics , Expressed Sequence Tags , Gene Expression Profiling , Humans , Molecular Sequence Data , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sus scrofaABSTRACT
The growth of Candida sp. 115 was investigated on the constituents of penicillin G hydrolysis reaction mixture. Neither penicillin G nor 6-aminopenicillanic acid was degraded or utilised for growth. The yeast accepted phenylacetic acid, sodium acetate and glucose as growth substrates. Phenylacetic acid was metabolised via p-hydroxy phenylacetic acid, which was the only accumulated metabolite. The enzymes responsible for hydroxylation of phenylacetic acid were induced by phenylacetic acid and sodium acetate.
