\n pBHR68 was used in all studies 10<\/a>]. The XL1-Blue strain of E. coli<\/em> was chosen for its ability to out-perform other E. coli<\/em> strains for PHB production 27<\/a>]. The plasmid pBHR68 was selected as it contained the lactose inducible phaCAB operon
\n and had demonstrated PHB accumulation up to approximately 50\u00a0% of the dry cell weight
\n after 48\u00a0h of growth in a minimal media 28<\/a>].\n <\/p>\n
Culture media<\/h4>\n
For all experiments single colonies were picked from Luria\u2013Bertani (LB) agar plates, BIOSTAT Q multi-fermentor bioreactor system (B. Braun Biotech International, Melsungen, A MBD generator was setup in-line with a 1\u00a0L BIOSTAT bioreactor running in batch-mode Glucose concentration was determined with a glucose assay reagent kit (Sigma Aldrich, PHB concentration was determined by an NMR-GC correlation 32<\/a>] at 12, 24, and 48\u00a0h respectively. The methods used in this study followed a procedure The volumetric oxygen transfer coefficient (k \n (1)<\/span><\/p>\n Integrating Eq.\u00a01 and solving for k \n (2)<\/span><\/p>\n Plotting ln (C*?C Strain selection E. coli strain XL1-Blue (recA1 endA1 gyrA96 thi–1 hsdR17 supE44 relA1 lac [F\u00b4 proAB lacIqZ?M15 Tn10 (TetR)]) (Agilent Technologies, Santa Clara, CA) harboring the ampicillin resistant plasmid pBHR68 was used in all studies 10]. The XL1-Blue strain of E. coli was chosen for its ability to out-perform other E. coli strains for PHB Read More<\/a><\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[],"tags":[],"class_list":["post-92232","post","type-post","status-publish","format-standard","hentry"],"_links":{"self":[{"href":"https:\/\/healthmedicinet.com\/i\/wp-json\/wp\/v2\/posts\/92232","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/healthmedicinet.com\/i\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/healthmedicinet.com\/i\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/healthmedicinet.com\/i\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/healthmedicinet.com\/i\/wp-json\/wp\/v2\/comments?post=92232"}],"version-history":[{"count":0,"href":"https:\/\/healthmedicinet.com\/i\/wp-json\/wp\/v2\/posts\/92232\/revisions"}],"wp:attachment":[{"href":"https:\/\/healthmedicinet.com\/i\/wp-json\/wp\/v2\/media?parent=92232"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/healthmedicinet.com\/i\/wp-json\/wp\/v2\/categories?post=92232"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/healthmedicinet.com\/i\/wp-json\/wp\/v2\/tags?post=92232"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}
\n inoculated in 5\u00a0mL LB media pre-cultures and grown overnight at 37\u00a0\u00b0C 29<\/a>]. These 5\u00a0mL cultures were then used to start larger 50\u00a0mL cultures. Larger cultures
\n consisted of a modified M9 minimal media containing: M9 salts (Na
\n 2<\/sub>
\n HPO
\n 4<\/sub>
\n , KH
\n 2<\/sub>
\n PO
\n 4<\/sub>
\n , NaCl, NH
\n 4<\/sub>
\n Cl, Becton, Dickinson and Co, Sparks, MD), supplemented with 1.75\u00a0% (w\/v) glucose
\n (ACS grade, Acros Organics, Fair Lawn, NJ), 0.2\u00a0% (w\/v) yeast extract (Becton, Dickinson
\n and Co, Sparks, MD), and 100\u00a0\u00b5g\/ml ampicillin (IBI Scientific, Peosta, IA). The addition
\n of small amounts of yeast extract to the culture has been shown to increase PHB yields
\n 27<\/a>], 30<\/a>]. The 50\u00a0mL culture was grown overnight in an orbital shaker table at 37\u00a0\u00b0C and 220\u00a0rpm
\n and was used to seed 1 L fermentors of the same media composition. For PHB production
\n studies using fermentors, 0.1\u00a0mM Isopropyl ?-D<\/small>-1-thiogalactopyranoside (IPTG) (Gold Biotechnology, Inc. St. Louis, MO) was added
\n at the start of the fermentation (t\u00a0=\u00a00\u00a0h).\n <\/p>\nConventional air-sparged fermentation<\/h4>\n
\n Germany) was used with a 1\u00a0L working volume similar to that used in a previous study
\n 31<\/a>]. Conventional air-sparged culturing was conducted for the production of PHB at agitation
\n rates of 350, 500, or 750\u00a0rpm with air-sparge rates of 0.4 or 0.8 vvm. The bioreactor
\n was equipped with pH, dissolved oxygen (DO), and temperature probes. Both the DO and
\n pH probes were calibrated after sterilization. The DO and pH of the media were not
\n controlled but allowed to fall freely during fermentation. Turbidity (OD
\n 600<\/sub>
\n ) and glucose consumption were measured at 0, 4, 8, 12, 24, and 48\u00a0h. PHB production
\n was measured at 12, 24, and 48\u00a0h. All experiments were duplicated.\n <\/p>\nMicrobubble dispersion sparged fermentation<\/h4>\n
\n similar to that seen in a previous study 31<\/a>]. In addition to bioreactor setup mentioned in the previous section, the MBD generator,
\n containing a stainless steel disc 5\u00a0cm in diameter and 3\u00a0mm thick, was connected to
\n a high-speed electrical motor that spun the disk at approximately 4000\u00a0rpm. Baffles
\n 5\u00a0mm from the spinning disk generated a high sheared zone and air was fed into the
\n MBD generator at 0.8 vvm to create microbubbles. Microbubbles were fed into the bioreactor
\n using a peristaltic pump and Masterflex
\n \u00ae<\/sup>
\n tubing at approximately 100-150\u00a0ml\/min. A second peristaltic pump was used to pump
\n fluid back to the MBD generator from the bioreactor at a similar flowrate to maintain
\n a constant bioreactor volume. The recycling of the fermentation broth also served
\n as a microbubble stabilizer because the natural surfactants generated by the E. coli<\/em> assisted in stabilizing the microbubbles. As with the conventional air-sparged bioreactor,
\n DO and pH were not controlled but allowed to fall freely. The media used in the MBD
\n study was the same as that used in the air-sparge fermentation studies and no additional
\n surfactants were used to stabilize the microbubbles generated. Turbidity (OD
\n 600<\/sub>
\n ), glucose consumption, and PHB production was measured at the same time points as
\n conventional air-sparged fermentation experiments. Bioreactor impeller speed of 350\u00a0rpm
\n was maintained over the course of the MBD study. All experiments were duplicated.\n <\/p>\nGlucose analysis<\/h4>\n
\n St Louis, MO) using a modified procedure similar to a previous study 13<\/a>]. Briefly, 120\u00a0\u00b5l glucose assay reagent was added to 60\u00a0\u00b5l sample and incubated at
\n 37\u00a0\u00b0C for 30\u00a0min. After incubation, 120\u00a0\u00b5l of 12\u00a0N H
\n 2<\/sub>
\n SO
\n 4<\/sub>
\n was added to stop the enzymatic reaction. Absorbance was measured at 540\u00a0nm using
\n a Synergy 2 microtiter plate reader (BioTek, Winooski, VT). Concentration calculations
\n were carried out according to a glucose standard curve.\n <\/p>\nPHB concentration determination<\/h4>\n
\n developed previously 32<\/a>]. After fermentation approximately 100\u00a0mL of culture was centrifuged, frozen to ?80\u00a0\u00b0C,
\n and lyophilized. 15\u00a0mg of lyophilized sample was mixed with equal volumes of 5\u00a0% sodium
\n hypochlorite and deuterated chloroform containing 0.03\u00a0% TMS (Cambridge Isotope Laboratories,
\n Inc. Andover, MA). Samples were centrifuged to promote phase separation and the organic
\n phase was analyzed using
\n 1<\/sup>
\n H NMR (Jeol ECX-300 NMR, Jeol USA, Inc. Peabody, MA). PHB concentration was determined
\n from an NMR-GC calibration standard 32<\/a>].\n <\/p>\nCalculation of oxygen transfer coefficient<\/h4>\n
\n L<\/sub>
\n a) was determined using a non-fermentative method (\u201cgas out-gas in\u201d) as discussed
\n by Tribe et al. 33<\/a>]. The advantage of this technique is that k
\n L<\/sub>
\n a can be determined directly from dissolved oxygen (DO) measurement 34<\/a>]. Briefly, this method requires gassing the bioreactor with nitrogen to displace the
\n dissolved O
\n 2.<\/sub>
\n Next, air was gassed into the system at sparging rate of 0.4, 0.8 vvm, or via MBD.
\n DO\u00a0% was recorded over time until DO\u00a0% reached approximately 90\u201395\u00a0%. Equation\u00a0133<\/a>] can be used for determining k
\n L<\/sub>
\n a, where: dC
\n L<\/sub>
\n \/dt is change in DO over time (t), C* is saturated DO concentration, and C
\n L<\/sub>
\n is DO concentration in the bioreactor.\n <\/p>\n![\"a](\"\/content\/inline\/s13104-016-2145-9-i1.gif\")
<\/a><\/p>\n
\n L<\/sub>
\n a gives Eq.\u00a02.\n <\/p>\n![\"a](\"\/content\/inline\/s13104-016-2145-9-i2.gif\")
<\/a><\/p>\n
\n L<\/sub>
\n ) verses time (t) produces a linear graph with a slope equal to the k
\n L<\/sub>
\n a in reciprocal time (h
\n ?1<\/sup>
\n ).\n <\/p>\n","protected":false},"excerpt":{"rendered":"