Bradley R. Ringeisen

Shewanella oneidensis MR-1 has conventionally been considered unable to use glucose as a carbon substrate for growth. The genome sequence of S. oneid

Two major challenges in tissue engineering are mimicking the native cell–cell arrangements of tissues and maintaining viability of three‐dimension (3D) tissues thicker than 300 µm. Cell printing and prevascularization of engineered tissues are promising approaches to meet these challenges. However, the printing technologies used in biofabrication must balance the competing parameters of resolution, speed, and volume, which limit the resolution of thicker 3D structures. We suggest that high‐resolution conformal printing techniques can be used to print 2D patterns of vascular cells onto biopaper substrates which can then be stacked to form a thicker tissue construct. Towards this end we created 1 cm × 1 cm × 300 µm biopapers to be used as the transferable, stackable substrate for cell printing. 3.6% w/v poly‐lactide‐co‐glycolide was dissolved in chloroform and poured into molds filled with NaCl crystals. The salt was removed with DI water and the scaffolds were dried and loaded with a Collagen Type I or Matrigel™. SEM of the biopapers showed extensive porosity and gel loading throughout. Biological laser printing (BioLP™) was used to deposit human umbilical vein endothelial cells (HUVEC) in a simple intersecting pattern to the surface of the biopapers. The cells differentiated and stretched to form networks preserving the printed pattern. In a separate experiment to demonstrate “stackability,” individual biopapers were randomly seeded with HUVECs and cultured for 1 day. The mechanically stable and viable biopapers were then stacked and cultured for 4 days. Three‐dimensional confocal microscopy showed cell infiltration and survival in the compound multilayer constructs. These results demonstrate the feasibility of stackable “biopapers” as a scaffold to build 3D vascularized tissues with a 2D cell‐printing technique. Biotechnol. Bioeng. 2012;109: 262–273.

Microbial fuel cells (MFCs) represent a promising approach for sustainable energy production as they generate electricity directly from metabolism of organic substrates without the need for catalysts. However, the mechanisms of electron transfer between microbes and electrodes, which could ultimately limit power extraction, remain controversial. Here we demonstrate optically transparent nanoelectrodes as a platform to investigate extracellular electron transfer in Shewanella oneidensis MR-1, where an array of nanoholes precludes or single window allows for direct microbe-electrode contacts. Following addition of cells, short-circuit current measurements showed similar amplitude and temporal response for both electrode configurations, while in situ optical imaging demonstrates that the measured currents were uncorrelated with the cell number on the electrodes. High-resolution imaging showed the presence of thin, 4- to 5-nm diameter filaments emanating from cell bodies, although these filaments do not appear correlated with current generation. Both types of electrodes yielded similar currents at longer times in dense cell layers and exhibited a rapid drop in current upon removal of diffusible mediators. Reintroduction of the original cell-free media yielded a rapid increase in current to ∼80% of original level, whereas imaging showed that the positions of > 70% of cells remained unchanged during solution exchange. Together, these measurements show that electron transfer occurs predominantly by mediated mechanism in this model system. Last, simultaneous measurements of current and cell positions showed that cell motility and electron transfer were inversely correlated. The ability to control and image cell/electrode interactions down to the single-cell level provide a powerful approach for advancing our fundamental understanding of MFCs.

We have constructed and successfully laboratory tested a device that can periodically change from a submerged to a buoyant state using gas generated by microbes alone. The duration of the buoyant state and the switching frequency from the submerged to buoyant state can be controlled. If the type of microbes used is native to the deployment location, gas generation will not create an identifiable acoustic signature disclose the presence and operation of the device. In this report, we discuss the operation and testing of the device, the microbes that we use to test the operation of the device, and the test results.

Electrochemically active bacteria (EAB) are prominently found in aquatic environmental sediment samples and wastewater streams, which are known to contain several different types of microorganisms. Even though microbial consortia are found to enhance both Coulombic efficiency and total power output in microbial fuel cells (MFCs), it is currently unknown how many different EAB contribute to current generation in these systems. It is also difficult to track the relative population of different species during MFC operation. We used biological laser printing (BioLP) to isolate different bacterium from complex environmental samples and MFC anolytes. BioLP can be used to print droplets containing a single cell directly from liquid culture, thereby enabling EAB to be sorted from unmodified environmental or MFC samples. Isolated species were identified through 16S rDNA analysis of pure cultures derived from the printed samples. These experiments demonstrate how cell printing can be used as a single‐step method to separate and identify microorganisms from complex environmental samples and operating MFCs.

The ability of the metal reducer Shewanella oneidensis MR‐1 to generate electricity in microbial fuel cells (MFCs) depends on the activity of a predicted type IV prepilin peptidase; PilD. Analysis of an S. oneidensis MR‐1 pilD mutant indicated that it was deficient in pili production (Msh and type IV) and type II secretion (T2S). The requirement for T2S in metal reduction has been previously identified, but the role of pili remains largely unexplored. To define the role of type IV or Msh pili in electron transfer, mutants that lack one or both pilus biogenesis systems were generated and analyzed; a mutant that lacked flagella was also constructed and tested. All mutants were able to reduce insoluble Fe(III) and to generate current in MFCs, in contrast to the T2S mutant that is deficient in both processes. Our results show that loss of metal reduction in a PilD mutant is due to a T2S deficiency, and therefore the absence of c cytochromes from the outer surface of MR‐1 cells, and not the loss of pili or flagella. Furthermore, MR‐1 mutants deficient in type IV pili or flagella generated more current than the wild type, even though extracellular riboflavin levels were similar in all strains. This enhanced current generating ability is in contrast to a mutant that lacks the outer membrane c cytochromes, MtrC and OmcA. This mutant generated significantly less current than the wild type in an MFC and was unable to reduce Fe(III). These results indicated that although nanofilaments and soluble mediators may play a role in electron transfer, surface exposure of outer membrane c cytochromes was the determining factor in extracellular electron transfer in S. oneidensis MR‐1.

Cell printing has been popularized over the past few years as a revolutionary advance in tissue engineering has potentially enabled heterogeneous 3‐D scaffolds to be built cell‐by‐cell. This review article summarizes the state‐of‐the‐art cell printing techniques that utilize fluid jetting phenomena to deposit 2‐ and 3‐D patterns of living eukaryotic cells. There are four distinct categories of jetbased approaches to printing cells. Laser guidance direct write (LG DW) was the first reported technique to print viable cells by forming patterns of embryonic‐chick spinal‐cord cells on a glass slide (1999). Shortly after this, modified laser‐induced forward transfer techniques (LIFT) and modified ink jet printers were also used to print viable cells, followed by the most recent demonstration using an electrohydrodynamic jetting (EHDJ) method. The low cost of some of these printing technologies has spurred debate as to whether they could be used on a large scale to manufacture tissue and possibly even whole organs. This review summarizes the published results of these cell printers (cell viability, retained genotype and phenotype), and also includes a physical description of the various jetting processes with a discussion of the stresses and forces that may be encountered by cells during printing. We conclude the review by comparing and contrasting the different jet‐based techniques, while providing a map for future experiments that could lead to significant advances in the field of tissue engineering.

A miniature microbial fuel cell (mini-MFC) is described that demonstrates high output power per device cross-section (2.0 cm²) and volume (1.2 cm³). Shewanella oneidensis DSP10 in growth medium with lactate and buffered ferricyanide solutions were used as the anolyte and catholyte, respectively. Maximum power densities of 24 and 10 mW/m² were measured using the true surface areas of reticulated vitreous carbon (RVC) and graphite felt (GF) electrodes without the addition of exogenous mediators in the anolyte. Current densities at maximum power were measured as 44 and 20 mA/m² for RVC and GF, while short circuit current densities reached 32 mA/m² for GF anodes and 100 mA/m² for RVC. When the power density for GF was calculated using the cross sectional area of the device or the volume of the anode chamber, we found values (3 W/m², 500 W/m³) similar to the maxima reported in the literature. The addition of electron mediators resulted in current and power increases of 30−100%. These power densities were surprisingly high considering a pure S. oneidensis culture was used. We found that the short diffusion lengths and high surface-area-to-chamber volume ratio utilized in the mini-MFC enhanced power density when compared to output from similar macroscopic MFCs.