Photobioreactor cultivation strategies for microalgae and cyanobacteria.

The current burden on fossil-derived chemicals and fuels combined with the rapidly increasing global population has led to a crucial need to develop renewable and sustainable sources of chemicals and biofuels. Photoautotrophic microorganisms, including cyanobacteria and microalgae, have garnered a great deal of attention for their capability to produce these chemicals from carbon dioxide, mineralized water, and solar energy. While there have been substantial amounts of research directed at scaling-up production from these microorganisms, several factors have proven difficult to overcome, including high costs associated with cultivation, photobioreactor construction, and artificial lighting. Decreasing these costs will substantially increase the economic feasibility of these production processes. Thus, the purpose of this review is to describe various photobioreactor designs, and then provide an overview on lighting systems, mixing, gas transfer, and the hydrodynamics of bubbles. These factors must be considered when the goal of a production process is economic feasibility. Targets for improving microalgae and cyanobacteria cultivation media, including water reduction strategies will also be described. As fossil fuel reserves continue to be depleted and the world population continues to increase, it is imperative that renewable chemical and biofuel production processes be developed toward becoming economically feasible. Thus, it is essential that future research is directed toward improving these processes. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:811-827, 2018.

Current and Potential Treatments for Reducing Campylobacter Colonization in Animal Hosts and Disease in Humans.

Campylobacter jejuni is the leading cause of bacteria-derived gastroenteritis worldwide. In the developed world, Campylobacter is usually acquired by consuming under-cooked poultry, while in the developing world it is often obtained through drinking contaminated water. Once consumed, the bacteria adhere to the intestinal epithelium or mucus layer, causing toxin-mediated inhibition of fluid reabsorption from the intestine and invasion-induced inflammation and diarrhea. Traditionally, severe or prolonged cases of campylobacteriosis have been treated with antibiotics; however, overuse of these antibiotics has led to the emergence of antibiotic-resistant strains. As the incidence of antibiotic resistance, emergence of post-infectious diseases, and economic burden associated with Campylobacter increases, it is becoming urgent that novel treatments are developed to reduce Campylobacter numbers in commercial poultry and campylobacteriosis in humans. The purpose of this review is to provide the current status of present and proposed treatments to combat Campylobacter infection in humans and colonization in animal reservoirs. These treatments include anti-Campylobacter compounds, probiotics, bacteriophage, vaccines, and anti-Campylobacter bacteriocins, all of which may be successful at reducing the incidence of campylobacteriosis in humans and/or colonization loads in poultry. In addition to reviewing treatments, we will also address several proposed targets that may be used in future development of novel anti-Campylobacter treatments.

Molecular genetic improvements of cyanobacteria to enhance the industrial potential of the microbe: A review.

The rapid increase in worldwide population coupled with the increasing demand for fossil fuels has led to an increased urgency to develop sustainable sources of energy and chemicals from renewable resources. Using microorganisms to produce high-value chemicals and next-generation biofuels is one sustainable option and is the focus of much current research. Cyanobacteria are ideal platform organisms for chemical and biofuel production because they can be genetically engineered to produce a broad range of products directly from CO2 , H2 O, and sunlight, and require minimal nutrient inputs. The purpose of this review is to provide an overview on advances that have been or could be made to improve strains of cyanobacteria for industrial purposes. First, the benefits of using cyanobacteria as a platform for chemical and biofuel production are discussed. Next, an overview of cyanobacterial strain improvements by genetic engineering is provided. Finally, mutagenesis techniques to improve the industrial potential of cyanobacteria are described. Along with providing an overview on various areas of research that are currently being investigated to improve the industrial potential of cyanobacteria, this review aims to elucidate potential targets for future research involving cyanobacteria as an industrial microorganism. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1357-1371, 2016.

Optimizing cyanobacteria growth conditions in a sealed environment to enable chemical inhibition tests with volatile chemicals.

Cyanobacteria are currently being engineered to photosynthetically produce next-generation biofuels and high-value chemicals. Many of these chemicals are highly toxic to cyanobacteria, thus strains with increased tolerance need to be developed. The volatility of these chemicals may necessitate that experiments be conducted in a sealed environment to maintain chemical concentrations. Therefore, carbon sources such as NaHCO3 must be used for supporting cyanobacterial growth instead of CO2 sparging. The primary goal of this study was to determine the optimal initial concentration of NaHCO3 for use in growth trials, as well as if daily supplementation of NaHCO3 would allow for increased growth. The secondary goal was to determine the most accurate method to assess growth of Anabaena sp. PCC 7120 in a sealed environment with low biomass titers and small sample volumes. An initial concentration of 0.5g/L NaHCO3 was found to be optimal for cyanobacteria growth, and fed-batch additions of NaHCO3 marginally improved growth. A separate study determined that a sealed test tube environment is necessary to maintain stable titers of volatile chemicals in solution. This study also showed that a SYTO® 9 fluorescence-based assay for cell viability was superior for monitoring filamentous cyanobacterial growth compared to absorbance, chlorophyll α (chl a) content, and biomass content due to its accuracy, small sampling size (100µL), and high throughput capabilities. Therefore, in future chemical inhibition trials, it is recommended that 0.5g/L NaHCO3 is used as the carbon source, and that culture viability is monitored via the SYTO® 9 fluorescence-based assay that requires minimum sample size.

Testing a dual-fluorescence assay to monitor the viability of filamentous cyanobacteria.

Filamentous cyanobacteria are currently being engineered to produce long-chain organic compounds, including 3rd generation biofuels. Because of their filamentous morphology, standard methods to quantify viability (e.g., plate counts) are not possible. This study investigated a dual-fluorescence assay based upon the LIVE/DEAD® BacLight™ Bacterial Viability Kit to quantify the percent viability of filamentous cyanobacteria using a microplate reader in a high throughput 96-well plate format. The manufacturer's protocol calls for an optical density normalization step to equalize the numbers of viable and non-viable cells used to generate calibration curves. Unfortunately, the isopropanol treatment used to generate non-viable cells released a blue pigment that altered absorbance readings of the non-viable cell solution, resulting in an inaccurate calibration curve. Thus we omitted this optical density normalization step, and carefully divided cell cultures into two equal fractions before the isopropanol treatment. While the resulting calibration curves had relatively high correlation coefficients, their use in various experiments resulted in viability estimates ranging from below 0% to far above 100%. We traced this to the apparent inaccuracy of the propidium iodide (PI) dye that was to stain only non-viable cells. Through further analysis via microplate reader, as well as confocal and wide-field epi-fluorescence microscopy, we observed non-specific binding of PI in viable filamentous cyanobacteria. While PI will not work for filamentous cyanobacteria, it is possible that other fluorochrome dyes could be used to selectively stain non-viable cells. This will be essential in future studies for screening mutants and optimizing photobioreactor system performance for filamentous cyanobacteria.

Type 3 fimbriae and biofilm formation are regulated by the transcriptional regulators MrkHI in Klebsiella pneumoniae.

Klebsiella pneumoniae is an opportunistic pathogen which frequently causes hospital-acquired urinary and respiratory tract infections. K. pneumoniae may establish these infections in vivo following adherence, using the type 3 fimbriae, to indwelling devices coated with extracellular matrix components. Using a colony immunoblot screen, we identified transposon insertion mutants which were deficient for type 3 fimbrial surface production. One of these mutants possessed a transposon insertion within a gene, designated mrkI, encoding a putative transcriptional regulator. A site-directed mutant of this gene was constructed and shown to be deficient for fimbrial surface expression under aerobic conditions. MrkI mutants have a significantly decreased ability to form biofilms on both abiotic and extracellular matrix-coated surfaces. This gene was found to be cotranscribed with a gene predicted to encode a PilZ domain-containing protein, designated MrkH. This protein was found to bind cyclic-di-GMP (c-di-GMP) and regulate type 3 fimbrial expression.