Biomedical Engineering Reference
In-Depth Information
changes that may affect the performance of the consortium during environmental applications
if an important member of the population is lost, for example.
During fed batch microbial production, cultures are grown from a low cell density to a high
cell density by controlling substrate addition and reactor conditions. This process allows
harvesting of cultures during their most active growth states, and minimizes the risk of
population changes that can occur during long-term culture maintenance. Likewise, the buildup
of recalcitrant toxic metabolic products in the medium is minimized.
The primary limitation of the fed batch growth approach is that cells may have to be
harvested before they are needed for field application. As such, it must be possible to store the
cultures until needed. The storage of large culture volumes, especially anaerobic cultures that
cannot be air dried because of oxygen toxicity, can require a large space or even a large
refrigerated space. Concentrating the cultures under strict anaerobic conditions before storage,
however, can reduce storage space requirements (see below).
For production of Dhc -containing bioaugmentation cultures, the fed batch growth process
appears most practical. This approach allows cells to be harvested in late log phase (i.e., when
cells are at their greatest growth rate and density) or early stationary phase (i.e., just at the end
of the rapid growth phase) to ensure the greatest cell numbers and highest possible activity in
the applied cultures, and to maintain culture consistency between batches. Fed batch growth also
prevents the accumulation of extracellular metabolic products (e.g., acetate and propionate)
which ultimately could affect culture activity. Experimentation has demonstrated that Dhc
cultures grown by the fed batch process can be concentrated by membrane filtration and can
be stored refrigerated for more than 30 days (d) without considerable loss of activity (Vainberg
et al., 2009 ). Inoculum concentration processes and storage studies are presented below.
3.2.2 Culture Growth Protocol
Production of bacterial cultures is typically accomplished in a series of vessels that increase
culture volume in a step-wise fashion. That is, an initial starter culture is grown in a vessel, and
that culture is used as a seed culture for inoculating a larger culture. For example, for growth of
Dhc -containing cultures, small serum vial enrichment cultures (160 milliliters [mL]) can be used
to inoculate 2-7-L flasks or reactors. Once high Dhc levels are achieved, this culture is used to
inoculate 10-20 L of culture medium, and so on. It is usually desirable to start a Dhc culture at an
optical density at 550 nanometers (nm) (OD 550 ) of approximately 0.1. This optical density equates
to approximately 10 11 total cells/L, and approximately 10 9 Dhc /L (note, 10 9 Dhc /L is equivalent to
10 6 Dhc /mL). Thus, it is important to plan seed culture steps to ensure a sufficient inoculum size
at each scale-up step. The following sections describe methods used to prepare Dhc -containing
cultures in Shaw Environmental Inc.'s laboratory and are derived from Vainberg et al. ( 2009 ).
3.2.2.1 Seed Cultures
Bench-scale experiments and seed culture production are performed in 3-L or 7-L Applicon
reactors (Cole Parmer, Vernon Hills, Illinois) equipped with pH, dissolved oxygen (DO) and
mixer controls. Substrate and sodium hydroxide (NaOH) feeds are controlled by using syringe
pumps (Harvard Apparatus, Holliston, Massachusetts) and low-flow peristaltic pumps (Cole
Parmer, Chicago, Illinois). Larger seed cultures are produced in a similarly equipped 20-L
Biolafitte reactor (Pierre Guerin, Inc., Spring Lake Park, Minnesota). Still larger cultures are
produced in a 750-L ABEC reactor (ABEC Inc., Bethlehem, Pennsylvania) or a custom built
4,000-L stainless steel reactor. In each case, anaerobic conditions are maintained by pressuriz-
ing the vessels with nitrogen. At the end of the growth cycle, cells in the reactor broth are
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