Production of single cell protein from agro-waste using

Rhodococcus opacus
Kristina M. Mahan,1 Rosemary K. Le,1 Tyrone Wells Jr.,1 Seth Anderson,1 Joshua S. Yuan,4
Ryan J. Stoklosa,5,6 Aditya Bhalla,6,7 David B. Hodge,5,6,8 Arthur J. Ragauskas1,2,3,9

1. Department of Chemical and Biomolecular Engineering, The University of Tennessee-

Knoxville, Knoxville, USA
2. Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, USA
3. Department of Forestry, Wildlife and Fisheries, Center of Renewable Carbon, University
of Tennessee, Institute of Agriculture, Knoxville, USA
4. Synthetic and Systems Biology Innovation Hub, Department of Plant Pathology and
Microbiology, Texas A&M University, College Station, USA
5. Department of Chemical Engineering and Materials Science, Michigan State University,
East Lansing, USA
6. Great Lakes Bioenergy Research Center, Michigan State University, East Lansing, USA
7. Department of Biochemistry, Michigan State University, East Lansing, USA
8. Department of Biosystems and Agricultural Engineering, Michigan State University, East
Lansing, USA
9. Systems Biology, Sandia National Laboratories, Livermore, USA

Abstract
Livestock and fish farming are rapidly growing industries facing the simultaneous pressure of
increasing production demands and limited protein required to produce feed. Bacteria that can
convert low-value non-food waste streams into singe cell protein (SCP) present an intriguing
route for rapid protein production. The oleaginous bacterium Rhodococcus opacus serves as a
model organism for understanding microbial lipid production. SCP production has not been
explored using an organism from this genus. In the present research, R. opacus strains DSM
1069 and PD630 were fed three agro-waste streams: (1) orange pulp, juice, and peel; (2) lemon
pulp, juice, and peel; and (3) corn stover effluent, to determine if these low-cost substrates would
be suitable for producing a value-added product, SCP for aquafarming or livestock feed. Both
strains used agro-waste carbon sources as a growth substrate to produce protein-rich cell biomass
suggesting that that R. opacus can be used to produce SCP using agro-wastes as low-cost
substrates.

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Introduction
Single cell protein (SCP) is a protein source from microbial cultures including algae, yeasts,
molds, or bacteria for feeding fish, animals and even humans [4, 13, 22, 29, 32, 42]. With
continuing increase in the world’s agricultural production, SCP is an important protein
substitute in the feed industry [2, 7]. Current industrial farming practices mostly use grains
(corn and soybeans), as animal and fish feed. Fish feed also includes fishmeal which target
food-grade fish that can also be used for human consumption [6]. As the human population
increases, it is crucial to identify sustainable, non-food protein sources. Microbes can be used
to ferment a variety of low-cost agro-wastes and convert them into value-added products [4].
The recycling of these waste materials can reduce the cost of SCP production significantly [ 4].
Reports have shown SCP production from agro-wastes such as citric waste [10, 21, 28, 31, 50],
pineapple cannery effluent [30], yam peels [5], whey concentrates [35, 40], corn stover [1],
rice effluent and polishings [36, 49], soy molasses [9, 12], hydrolyzed sugar cane bagasse [34]
and others. Companies have been working on non-animal, non-vegetable feed for livestock and
fish farms using various strains of bacteria and other single-celled organisms [26, 33].
Microbial SCPs represent a potential future nutrient source because bacteria have high protein
content and can grow rapidly on low-cost substrates with minimum requirements of soil, water,
and temperature conditions [4, 41]. R. opacus strains PD630 and DSM 1069 can aerobically
grow on lignin and sugar compounds [18, 19]. Reports indicate that citrus fruit peels, which
are by-products of the citrus industry, represent about 65% of the total weight of the processed
produce [38] and could become a valuable feed commodity. The agro-waste contains water-
soluble carbohydrates such as glucose, and water-insoluble ones such as pectin and cellulose.
The pectin can be extracted and used for other industrial and pharmaceutical purposes [ 27].
Citrus fruit peels are a currently underutilized resource, due to their production volume. The
residual peel waste is eliminated at disposal sites since the waste is not usable in the wet state
and drying is too costly [38]. Citrus waste has a very low crude protein content (6% on dry
basis), therefore a way to enrich the waste nutrient value must be increased if it is to be used
for animal or fish feed [31]. Another important agro-waste is corn stover which refers to stalks,
leaves and cobs that remain in fields after harvest. Corn stover is one of the primary biomass
resources being used for producing cellulosic ethanol in the United States. It has been
previously shown that pre-treated corn stover effluents containing lignin can be converted to

2
lipids using Rhodococcus [15, 22]. Rhodococcus organisms are heavily studied and used
industrially, but there have been no reports using any Rhodococcus strains to convert agro-
waste into SCP. In this paper, we describe SCP production by R. opacus PD630 and DSM
1069 using agro-waste as a potential low-cost substrate.

Materials and Methods

Organisms and Chemicals
All chemicals were obtained from Fisher Scientific (Hampton, NH, USA) or Sigma Aldrich
(St. Louis, MO, USA). R. opacus DSM1069 and PD630 (DSMZ 44193) strains were purchased
from the German Collection of Microorganisms and Cell Cultures (DSMZ,
http://www.dsmz.de). Corn stover was sourced from Zea mays L. Pioneer hybrid 36H56 by the
Hodge Group at Michigan State University, as described by Liu et al. [23]. Eureka lemons and
Valencia oranges were purchased from the grocery store; size reduced, and blended in an
industrial blender resulting in a mixture of pulp and juice. The orange and lemon mixtures
were frozen and freeze-dried before use.

Media
Two types of media were used during shake flask fermentations in case of both strains: a full
media and a minimal media with adjusted carbon and nitrogen source concentrations. Full
media were prepared according to DSMZ recommendations: DSMZ 65 for strain DSM 1069
and DSMZ 535 for PD630. Minimal media for DSM 1069 contained the following: 0.4 g
KH2PO4, 1.6 g K2HPO4, 0.2 g MgSO4·7H2O, 0.015 g FeCl3, 0.5 mg MnSO4·H2O, 1.0 mg
CuSO4·5H2O, 1.0 mg ZnSO4·7H2O, 0.5 mg CaCl2, 0.1 mg KCl, and 0.5 mg H3BO3 per liter
distilled water [11]. In case of PD630, it contained the following: 9.0 g Na2HPO4·12H2O, 1.5 g
KH2PO4, 0.2 g MgSO4·7H2O, 1.2 mg FeNH4-citrate, 20 mg CaCl2, 2 mL Hoagland solution
(Sigma, H2395), and 0.5 g NaHCO3 per liter distilled water [39]. Added to minimal media
were nitrogen (DSM1069: NH 4NO3 [11]; PD630: NH4Cl [39]) (1% w/v). The pH was set to 7
in all cases, then the media was autoclaved before use. Cells were first inoculated into aerobic
shaker tubes with 10.0 mL full media, and when the absorbance at 600 nm (OD) reached ~ 0.6
the cells were centrifuged and washed twice with minimal media. Cells were then re-suspended
in 10 mL minimal media, and 0.10–1.5 mL was inoculated into the adaptation flasks.

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Adaptation proceeded in minimal media containing 0.1% w/v nitrogen source and the indicated
agro-waste carbon source.

Shake Tube and Flask Fermentation

One loop of cells of the specified strain was transferred to 50 mL full medium (pH 7.0) in a
250 mL flask and cultivated by shaking at 150 rpm and 28°C until the absorbance at 600 nm
(OD) reached ~ 0.6. Subsequently, cells were centrifuged and washed twice with minimal
media. Cells were then re-suspended in minimal media and used to inoculate the 250 mL
adaptation flasks containing 50 mL minimal medium (pH 7.0), 0.1% of the indicated nitrogen
source (w/v), and the indicated agro-waste carbon source. The initial adaptation proceeded for
48 h. Cells were harvested by centrifugation and used to inoculate 250 mL flasks containing
100 mL minimal media, 0.1% indicated nitrogen source (w/v), and the indicated carbon source
[orange waste (12.2 mg/mL), lemon waste (12.2 mg/mL), or corn stover effluent (40 mg/mL)]
(100 mL). Samples were acquired every 24 h for at least 4 days. Minimal medium without
carbon source was used as a negative control.

Agro-waste
Juice, pulp, and peel from oranges and lemons were blended, separately, and frozen at − 20°C
until ready for use. A sample of each blend was lyophilized to determine the solid content of
the material. The lemon blend resulted in a solid content of 19.4 mg/mL and the orange
resulted in 12.2 mg/mL. The lemon blend was diluted to 12.2 mg/mL to be comparable to the
orange substrate directly. Both blends were neutralized to pH 7.0 and autoclave-sterilized, with
0.1% (final) nitrogen content before use. The corn stover solid loading was 40.0 mg/mL. The
effluent was neutralized to pH 7.0 and autoclave-sterilized, with 0.1% (final) nitrogen content
before use.

Analytical Methods
After each sampling, cells were pelleted by centrifugation (5000 rpm, 10 min) and washed in
physiological salt solution twice, then the pellets were freeze-dried and cell dry weights
(CDW) were recorded to track cell growth. To follow the living cell number, serial dilution
and plating (SDP) experiments were done until 10 −8 dilution; then, the results were averaged
and converted to colony forming units per mL (CFU/mL).
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A Coomassie Plus/Bradford protein assay (Pierce/Thermo Fisher Scientific) was used to
determine the total protein concentration in the supernatant/cytosolic material from cell lysate
of a pellet resulting from centrifuging 10 mL of the fermentation broth at 24, 48, 72 and 96 h.
The cells were lysed using the Qproteome Bacterial Protein Kit (Qiagen). The 0 h protein
concentration is the result of assaying the initial, undiluted starting material. Total protein
(g/L) was expressed as the amount of protein produced by either R. opacus PD630 or
DSM1069 in 100 mL medium.

The distribution of monomeric sugars (arabinose, fructose, galactose, glucose, mannose, and
xylose) was detected in the fermentation broth over the course of the experiment. The sugars
were not hydrolyzed prior to detection with Dionex/HPLC and the broths were measured
unmodified. Individual sugar concentrations were calculated by standard curves relating
concentration to peak area. All assays were performed in duplicate, and the averages are
reported. The standard deviation was less than 10%.

Quantification of monosaccharides in the respective fermentation broths were performed using

high-performance anion exchange chromatography with pulsed amperometric detection
(HPAEC-PAD). Specifically, a Dionex ICS-3000 ion chromatograph with CarboPac PA-1
column was utilized. The temperature of the column was set to 30°C and the eluents A and B
were 100% DI water and 200 mM NaOH, respectively. The flow rate was set to 0.35 mL/min.
Duplicate samples of the fermentation broths were analyzed and each analyte was quantified
using linear regression based on calibration curves of an external standard of glucose, xylose,
mannose, galactose, arabinose for the corn stover effluent and fructose were also included for
the lemon and orange substrates (following NREL/TP-510-42618). Disaccharides were not
reported.

Results and Discussion

R. opacus DSM 1069 and PD630 were both able to grow on all three different agro-waste
substrates which included lemon waste, orange waste, and corn stover effluent similar to the
full media condition (Fig. 1a, b). Aerobic shake flasks (250 mL) containing 100 mL minimal

5
media (pH 7.0) with 0.1% w/v nitrogen source and the indicated amounts of given agro-waste
carbon sources were inoculated according to materials and methods. Subsequent growth was
monitored by CDW and SDP (living cell number) measurements. Strains were also grown in a
full media (rich) or a minimal media with no carbon source as controls. The full media
condition allowed for the most growth of both R. opacus strains (PD630: 12.0 log CFU/mL;
DSM 1069: 13.5 log CFU/mL) as compared to the fermentations using the agro-waste
substrates after 96 h (Fig. 1). Growth on corn stover effluent was similar for both strains
(PD630: 9.7 log CFU/mL; DSM 1069: 9.5 log CFU/mL), but the PD630 strain grew better on
orange ago-waste (10.4 log CFU/mL) while the DSM 1069 strain grew better on lemon agro-
waste (11.8 log CFU/mL). R. opacus PD630 had higher protein contents compared to R.
opacusDSM 1069 (Table 1; Fig. 2). The condition that yielded the highest protein content was
growing R. opacus PD630 using lemon agro-waste for 24 h which yielded 2 g/L cell dry
weight and 1.25 g/L total protein for a final protein content of 62.6% (Fig. 2a). The maximum
cell dry weight and total protein yielded 3.55 and 1.77 g/L in a 250 mL shake flask after 48 h
of fermentation using orange agro-waste as growth substrate by the DSM 1069 strain (Fig. 2d).
The protein contents obtained in this study are comparable to what is found in other industrial
feed sources (≤ 72%) [25, 33, 37, 43] with similar amino acid compositions [16, 32, 45]
suggesting that R. opacus strains can adequately be used as a protein supplement for animal
feed.

Monomeric sugar analysis revealed that the citric waste substrates contained differing
concentrations of arabinose, fructose, galactose, glucose, mannose, and xylose (Fig. 2). The
sugar composition indicated that corn stover was mainly composed of: 53% glucose, 25%
fructose, 17% mannose, and 5% galactose. The orange agro-waste substrate was composed
mainly of: 59% glucose, 37% fructose, and 3% mannose, while the lemon agro-waste was
mainly composed of: 66% glucose, 32% fructose, and 2% mannose. Arabinose and xylose
were detected in all samples at concentrations below 1% as previously reported [8, 22].
Fermentations using orange, lemon or corn stover agro-waste as the sole carbon source utilized
glucose, fructose, and mannose (Fig. 2a–f). Sugar consumption generally followed same
pattern for both strains on all three agro-waste substrates. Between 92 and 99% of glucose was
consumed after 24 h, 100% of fructose was consumed between 24 and 72 h. A similar sugar
consumption pattern was seen with the other agro-waste substrates as well as with
6
fermentations using the DSM 1069 strain. The total sugar concentrations became depleted
during the first 24 h of the agro-waste fermentations, while CDW and protein concentrations
increased; resulting in between 42.2 and 68.2% total protein after the 96 h fermentation (Fig. 3;
Table 1). R. opacus DSM 1069 and PD630 were both able to consume sugars from the agro-
waste substrates to produce microbial biomass protein during fermentation.

The pH was also monitored during the 96 h fermentations. R. opacus cells grown using orange
agro-waste resulted in a drastic drop in pH (PD630 pH 5.1; DSM 1069 pH 4.8) which was
unlike what was observed with cells grown using full media (PD630 pH 8.8; DSM 1069 pH
8.0), media using lemon agro-waste (PD630 pH 7.3; DSM 1069 pH 7.0), or media using corn
stover (PD630 pH 7.6; DSM 1069 pH 7.1) as a carbon source (Fig. 3). The pH was not
controlled during the shake flask fermentations indicating that any changes in pH result from a
change in the cell’s metabolism. Fermentations using R. opacus strains growing on different
lignin substrates have typically resulted in an increase in pH. Fermentations resulting in
decreases in pH have also previously been associated with increases in unsaturated lipid
production [18]. Previous experiments have shown that both strains can successfully grow on
various lignin-like compounds and can accumulate more than 20% of their own weight in
lipids under nitrogen-limited shake flask fermentations [18, 24]. The goal of this study was to
maximize protein production and therefore lipid production was not monitored. To minimize
lipid production, cells were grown under non-nitrogen-limited conditions.

Rhodococcus species can grow on various agricultural by-products and lignin-like aromatic
compounds [14, 18, 19]. Much work has been done in using R. opacus PD630 and DSM 1069
to bioconvert lignin compounds to produce lipids for use as biofuels
[3, 17, 18, 19, 20, 44, 46, 47, 48], but no studies have tried to use these organisms to produce
proteins for use in fish or animal feeds. The results of this study demonstrate that citric and
corn stover agro-wastes have the potential as low-cost fermentation substrates for SCP
production using these R. opacusstrains. This study determined that corn stover and citric agro-
waste can be used to generate microbial biomass protein by culture fermentation without
extensive pretreatment or nutrient supplementation. Rhodococcus possess a unique pathway for
sugar catabolism and is able to use multiple aromatic carbon substrates. The species is suited to
grow on agricultural or industrial waste to produce biofuels, SCP, or other value-added
7
products. Results from this study contribute to the body of knowledge concerning microbial
biomass protein production from low-cost agro-waste residues. Fermentations in this study
were done in small shake flasks without controlling for pH and other conditions. Protein
production could be dramatically increased if conditions (i.e., pH, temperature, substrate
concentrations, oxygen concentrations, and incubation time) were optimized and scaled up
using bioreactors. The research indicated that biomass protein could be produced from culture
fermentations using R. opacus strains growing on citric or corn stover agro-waste. The
produced microbial protein can be used for fortification to livestock and poultry feed or be
used as a food source in fish farming.

Acknowledgments
The research team acknowledges the Department of Energy (DE—EE0006112) for funding the
microbial conversion results reported in this manuscript. We thank Dr. Matyas Kosa for
critically reviewing this manuscript.

References

Ahmed S, Ahmad F, Hashmi A (2010) Production of microbial biomass protein by sequential

culture fermentation of Arachniotus Sp., and Candida utilis. Pak J Bot 42:1225–1234

Alexandratos N, Bruinsma J (2012) World agriculture towards 2030/2050: the 2012 revision,
ESA Working paper. FAO, Rome

Alvarez HM, Kalscheuer R, Steinbüchel A (1997) Accumulation of storage lipids in species

of Rhodococcus and Nocardia and effect of inhibitors and polyethylene glycol. Eur J Lipid
Sci Technol 99:239–246

Anupama Ravindra P (2000) Value-added food: single cell protein. Biotechnol Adv 18:459–
479

Aruna TE, Aworh OC, Raji AO, Olagunju AI (2017) Protein enrichment of yam peels by
fermentation with Saccharomyces cerevisiae (BY4743). Ann Agric Sci 62:33–
37. https://doi.org/10.1016/j.aoas.2017.01.002

Cashion T, Le Manach F, Zeller D, Pauly D (2017) Most fish destined for fishmeal production
are food-grade fish. Fish Fish 18:837–844. https://doi.org/10.1111/faf.12209

Chanda S, Chakrabarti S (1996) Plant origin liquid waste: a resource for single-cell protein
production by yeast. Biores Technol 57:51–54

8
Chen SF, Mowery RA, Scarlata CJ, Chambliss CK (2007) Compositional analysis of water-
soluble materials in corn stover. J Agric Food Chem 55:5912–
5918. https://doi.org/10.1021/jf0700327

Cheung PCK (1997) Chemical evaluation of some lesser known edible mushroom mycelia
produced in submerged culture from soy milk waste. Food Chem 60:61–65

De Gregorio A, Mandalari G, Arena N, Nucita F, Tripodo MM, Lo Curto RB (2002) SCP and
crude pectinase production by slurry-state fermentation of lemon pulps. Biores Technol
83:89–94. https://doi.org/10.1016/S0960-8524(01)00209-7

Eggeling L, Sahm H (1980) Degradation of coniferyl alcohol and other lignin-related aromatic-
compounds by Nocardia sp DSM-1069. Arch Microbiol 126:141–148

Gao YR, Li DP, Liu Y (2012) Production of single cell protein from soy molasses
using Candida tropicalis. Ann Microbiol 62:1165–1172

Goldberg I (2013) Single cell protein, vol 1. Springer, New York

Harwood CS, Parales RE (1996) The beta-ketoadipate pathway and the biology of self-identity.
Annu Rev Microbiol 50:553–590

He Y, Li X, Ben H, Xue X, Yang B (2017) Lipid production from dilute alkali corn stover
lignin by Rhodococcus strains. ACS Sustain Chem Eng 5:2302–
2311. https://doi.org/10.1021/acssuschemeng.6b02627

Holder JW, Ulrich JC, DeBono AC, Godfrey PA, Desjardins CA, Zucker J, Zeng Q, Leach AL,
Ghiviriga I, Dancel C, Abeel T, Gevers D, Kodira CD, Desany B, Affourtit JP, Birren BW,
Sinskey AJ (2011) Comparative and functional genomics of Rhodococcus opacusPD630
for biofuels development. PLoS Genet
7:e1002219. https://doi.org/10.1371/journal.pgen.1002219

Klatte S, Kroppenstedt RM, Rainey FA (1994) Rhodococcus opacus sp. nov., an unusual
nutritionally versatile Rhodococcus-species. Syst Appl Microbiol 17:355–360

Kosa M, Ragauskas AJ (2012) Bioconversion of lignin model compounds with

oleaginous Rhodococci. Appl Microbiol Biotechnol 93:891–900

Kosa M, Ragauskas AJ (2013) Lignin to lipid bioconversion by oleaginous Rhodococci. Green

Chem 15:2070–2074

Lancefield CS, Rashid GM, Bouxin F, Wasak A, Tu W-C, Hallett J, Zein S, Rodriguez J,
Jackson SD, Westwood NJ (2016) Investigation of the chemocatalytic and biocatalytic
valorization of a range of different lignin preparations: the importance of β-O-4 content.
ACS Sustain Chem Eng 4:6921–6930

Lanuzza F, Mondello F, Tripodo MM (2014) Studies About the Utilization of Citrus Wastes in
View of Environment Protection. In: Pathways to Environmental Sustainability. Springer,
pp 147-156
9
Le RK, Wells T, Das P, Meng XZ, Stoklosa RJ, Bhalla A, Hodge DB, Yuan JS, Ragauskas AJ
(2017) Conversion of corn stover alkaline pre-treatment waste streams into biodiesel
via Rhodococci. RSC Adv 7:4108–4115

Liu T, Williams DL, Pattathil S, Li M, Hahn MG, Hodge DB (2014) Coupling alkaline pre-
extraction with alkaline-oxidative post-treatment of corn stover to enhance enzymatic
hydrolysis and fermentability. Biotechnol Biofuels 7:48. https://doi.org/10.1186/1754-
6834-7-48

Mahan KM, Le RK, Yuan J, Ragauskas AJ (2017) A review on the bioconversion of lignin to
microbial lipid with oleaginous Rhodococcus opacus. J Biotechnol Biomater 7:262

Makkar HP, Tran G, Heuzé V, Ankers P (2014) State-of-the-art on use of insects as animal
feed. Anim Feed Sci Technol 197:1–33

Matassa S, Boon N, Pikaar I, Verstraete W (2016) Microbial protein: future sustainable food
supply route with low environmental footprint. Microb Biotechnol 9:568–575

May CD (1990) Industrial pectins—sources, production and applications. Carbohyd Polym

12:79–99

Mondal AK, Sengupta S, Bhowal J, Bhattacharya D (2012) Utilization of fruit wastes in

producing single cell protein. Int J Sci Environ Technol 1:430–438

Nasseri A, Rasoul-Amini S, Morowvat M, Ghasemi Y (2011) Single cell protein: production

and process. Am J Food Technol 6:103–116

Nigam JN (1998) Single cell protein from pineapple cannery effluent. World J Microbiol
Biotechnol 14:693–696

Nishio N, Nagai S (1981) Single cell protein-production from mandarin orange peel. Eur J
Appl Microbiol Biotechnol 11:156–160

Overland M, Skrede A (2017) Yeast derived from lignocellulosic biomass as a sustainable feed
resource for use in aquaculture. J Sci Food Agric 97:733–
742. https://doi.org/10.1002/jsfa.8007

Overland M, Tauson AH, Shearer K, Skrede A (2010) Evaluation of methane-utilising bacteria

products as feed ingredients for monogastric animals. Arch Anim Nutr 64:171–
189. https://doi.org/10.1080/17450391003691534

Pandey A, Soccol CR, Nigam P, Soccol VT (2000) Biotechnological potential of agro-

industrial residues. I: sugarcane bagasse. Biores Technol 74:69–80

Paraskevopoulou A, Athanasiadis I, Kanellaki M, Bekatorou A, Blekas G, Kiosseoglou V

(2003) Functional properties of single cell protein produced by kefir microflora. Food Res
Int 36:431–438

10
Rajoka MI, Kiani MAT, Khan S, Awan MS, Hashmi AS (2004) Production of single cell
protein from rice polishings using Candida utilis. World J Microbiol Biotechnol 20:297–
301

Safi C, Zebib B, Merah O, Pontalier P-Y, Vaca-Garcia C (2014) Morphology, composition,

production, processing and applications of Chlorella vulgaris: a review. Renew Sustain
Energy Rev 35:265–278

Scerra V, Caridi A, Foti F, Sinatra MC (1999) Influence of dairy Penicillium spp. on nutrient
content of citrus fruit peel. Anim Feed Sci Technol 78:169–176

Schlegel HG, Kaltwasser H, Gottschalk G (1961) Ein submersverfahren zur kultur

wasserstoffoxydierender bakterien—wachstumsphysiologische untersuchungen. Archiv Fur
Mikrobiologie 38:209

Schultz N, Chang LF, Hauck A, Reuss M, Syldatk C (2006) Microbial production of single-
cell protein from deproteinized whey concentrates. Appl Microbiol Biotechnol 69:515–520

Stringer DA (1982) Industrial-development and evaluation of new protein-sources—

microorganisms. Proc Nutr Soc 41:289–300

Suman G, Nupur M, Anuradha S, Pradeep B (2015) Single cell protein production: a review.
Int J Curr Microbiol Appl Sci 4:251–262

Tacon AG, Metian M, Hasan (2009) Feed ingredients and fertilizers for farmed aquatic
animals: sources and composition, vol 540. Food and Agriculture Organization of the
United Nations (FAO), Rome

Wältermann M, Luftmann H, Baumeister D, Kalscheuer R, Steinbüchel A (2000) Rhodococcus

opacus strain PD630 as a new source of high-value single-cell oil? Isolation and
characterization of triacylglycerols and other storage lipids. Microbiology 146:1143–1149

Wang JP, Kim JD, Kim JE, Kim IH (2013) Amino acid digestibility of single cell protein
from Corynebacterium ammoniagenes in growing pigs. Anim Feed Sci Technol 180:111–
114. https://doi.org/10.1016/j.anifeedsci.2012.12.006

Wei Z, Zeng G, Huang F, Kosa M, Sun Q, Meng X, Huang D, Ragauskas AJ (2015) Microbial
lipid production by oleaginous Rhodococci cultured in lignocellulosic autohydrolysates.
Appl Microbiol Biotechnol 99:7369–7377

Wei Z, Zeng G, Kosa M, Huang D, Ragauskas AJ (2015) Pyrolysis oil-based lipid production
as biodiesel feedstock by Rhodococcus opacus. Appl Biochem Biotechnol 175:1234–1246

Wells T, Wei Z, Ragauskas A (2015) Bioconversion of lignocellulosic pretreatment effluent

via oleaginous Rhodococcus opacus DSM 1069. Biomass Bioenerg 72:200–205

Zepka LQ, Jacob-Lopes E, Goldbeck R, Souza-Soares LA, Queiroz MI (2010) Nutritional

evaluation of single-cell protein produced by Aphanothece microscopica Nageli. Biores
Technol 101:7107–7111
11
Zhou Y-M, Chen Y-P, Shen Y (2017) Single cell protein-feed: Taking orange waste as raw
material for fermentation. In: Advanced materials and energy sustainability: proceedings of
the 2016 international conference on advanced materials and energy sustainability
(AMES2016). World Scientific, Singapore, pp 323–335

12